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Sample records for aminoglycoside resistance methyltransferase

  1. Intrinsic resistance to aminoglycosides in Enterococcus faecium is conferred by the 16S rRNA m5C1404-specific methyltransferase EfmM

    DEFF Research Database (Denmark)

    Galimand, Marc; Schmitt, Emmanuelle; Panvert, Michel

    2011-01-01

    methyltransferase, as well as by the previously characterized aac(6')-Ii that encodes a 6'-N-aminoglycoside acetyltransferase. Inactivation of efmM in E. faecium increases susceptibility to the aminoglycosides kanamycin and tobramycin, and, conversely, expression of a recombinant version of efmM in Escherichia coli...... confers resistance to these drugs. The EfmM protein shows significant sequence similarity to E. coli RsmF (previously called YebU), which is a 5-methylcytidine (m(5)C) methyltransferase modifying 16S rRNA nucleotide C1407. The target for EfmM is shown by mass spectrometry to be a neighboring 16S r...

  2. Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin.

    Science.gov (United States)

    McGann, Patrick; Chahine, Sarah; Okafor, Darius; Ong, Ana C; Maybank, Rosslyn; Kwak, Yoon I; Wilson, Kerry; Zapor, Michael; Lesho, Emil; Hinkle, Mary

    2016-01-01

    16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  3. Association of the novel aminoglycoside resistance determinant RmtF with NDM carbapenemase in Enterobacteriaceae isolated in India and the UK

    DEFF Research Database (Denmark)

    Hidalgo, Laura; Hopkins, Katie L; Gutierrez, Belen

    2013-01-01

    16S rRNA methyltransferases are an emerging mechanism conferring high-level resistance to clinically relevant aminoglycosides and have been associated with important mechanisms such as NDM-1. We sought genes encoding these enzymes in isolates highly resistant (MIC >200 mg/L) to gentamicin and ami...

  4. Rapid Aminoglycoside NP Test for Rapid Detection of Multiple Aminoglycoside Resistance in Enterobacteriaceae.

    Science.gov (United States)

    Nordmann, Patrice; Jayol, Aurélie; Dobias, Jan; Poirel, Laurent

    2017-04-01

    The rapid aminoglycoside NP (Nordmann/Poirel) test was developed to rapidly identify multiple aminoglycoside (AG) resistance in Enterobacteriaceae It is based on the detection of the glucose metabolism related to enterobacterial growth in the presence of a defined concentration of amikacin plus gentamicin. Formation of acid metabolites was evidenced by a color change (orange to yellow) of the red phenol pH indicator. The rapid aminoglycoside NP test was evaluated by using bacterial colonies of 18 AG-resistant isolates producing 16S rRNA methylases, 20 AG-resistant isolates expressing AG-modifying enzymes (acetyl-, adenyl-, and phosphotransferases), and 10 isolates susceptible to AG. Its sensitivity and specificity were 100% and 97%, respectively, compared to the broth dilution method, which was taken as the gold standard for determining aminoglycoside resistance. The test is inexpensive, rapid (<2 h), and implementable worldwide. Copyright © 2017 American Society for Microbiology.

  5. AMINOGLYCOSIDE RESISTANCE GENES IN Pseudomonas aeruginosa ISOLATES FROM CUMANA, VENEZUELA

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    Bertinellys TEIXEIRA

    2016-01-01

    Full Text Available The enzymatic modification of aminoglycosides by aminoglycoside-acetyltransferases (AAC, aminoglycoside-adenyltransferases (AAD, and aminoglycoside-phosphotransferases (APH, is the most common resistance mechanism in P. aeruginosa and these enzymes can be coded on mobile genetic elements that contribute to their dispersion. One hundred and thirty seven P. aeruginosa isolates from the University Hospital, Cumana, Venezuela (HUAPA were evaluated. Antimicrobial susceptibility was determined by the disk diffusion method and theaac, aadB and aph genes were detected by PCR. Most of the P. aeruginosa isolates (33/137 were identified from the Intensive Care Unit (ICU, mainly from discharges (96/137. The frequency of resistant P. aeruginosaisolates was found to be higher for the aminoglycosides tobramycin and amikacin (30.7 and 29.9%, respectively. Phenotype VI, resistant to these antibiotics, was the most frequent (14/49, followed by phenotype I, resistant to all the aminoglycosides tested (12/49. The aac(6´-Ib,aphA1 and aadB genes were the most frequently detected, and the simultaneous presence of several resistance genes in the same isolate was demonstrated. Aminoglycoside resistance in isolates ofP. aeruginosa at the HUAPA is partly due to the presence of the aac(6´-Ib, aphA1 andaadB genes, but the high rates of antimicrobial resistance suggest the existence of several mechanisms acting together. This is the first report of aminoglycoside resistance genes in Venezuela and one of the few in Latin America.

  6. AMINOGLYCOSIDE RESISTANCE GENES IN Pseudomonas aeruginosa ISOLATES FROM CUMANA, VENEZUELA.

    Science.gov (United States)

    Teixeira, Bertinellys; Rodulfo, Hectorina; Carreño, Numirin; Guzmán, Militza; Salazar, Elsa; De Donato, Marcos

    2016-01-01

    The enzymatic modification of aminoglycosides by aminoglycoside-acetyltransferases (AAC), aminoglycoside-adenyltransferases (AAD), and aminoglycoside-phosphotransferases (APH), is the most common resistance mechanism in P. aeruginosa and these enzymes can be coded on mobile genetic elements that contribute to their dispersion. One hundred and thirty seven P. aeruginosa isolates from the University Hospital, Cumana, Venezuela (HUAPA) were evaluated. Antimicrobial susceptibility was determined by the disk diffusion method and theaac, aadB and aph genes were detected by PCR. Most of the P. aeruginosa isolates (33/137) were identified from the Intensive Care Unit (ICU), mainly from discharges (96/137). The frequency of resistant P. aeruginosaisolates was found to be higher for the aminoglycosides tobramycin and amikacin (30.7 and 29.9%, respectively). Phenotype VI, resistant to these antibiotics, was the most frequent (14/49), followed by phenotype I, resistant to all the aminoglycosides tested (12/49). The aac(6´)-Ib,aphA1 and aadB genes were the most frequently detected, and the simultaneous presence of several resistance genes in the same isolate was demonstrated. Aminoglycoside resistance in isolates ofP. aeruginosa at the HUAPA is partly due to the presence of the aac(6´)-Ib, aphA1 andaadB genes, but the high rates of antimicrobial resistance suggest the existence of several mechanisms acting together. This is the first report of aminoglycoside resistance genes in Venezuela and one of the few in Latin America.

  7. In Silico Assigned Resistance Genes Confer Bifidobacterium with Partial Resistance to Aminoglycosides but Not to Β-Lactams

    Science.gov (United States)

    Fouhy, Fiona; O’Connell Motherway, Mary; Fitzgerald, Gerald F.; Ross, R. Paul; Stanton, Catherine; van Sinderen, Douwe; Cotter, Paul D.

    2013-01-01

    Bifidobacteria have received significant attention due to their contribution to human gut health and the use of specific strains as probiotics. It is thus not surprising that there has also been significant interest with respect to their antibiotic resistance profile. Numerous culture-based studies have demonstrated that bifidobacteria are resistant to the majority of aminoglycosides, but are sensitive to β-lactams. However, limited research exists with respect to the genetic basis for the resistance of bifidobacteria to aminoglycosides. Here we performed an in-depth in silico analysis of putative Bifidobacterium-encoded aminoglycoside resistance proteins and β-lactamases and assess the contribution of these proteins to antibiotic resistance. The in silico-based screen detected putative aminoglycoside and β-lactam resistance proteins across the Bifidobacterium genus. Laboratory-based investigations of a number of representative bifidobacteria strains confirmed that despite containing putative β-lactamases, these strains were sensitive to β-lactams. In contrast, all strains were resistant to the aminoglycosides tested. To assess the contribution of genes encoding putative aminoglycoside resistance proteins in Bifidobacterium sp. two genes, namely Bbr_0651 and Bbr_1586, were targeted for insertional inactivation in B. breve UCC2003. As compared to the wild-type, the UCC2003 insertion mutant strains exhibited decreased resistance to gentamycin, kanamycin and streptomycin. This study highlights the associated risks of relying on the in silico assignment of gene function. Although several putative β-lactam resistance proteins are located in bifidobacteria, their presence does not coincide with resistance to these antibiotics. In contrast however, this approach has resulted in the identification of two loci that contribute to the aminoglycoside resistance of B. breve UCC2003 and, potentially, many other bifidobacteria. PMID:24324818

  8. In silico assigned resistance genes confer Bifidobacterium with partial resistance to aminoglycosides but not to β-lactams.

    Directory of Open Access Journals (Sweden)

    Fiona Fouhy

    Full Text Available Bifidobacteria have received significant attention due to their contribution to human gut health and the use of specific strains as probiotics. It is thus not surprising that there has also been significant interest with respect to their antibiotic resistance profile. Numerous culture-based studies have demonstrated that bifidobacteria are resistant to the majority of aminoglycosides, but are sensitive to β-lactams. However, limited research exists with respect to the genetic basis for the resistance of bifidobacteria to aminoglycosides. Here we performed an in-depth in silico analysis of putative Bifidobacterium-encoded aminoglycoside resistance proteins and β-lactamases and assess the contribution of these proteins to antibiotic resistance. The in silico-based screen detected putative aminoglycoside and β-lactam resistance proteins across the Bifidobacterium genus. Laboratory-based investigations of a number of representative bifidobacteria strains confirmed that despite containing putative β-lactamases, these strains were sensitive to β-lactams. In contrast, all strains were resistant to the aminoglycosides tested. To assess the contribution of genes encoding putative aminoglycoside resistance proteins in Bifidobacterium sp. two genes, namely Bbr_0651 and Bbr_1586, were targeted for insertional inactivation in B. breve UCC2003. As compared to the wild-type, the UCC2003 insertion mutant strains exhibited decreased resistance to gentamycin, kanamycin and streptomycin. This study highlights the associated risks of relying on the in silico assignment of gene function. Although several putative β-lactam resistance proteins are located in bifidobacteria, their presence does not coincide with resistance to these antibiotics. In contrast however, this approach has resulted in the identification of two loci that contribute to the aminoglycoside resistance of B. breve UCC2003 and, potentially, many other bifidobacteria.

  9. Aminoglycoside resistance among isolates of nosocomial Enterobacteriaceae

    International Nuclear Information System (INIS)

    Botha, P.L.; Elisha, G.; Pratt, K.

    1981-01-01

    Fifty-seven gentamicin-resistant isolates of Enterobacteriaceae, obtained from patients attending hospital, were examined for the production of aminoglycoside-modifying enzymes. Of the 51 strains producing such enzymes, 34 were presumptively plasmid-mediated as indicated by conjugation experiments

  10. Cytosolic proteome profiling of aminoglycosides resistant Mycobacterium tuberculosis clinical isolates using MALDI-TOF/MS

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    Divakar Sharma

    2016-11-01

    Full Text Available Emergence of extremely drug resistant tuberculosis (XDR-TB is the consequence of the failure of second line TB treatment. Aminoglycosides are the important second line anti-TB drugs used to treat the multi drug resistant tuberculosis (MDR-TB. Main known mechanism of action of aminoglycosides is to inhibit the protein synthesis by inhibiting the normal functioning of ribosome. Primary target of aminoglycosides are the ribosomal RNA and its associated proteins. Various mechanisms have been proposed for aminoglycosides resistance but still some are unsolved. As proteins are involved in most of the biological processes, these act as a potential diagnostic markers and drug targets. In the present study we analyzed the purely cytosolic proteome of amikacin (AK and kanamycin (KM resistant Mycobacterium tuberculosis isolates by proteomic and bioinformatic approaches. Twenty protein spots were found to have over expressed in resistant isolates and were identified. Among these Rv3208A, Rv2623, Rv1360, Rv2140c, Rv1636 and Rv2185c are six proteins with unknown functions or undefined role. Docking results showed that AK and KM binds to the conserved domain (DUF, USP-A, Luciferase, PEBP and Polyketidecyclase/dehydrase domain of these hypothetical proteins and over expression of these proteins might neutralize/modulate the effect of drug molecules. TBPred and GPS-PUP predicted cytoplasmic nature and potential pupylation sites within these identified proteins respectively. String analysis also suggested that over expressed proteins along with their interactive partners might be involved in aminoglycosides resistance. Cumulative effect of these over expressed proteins could be involved in AK and KM resistance by mitigating the toxicity, repression of drug target and neutralizing affect. These findings need further exploitation for the expansion of newer therapeutics or diagnostic markers against AK and KM resistance so that an extreme condition like XDR-TB can

  11. Cytosolic Proteome Profiling of Aminoglycosides Resistant Mycobacterium tuberculosis Clinical Isolates Using MALDI-TOF/MS.

    Science.gov (United States)

    Sharma, Divakar; Lata, Manju; Singh, Rananjay; Deo, Nirmala; Venkatesan, Krishnamurthy; Bisht, Deepa

    2016-01-01

    Emergence of extensively drug resistant tuberculosis (XDR-TB) is the consequence of the failure of second line TB treatment. Aminoglycosides are the important second line anti-TB drugs used to treat the multi drug resistant tuberculosis (MDR-TB). Main known mechanism of action of aminoglycosides is to inhibit the protein synthesis by inhibiting the normal functioning of ribosome. Primary target of aminoglycosides are the ribosomal RNA and its associated proteins. Various mechanisms have been proposed for aminoglycosides resistance but still some are unsolved. As proteins are involved in most of the biological processes, these act as a potential diagnostic markers and drug targets. In the present study we analyzed the purely cytosolic proteome of amikacin (AK) and kanamycin (KM) resistant Mycobacterium tuberculosis isolates by proteomic and bioinformatic approaches. Twenty protein spots were found to have over expressed in resistant isolates and were identified. Among these Rv3208A, Rv2623, Rv1360, Rv2140c, Rv1636, and Rv2185c are six proteins with unknown functions or undefined role. Docking results showed that AK and KM binds to the conserved domain (DUF, USP-A, Luciferase, PEBP and Polyketidecyclase/dehydrase domain) of these hypothetical proteins and over expression of these proteins might neutralize/modulate the effect of drug molecules. TBPred and GPS-PUP predicted cytoplasmic nature and potential pupylation sites within these identified proteins, respectively. String analysis also suggested that over expressed proteins along with their interactive partners might be involved in aminoglycosides resistance. Cumulative effect of these over expressed proteins could be involved in AK and KM resistance by mitigating the toxicity, repression of drug target and neutralizing affect. These findings need further exploitation for the expansion of newer therapeutics or diagnostic markers against AK and KM resistance so that an extreme condition like XDR-TB can be prevented.

  12. Novel Aminoglycoside Resistance Transposons and Transposon-Derived Circular Forms Detected in Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates

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    Dwibedi, Chinmay Kumar; Sjöström, Karin; Edquist, Petra; Wai, Sun Nyunt; Uhlin, Bernt Eric

    2016-01-01

    Acinetobacter baumannii has emerged as an important opportunistic pathogen equipped with a growing number of antibiotic resistance genes. Our study investigated the molecular epidemiology and antibiotic resistance features of 28 consecutive carbapenem-resistant clinical isolates of A. baumannii collected throughout Sweden in 2012 and 2013. The isolates mainly belonged to clonal complexes (CCs) with an extensive international distribution, such as CC2 (n = 16) and CC25 (n = 7). Resistance to carbapenems was related to blaOXA-23 (20 isolates), blaOXA-24/40-like (6 isolates), blaOXA-467 (1 isolate), and ISAba1-blaOXA-69 (1 isolate). Ceftazidime resistance was associated with blaPER-7 in the CC25 isolates. Two classical point mutations were responsible for resistance to quinolones in all the isolates. Isolates with high levels of resistance to aminoglycosides carried the 16S rRNA methylase armA gene. The isolates also carried a variety of genes encoding aminoglycoside-modifying enzymes. Several novel structures involved in aminoglycoside resistance were identified, including Tn6279, ΔTn6279, Ab-ST3-aadB, and different assemblies of Tn6020 and TnaphA6. Importantly, a number of circular forms related to the IS26 or ISAba125 composite transposons were detected. The frequent occurrence of these circular forms in the populations of several isolates indicates a potential role of these circular forms in the dissemination of antibiotic resistance genes. PMID:26824943

  13. Structural and molecular basis for resistance to aminoglycoside antibiotics by the adenylyltransferase ANT(2″)-Ia.

    Science.gov (United States)

    Cox, Georgina; Stogios, Peter J; Savchenko, Alexei; Wright, Gerard D

    2015-01-06

    The aminoglycosides are highly effective broad-spectrum antimicrobial agents. However, their efficacy is diminished due to enzyme-mediated covalent modification, which reduces affinity of the drug for the target ribosome. One of the most prevalent aminoglycoside resistance enzymes in Gram-negative pathogens is the adenylyltransferase ANT(2″)-Ia, which confers resistance to gentamicin, tobramycin, and kanamycin. Despite the importance of this enzyme in drug resistance, its structure and molecular mechanism have been elusive. This study describes the structural and mechanistic basis for adenylylation of aminoglycosides by the ANT(2″)-Ia enzyme. ANT(2″)-Ia confers resistance by magnesium-dependent transfer of a nucleoside monophosphate (AMP) to the 2″-hydroxyl of aminoglycoside substrates containing a 2-deoxystreptamine core. The catalyzed reaction follows a direct AMP transfer mechanism from ATP to the substrate antibiotic. Central to catalysis is the coordination of two Mg(2+) ions, positioning of the modifiable substrate ring, and the presence of a catalytic base (Asp86). Comparative structural analysis revealed that ANT(2″)-Ia has a two-domain structure with an N-terminal active-site architecture that is conserved among other antibiotic nucleotidyltransferases, including Lnu(A), LinB, ANT(4')-Ia, ANT(4″)-Ib, and ANT(6)-Ia. There is also similarity between the nucleotidyltransferase fold of ANT(2″)-Ia and DNA polymerase β. This similarity is consistent with evolution from a common ancestor, with the nucleotidyltransferase fold having adapted for activity against chemically distinct molecules. IMPORTANCE  : To successfully manage the threat associated with multidrug-resistant infectious diseases, innovative therapeutic strategies need to be developed. One such approach involves the enhancement or potentiation of existing antibiotics against resistant strains of bacteria. The reduction in clinical usefulness of the aminoglycosides is a particular

  14. Comprehensive study to investigate the role of various aminoglycoside resistance mechanisms in clinical isolates of Acinetobacter baumannii.

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    Sheikhalizadeh, Vajihe; Hasani, Alka; Ahangarzadeh Rezaee, Mohammad; Rahmati-Yamchi, Mohammad; Hasani, Akbar; Ghotaslou, Reza; Goli, Hamid Reza

    2017-02-01

    Therapeutic resistance towards most of the current treatment regime by Acinetobacter baumannii has reduced the prescribing antibiotic pattern and option is being re-shifted towards more toxic agents including aminoglycosides. The present investigation aimed at to study various mechanisms towards aminoglycoside non-susceptibility in clinical isolates of A. baumannii. The bacteria were subjected to genetic basis assessment for the presence of aminoglycoside modifying enzymes (AME), 16S rRNA methylase encoding genes and relative expression of AdeABC and AbeM efflux pumps in relation to their susceptibility to five aminoglycosides. When isolates were subjected to typing by repetitive extragenic palindromic (REP) PCR, isolates could be separated into thirteen definite clones. The majority of isolates (94%) were positive for AME encoding genes. Possession of ant(2')-Ia correlated with non-susceptibility towards gentamicin, amikacin, kanamycin, tobramycin; while, presence of aph(3')-VIa attributed to resistance towards amikacin, kanamycin; possession of aac(3')-Ia allied with non-susceptibility to amikacin, tobramycin and presence of aac(3')IIa correlated with kanamycin non-susceptibility. Presence of armA was detected in 34.4%, 34.2%, 29.2%, 40.3%, and 64.2% of isolates showing non-susceptibility to gentamicin, amikacin, kanamycin, tobramycin and netilmicin, respectively. No isolates were found to carry rmtB or rmtC. Amikacin non-susceptibility in comparison to other aminoglycosides correlated with over production of adeB. Overall, the results represented a definitive correlation between presence of AME encoding genes as well as armA and resistance of A. baumannii towards aminoglycosides. On the other hand, the up-regulation of AdeABC and AbeM systems was found to have only the partial role in development of aminoglycoside resistance. Copyright © 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All

  15. Distinction between the Cfr Methyltransferase Conferring Antibiotic Resistance and the Housekeeping RlmN Methyltransferase

    DEFF Research Database (Denmark)

    Atkinson, Gemma C; Hansen, Lykke H; Tenson, Tanel

    2013-01-01

    The cfr gene encodes the Cfr methyltransferase that primarily methylates C-8 in A2503 of 23S rRNA in the peptidyl transferase region of bacterial ribosomes. The methylation provides resistance to six classes of antibiotics of clinical and veterinary importance. The rlmN gene encodes the Rlm......N methyltransferase that methylates C-2 in A2503 in 23S rRNA and A37 in tRNA, but RlmN does not significantly influence antibiotic resistance. The enzymes are homologous and use the same mechanism involving radical S-adenosyl methionine to methylate RNA via an intermediate involving a methylated cysteine....... The differentiation between the two classes is supported by previous and new experimental evidence from antibiotic resistance, primer extensions, and mass spectrometry. Finally, evolutionary aspects of the distribution of Cfr- and RlmN-like enzymes are discussed....

  16. A study of gram-negative bacterial resistance to Aminoglycosides

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    Maleknejad P

    1993-05-01

    Full Text Available From hygienic and economical point of view, drug therapy and prophylaxy in infectious diseases are of great importance. After the world war II, a reduction in the efficacy of sulfonamide in the treatment of shigellosis was observed and later on it led to a survey on drug resistance and the way of its transmission. The aim of this survey, during which 100 cases of gram-negative bacteria were identified, is to study the drug resistance of this bacteria against five types of aminoglycosides by antibiotic sensitivity test (disc-diffusion. Out of 100 strains, 47% were resistant to gentamycin, 70% to kanamycin, 82% to streptomycin, 53% to tobramycin, and 8% to amikacin

  17. Chaperonin GroEL/GroES Over-Expression Promotes Aminoglycoside Resistance and Reduces Drug Susceptibilities in Escherichia coli Following Exposure to Sublethal Aminoglycoside Doses

    DEFF Research Database (Denmark)

    Goltermann, Lise; Sarusie, Menachem V; Bentin, Thomas

    2016-01-01

    Antibiotic resistance is an increasing challenge to modern healthcare. Aminoglycoside antibiotics cause translation corruption and protein misfolding and aggregation in Escherichia coli. We previously showed that chaperonin GroEL/GroES depletion and over-expression sensitize and promote short...

  18. An overview of strategies for synthetic modifications of aminoglycosides

    International Nuclear Information System (INIS)

    Aslam, M.W.

    2013-01-01

    Aminoglycosides, a family of structurally related broad-spectrum bactericidal compounds, have been used extensively for the treatment of aerobic gram-negative bacterial infections because of their rapid anti-bacterial activity, chemical stability and ability to work in combination with other drugs. However, toxic side-effects and growing bacterial resistance have narrowed the significance of aminoglycosides as antibiotics. Due to these limitations, extensive research on aminoglycosides has resulted in the development of a wide range of synthetic strategies to improve the overall characteristics of aminoglycosides. Herein, an overview of the various approaches reported in the literature to enhance binding affinity and to overcome resistance of aminoglycosides is provided. (author)

  19. Chromosomal mechanisms of aminoglycoside resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients

    DEFF Research Database (Denmark)

    Islam, S; Oh, H; Jalal, S

    2009-01-01

    pump protein MexY was determined by real-time PCR and correlated with susceptibilities to amikacin and tobramycin. The chromosomal genes mexZ, rplY, galU, PA5471 and nuoG, which were found to have a role in the gradual increase in MICs of aminoglycoside antibiotics in laboratory mutants of P....... aeruginosa, were analysed. MexY mRNA overproduction was found in 17/20 isolates collected in 1994 and 1997, and was correlated with decreased susceptibility to aminoglycosides. Alteration of the MexXY-OprM efflux system has been the main mechanism of resistance to aminoglycoside antibiotics in CF P......In total, 40 Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients were included in this study. Twenty of these were collected in 1994 and 1997, from six CF patients, and the rest were collected from different CF patients in 2000 and 2001. The relative expression of mRNA for the efflux...

  20. Novel Tn916-like elements confer aminoglycoside/macrolide co-resistance in clinical isolates of Streptococcus gallolyticus ssp. gallolyticus.

    Science.gov (United States)

    Kambarev, Stanimir; Pecorari, Frédéric; Corvec, Stéphane

    2018-02-09

    Streptococcus gallolyticus ssp. gallolyticus (Sgg) is a commensal bacterium and an opportunistic pathogen. In humans it has been clinically associated with the incidence of colorectal cancer (CRC) and epidemiologically recognized as an emerging cause of infective endocarditis (IE). The standard therapy of Sgg includes the administration of a penicillin in combination with an aminoglycoside. Even though penicillin-resistant isolates have still not been reported, epidemiological studies have shown that this microbe is a reservoir of multiple acquired genes, conferring resistance to tetracyclines, aminoglycosides, macrolides and glycopeptides. However, the underlying antibiotic resistance mobilome of Sgg remains poorly understood. To investigate the mobile genetic basis of antibiotic resistance in multiresistant clinical Sgg. Isolate NTS31106099 was recovered from a patient with IE and CRC at Nantes University Hospital, France and studied by Illumina WGS and comparative genomics. Molecular epidemiology of the identified mobile element(s) was performed using antibiotic susceptibility testing (AST), PCR, PFGE and WGS. Mobility was investigated by PCR and filter mating. Two novel conjugative transposons, Tn6263 and Tn6331, confer aminoglycoside/macrolide co-resistance in clinical Sgg. They display classical family Tn916/Tn1545 modular architecture and harbour an aph(3')-III→sat4→ant(6)-Ia→erm(B) multiresistance gene cluster, related to pRE25 of Enterococcus faecium. These and/or closely related elements are highly prevalent among genetically heterogeneous clinical isolates of Sgg. Previously unknown Tn916-like mobile genetic elements conferring aminoglycoside/macrolide co-resistance make Sgg, collectively with other gut Firmicutes such as enterococci and eubacteria, a potential laterally active reservoir of these antibiotic resistance determinants among the mammalian gastrointestinal microbiota. © The Author(s) 2018. Published by Oxford University Press on behalf

  1. In vitro studies with UK-18,892, a new aminoglycoside antibiotic.

    Science.gov (United States)

    Jevons, S; Cheeseman, H E; Brammer, K W

    1978-09-01

    The antibacterial activity of UK-18,892, a new semisynthetic aminoglycoside, was examined against aminoglycoside-susceptible and aminoglycoside-resistant clinical isolates of gram-negative bacilli and Staphylococcus aureus. UK-18,892 had a similar degree of activity to those of amikacin and kanamycin A against aminoglycoside-susceptible bacteria but was less potent than gentamicin against all isolates except Providencia spp. UK-18,892 was highly active against aminoglycoside-resistant bacteria, inhibiting 93% of the 268 isolates examined at 12.5 mug/ml. Amikacin was similarly active, whereas gentamicin inhibited only 14% of these isolates at 12.5 mug/ml.

  2. Characterization of resistance to tetracyclines and aminoglycosides of sheep mastitis pathogens: study of the effect of gene content on resistance.

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    Lollai, S A; Ziccheddu, M; Duprè, I; Piras, D

    2016-10-01

    Mastitis causes economic losses and antimicrobials are frequently used for mastitis treatment. Antimicrobial resistance surveys are still rare in the ovine field and characterization of strains is important in order to acquire information about resistance and for optimization of therapy. Bacterial pathogens recovered in milk samples from mastitis-affected ewes were characterized for resistance to tetracyclines and aminoglycosides, members of which are frequently used antimicrobials in small ruminants. A total of 185 strains of staphylococci, streptococci, and enterococci, common mastitis pathogens, were tested for minimal inhibitory concentration (MIC) to tetracycline, doxycycline, minocycline, gentamicin, kanamycin, streptomycin, and for resistance genes by PCR. Effects of different tet genes arrangements on MICs were also investigated. Staphylococci expressed the lowest MIC for tetracycline and tet(K) was the most common gene recovered; tet(M) and tet(O) were also found. Gene content was shown to influence the tetracycline MIC values. Enterococci and streptococci showed higher MICs to tetracyclines and nonsusceptible strains always harboured at least one ribosomal protection gene (MIC above 8 μg ml(-1) ). Streptococci often harboured two or more tet determinants. As regards the resistance to aminoglycosides, staphylococci showed the lowest gentamicin and kanamycin median MIC along with streptomycin high level resistant (HLR) strains (MIC >1024 μg ml(-1) ) all harbouring str gene. The resistance determinant aac(6')-Ie-aph(2″)-Ia was present in few strains. Streptococci were basically nonsusceptible to aminoglycosides but neither HLR isolates nor resistance genes were detected. Enterococci revealed the highest MICs for gentamicin; two str harbouring isolates were shown to be HLR to streptomycin. Evidence was obtained for the circulation of antimicrobial-resistant strains and genes in sheep dairy farming. Tetracycline MIC of 64 μg ml(-1) and high

  3. Aminoglycoside-resistant Aeromonas hydrophila as part of a polymicrobial infection following a traumatic fall into freshwater.

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    Shak, Joshua R; Whitaker, Jennifer A; Ribner, Bruce S; Burd, Eileen M

    2011-03-01

    Amikacin is a first-line treatment for Aeromonas infection due to high efficacy. There are few reports of aminoglycoside-resistant Aeromonas spp. We report a soft tissue infection containing multiple pathogens, including a strain of Aeromonas hydrophila resistant to amikacin, tobramycin, and multiple cephalosporins.

  4. Aminoglycoside-Resistant Aeromonas hydrophila as Part of a Polymicrobial Infection following a Traumatic Fall into Freshwater▿

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    Shak, Joshua R.; Whitaker, Jennifer A.; Ribner, Bruce S.; Burd, Eileen M.

    2011-01-01

    Amikacin is a first-line treatment for Aeromonas infection due to high efficacy. There are few reports of aminoglycoside-resistant Aeromonas spp. We report a soft tissue infection containing multiple pathogens, including a strain of Aeromonas hydrophila resistant to amikacin, tobramycin, and multiple cephalosporins. PMID:21209173

  5. Clonal origin of aminoglycoside-resistant Citrobacter freundii isolates in a Danish county

    DEFF Research Database (Denmark)

    Norskov-Lauritsen, N.; Sandvang, Dorthe; Hedegaard, J.

    2001-01-01

    During 1997, attention was drawn to an increased frequency of aminoglycoside-resistant Citrobacter freundii in a Danish county, when a total of 24 resistant C. freundii isolates was detected. In this study, 15 such isolates were typed by pulsed-field gel electrophoresis, riboprinting and partial...... with a dihydrofolate reductase gene in a class I integron. The source of the strain remains unresolved. Representative isolates were obtained from various specimens from hospitals and general practice throughout the county, with no evidence of patient-to-patient transmission....

  6. Molecular genetics of Mycobacterium tuberculosis resistant to aminoglycosides and cyclic peptide testing by MTBDRsl in Armenia

    Directory of Open Access Journals (Sweden)

    Hasmik Margaryan

    2016-01-01

    Conclusion: Isolates with rrs structural gene mutations were cross-resistant to streptomycin, KAN, CAP, and AMK. Detection of the A1401G mutation appeared to be 100% specific for the detection of resistance to KAN and AMK. Being the first assessment, these data establish the presence of phenotypic drug-resistant and extensively drug-resistant strains using molecular profiling and are helpful in understanding aminoglycoside resistance on a molecular level.

  7. Antibiotic stress-induced modulation of the endoribonucleolytic activity of RNase III and RNase G confers resistance to aminoglycoside antibiotics in Escherichia coli.

    Science.gov (United States)

    Song, Wooseok; Kim, Yong-Hak; Sim, Se-Hoon; Hwang, Soonhye; Lee, Jung-Hyun; Lee, Younghoon; Bae, Jeehyeon; Hwang, Jihwan; Lee, Kangseok

    2014-04-01

    Here, we report a resistance mechanism that is induced through the modulation of 16S ribosomal RNA (rRNA) processing on the exposure of Escherichia coli cells to aminoglycoside antibiotics. We observed decreased expression levels of RNase G associated with increased RNase III activity on rng mRNA in a subgroup of E. coli isolates that transiently acquired resistance to low levels of kanamycin or streptomycin. Analyses of 16S rRNA from the aminoglycoside-resistant E. coli cells, in addition to mutagenesis studies, demonstrated that the accumulation of 16S rRNA precursors containing 3-8 extra nucleotides at the 5' terminus, which results from incomplete processing by RNase G, is responsible for the observed aminoglycoside resistance. Chemical protection, mass spectrometry analysis and cell-free translation assays revealed that the ribosomes from rng-deleted E. coli have decreased binding capacity for, and diminished sensitivity to, streptomycin and neomycin, compared with wild-type cells. It was observed that the deletion of rng had similar effects in Salmonella enterica serovar Typhimurium strain SL1344. Our findings suggest that modulation of the endoribonucleolytic activity of RNase III and RNase G constitutes a previously uncharacterized regulatory pathway for adaptive resistance in E. coli and related gram-negative bacteria to aminoglycoside antibiotics.

  8. UK-18,892: resistance to modification by aminoglycoside-inactivating enzymes.

    Science.gov (United States)

    Andrews, R J; Brammer, K W; Cheeseman, H E; Jevons, S

    1978-12-01

    UK-18,892, a new semisynthetic aminoglycoside, was active against bacteria possessing aminoglycoside-inactivating enzymes, with the exception of some known to possess AAC(6') or AAD(4') enzymes. This activity has been rationalized by using cell-free extracts of bacteria containing known inactivating enzymes, where it was shown that UK-18,892 was not a substrate for the APH(3'), AAD(2''), AAC(3), and AAC(2') enzymes. It was also demonstrated that UK-18,892 protected mice against lethal infections caused by organisms possessing aminoglycoside-inactivating enzymes.

  9. Structure of the phosphotransferase domain of the bifunctional aminoglycoside-resistance enzyme AAC(6')-Ie-APH(2'')-Ia.

    Science.gov (United States)

    Smith, Clyde A; Toth, Marta; Bhattacharya, Monolekha; Frase, Hilary; Vakulenko, Sergei B

    2014-06-01

    The bifunctional acetyltransferase(6')-Ie-phosphotransferase(2'')-Ia [AAC(6')-Ie-APH(2'')-Ia] is the most important aminoglycoside-resistance enzyme in Gram-positive bacteria, conferring resistance to almost all known aminoglycoside antibiotics in clinical use. Owing to its importance, this enzyme has been the focus of intensive research since its isolation in the mid-1980s but, despite much effort, structural details of AAC(6')-Ie-APH(2'')-Ia have remained elusive. The structure of the Mg2GDP complex of the APH(2'')-Ia domain of the bifunctional enzyme has now been determined at 2.3 Å resolution. The structure of APH(2'')-Ia is reminiscent of the structures of other aminoglycoside phosphotransferases, having a two-domain architecture with the nucleotide-binding site located at the junction of the two domains. Unlike the previously characterized APH(2'')-IIa and APH(2'')-IVa enzymes, which are capable of utilizing both ATP and GTP as the phosphate donors, APH(2'')-Ia uses GTP exclusively in the phosphorylation of the aminoglycoside antibiotics, and in this regard closely resembles the GTP-dependent APH(2'')-IIIa enzyme. In APH(2'')-Ia this GTP selectivity is governed by the presence of a `gatekeeper' residue, Tyr100, the side chain of which projects into the active site and effectively blocks access to the adenine-binding template. Mutation of this tyrosine residue to a less bulky phenylalanine provides better access for ATP to the NTP-binding template and converts APH(2'')-Ia into a dual-specificity enzyme.

  10. Dissemination of Genes Encoding Aminoglycoside-Modifying Enzymes and armA Among Enterobacteriaceae Isolates in Northwest Iran.

    Science.gov (United States)

    Ghotaslou, Reza; Yeganeh Sefidan, Fatemeh; Akhi, Mohammad Taghi; Asgharzadeh, Mohammad; Mohammadzadeh Asl, Yalda

    2017-10-01

    Enzymatic inactivation is one of the most important mechanisms of resistance to aminoglycosides. The aim of this study was to investigate the prevalence of armA and diversity of the genes encoding aminoglycoside-modifying enzymes (AMEs) and their associations with resistance phenotypes in Enterobacteriaceae isolates. Three hundred and seven Enterobacteriaceae isolates were collected from five hospitals in northwest Iran. The disk diffusion method for amikacin, gentamicin, tobramycin, kanamycin, and streptomycin, as well as the minimum inhibitory concentration for amikacin, gentamicin, tobramycin, and kanamycin were done for susceptibility testing. Thirteen AME genes and armA methylase were screened using the PCR and sequencing assays. Two hundred and twenty (71.7%) of isolates were resistant to aminoglycosides and 155 (70.5%) of them were positive for aminoglycoside resistance genes. The most prevalent AME genes were ant(3″)-Ia and aph(3″)-Ib with the frequency 35.9% and 30.5%, respectively. Also, 21 (9.5%) of resistant isolates were positive for armA methylase gene. The prevalence of resistance to aminoglycoside is high and AME genes frequently are disseminated in Enterobacteriaceae isolates. There is an association between phenotypic resistance and the presence of some aminoglycoside genes.

  11. Application of protein typing in molecular epidemiological investigation of nosocomial infection outbreak of aminoglycoside-resistant Pseudomonas aeruginosa.

    Science.gov (United States)

    Song, Min; Tang, Min; Ding, Yinghuan; Wu, Zecai; Xiang, Chengyu; Yang, Kui; Zhang, Zhang; Li, Baolin; Deng, Zhenghua; Liu, Jinbo

    2017-12-16

    Pseudomonas aeruginosan has emerged as an important pathogen elated to serious infections and nosocomial outbreaks worldwide. This study was conducted to understand the prevalence of aminoglycoside (AMG)-resistant P. aeruginosa in our hospital and to provide a scientific basis for control measures against nosocomial infections. Eighty-two strains of P. aeruginosa were isolated from clinical departments and divided into AMG-resistant strains and AMG-sensitive strains based on susceptibility test results. AMG-resistant strains were typed by drug resistance gene typing (DRGT) and protein typing. Five kinds of aminoglycoside-modifying enzyme (AME) genes were detected in the AMG-resistant group. AMG-resistant P. aeruginosa strains were classified into three types and six subtypes by DRGT. Four protein peaks, namely, 9900.02, 7600.04, 9101.25 and 10,372.87 Da, were significantly and differentially expressed between the two groups. AMG-resistant P. aeruginosa strains were also categorised into three types and six subtypes at the distance level of 10 by protein typing. AMG-resistant P. aeruginosa was cloned spread in our hospital; the timely implementation of nosocomial infection prevention and control strategies were needed in preventing outbreaks and epidemic of AMG-resistant P. aeruginosa. SELDI-TOF MS technology can be used for bacterial typing, which provides a new method of clinical epidemiological survey and nosocomial infection control.

  12. Synergy of aminoglycoside antibiotics by 3-Benzylchroman derivatives from the Chinese drug Caesalpinia sappan against clinical methicillin-resistant Staphylococcus aureus (MRSA).

    Science.gov (United States)

    Zuo, G Y; Han, Z Q; Hao, X Y; Han, J; Li, Z S; Wang, G C

    2014-06-15

    The in vitro antimicrobial activities of three 3-Benzylchroman derivatives, i.e. Brazilin (1), Brazilein (2) and Sappanone B (3) from Caesalpinia sappan L. (Leguminosae) were assayed, which mainly dealt with synergistic evaluation of aminoglycoside and other type of antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) by the three compounds through the Chequerboard and Time-kill curve methods. The results showed that Compounds 1-3 alone exhibited moderate to weak activity against methicillin-susceptible S. aureus (MSSA) and other standard strains by MICs/MBCs ranged from 32/64 to >1024/>1024 μg/ml, with the order of activity as 1>2>3. Chequerboard method showed significant anti-MRSA synergy of 1/Aminoglycosides (Gentamicin, Amikacin, Etimicin and Streptomycin) combinations with (FICIs)50 at 0.375-0.5. The combined (MICs)50 values (μg/ml) reduced from 32-128/16-64 to 4-8/4-16, respectively. The percent of reduction by MICs ranged from 50% to 87.5%, with a maximum of 93.8% (1/16 of the alone MIC). Combinations of 2 and 3 with Aminoglycosides and the other antibiotics showed less potency of synergy. The dynamic Time-killing experiment further demonstrated that the combinations of 1/aminoglycoside were synergistically bactericidal against MRSA. The anti-MRSA synergy results of the bacteriostatic (Chequerboard method) and bactericidal (time-kill method) efficiencies of 1/Aminoglycoside combinations was in good consistency, which made the resistance reversed by CLSI guidelines. We concluded that the 3-Benzylchroman derivative Brazilin (1) showed in vitro synergy of bactericidal activities against MRSA when combined with Aminoglycosides, which might be beneficial for combinatory therapy of MRSA infection. Copyright © 2014. Published by Elsevier GmbH.

  13. Identification of aminoglycoside and β-lactam resistance genes from within an infant gut functional metagenomic library.

    Directory of Open Access Journals (Sweden)

    Fiona Fouhy

    Full Text Available The infant gut microbiota develops rapidly during the first 2 years of life, acquiring microorganisms from diverse sources. During this time, significant opportunities exist for the infant to acquire antibiotic resistant bacteria, which can become established and constitute the infant gut resistome. With increased antibiotic resistance limiting our ability to treat bacterial infections, investigations into resistance reservoirs are highly pertinent. This study aimed to explore the nascent resistome in antibiotically-naïve infant gut microbiomes, using a combination of metagenomic approaches. Faecal samples from 22 six-month-old infants without previous antibiotic exposure were used to construct a pooled metagenomic library, which was functionally screened for ampicillin and gentamicin resistance. Our library of ∼220Mb contained 0.45 ampicillin resistant hits/Mb and 0.059 gentamicin resistant hits/Mb. PCR-based analysis of fosmid clones and uncloned metagenomic DNA, revealed a diverse and abundant aminoglycoside and β-lactam resistance reservoir within the infant gut, with resistance determinants exhibiting homology to those found in common gut inhabitants, including Escherichia coli, Enterococcus sp., and Clostridium difficile, as well as to genes from cryptic environmental bacteria. Notably, the genes identified differed from those revealed when a sequence-driven PCR-based screen of metagenomic DNA was employed. Carriage of these antibiotic resistance determinants conferred substantial, but varied (2-512x, increases in antibiotic resistance to their bacterial host. These data provide insights into the infant gut resistome, revealing the presence of a varied aminoglycoside and β-lactam resistance reservoir even in the absence of selective pressure, confirming the infant resistome establishes early in life, perhaps even at birth.

  14. Environmental and genetic factors affecting mutability to aminoglycoside antibiotics among Escherichia coli K12 strains

    Directory of Open Access Journals (Sweden)

    Monteiro A.C.M.

    2003-01-01

    Full Text Available Environmental and genetic factors affecting the in vitro spontaneous mutation frequencies to aminoglycoside resistance in Escherichia coli K12 were investigated. Spontaneous mutation frequencies to kanamycin resistance were at least 100 fold higher on modified Luria agar (L2 plates, when compared to results obtained in experiments carried out with Nutrient agar (NA plates. In contrast to rifampincin, the increased mutability to kanamycin resistance could not be attributed to a mutator phenotype expressed by DNA repair defective strains. Kanamycin mutant selection windows and mutant preventive concentrations on L2 plates were at least fourfold higher than on NA plates, further demonstrating the role of growth medium composition on the mutability to aminoglycosides. Mutability to kanamycin resistance was increased following addition of sorbitol, suggesting that osmolarity is involved on the spontaneous mutability of E. coli K12 strains to aminoglycosides. The spontaneous mutation rates to kanamycin resistance on both L2 and NA plates were strictly associated with the selective antibiotic concentrations. Moreover, mutants selected at different antibiotic concentrations expressed heterogeneous resistance levels to kanamycin and most of them expressing multiple resistance to all tested aminoglycoside antibiotics (gentamicin, neomycin, amykacin and tobramycin. These results will contribute to a better understanding of the complex nature of aminoglycoside resistance and the emergence of spontaneous resistant mutants among E. coli K12 strains.

  15. DNA-Aptamers Binding Aminoglycoside Antibiotics

    Directory of Open Access Journals (Sweden)

    Nadia Nikolaus

    2014-02-01

    Full Text Available Aptamers are short, single stranded DNA or RNA oligonucleotides that are able to bind specifically and with high affinity to their non-nucleic acid target molecules. This binding reaction enables their application as biorecognition elements in biosensors and assays. As antibiotic residues pose a problem contributing to the emergence of antibiotic-resistant pathogens and thereby reducing the effectiveness of the drug to fight human infections, we selected aptamers targeted against the aminoglycoside antibiotic kanamycin A with the aim of constructing a robust and functional assay that can be used for water analysis. With this work we show that aptamers that were derived from a Capture-SELEX procedure targeting against kanamycin A also display binding to related aminoglycoside antibiotics. The binding patterns differ among all tested aptamers so that there are highly substance specific aptamers and more group specific aptamers binding to a different variety of aminoglycoside antibiotics. Also the region of the aminoglycoside antibiotics responsible for aptamer binding can be estimated. Affinities of the different aptamers for their target substance, kanamycin A, are measured with different approaches and are in the micromolar range. Finally, the proof of principle of an assay for detection of kanamycin A in a real water sample is given.

  16. Triclosan-Induced Aminoglycoside-Tolerant Listeria monocytogenes Isolates Can Appear as Small-Colony Variants

    DEFF Research Database (Denmark)

    Kastbjerg, Vicky Gaedt; Hein-Kristensen, Line; Gram, Lone

    2014-01-01

    Exposure of the human food-borne pathogen Listeria monocytogenes to sublethal concentrations of triclosan can cause resistance to several aminoglycosides. Aminoglycoside-resistant isolates exhibit two colony morphologies: normal-size and pinpoint colonies. The purposes of the present study were...... to characterize the small colonies of L. monocytogenes and to determine if specific genetic changes could explain the triclosan-induced aminoglycoside resistance in both pinpoint and normal-size isolates. Isolates from the pinpoint colonies grew poorly under aerated conditions, but growth was restored by addition......I and that exposure to triclosan can cause resistance to antibiotics that enters the cell via active transport. Further studies are needed to elucidate if L. monocytogenes pinpoint isolates could have any clinical impact, e.g., in persistent infections....

  17. Structural Analysis of the Tobramycin and Gentamicin Clinical Resistome Reveals Limitations for Next-generation Aminoglycoside Design.

    Science.gov (United States)

    Bassenden, Angelia V; Rodionov, Dmitry; Shi, Kun; Berghuis, Albert M

    2016-05-20

    Widespread use and misuse of antibiotics has allowed for the selection of resistant bacteria capable of avoiding the effects of antibiotics. The primary mechanism for resistance to aminoglycosides, a broad-spectrum class of antibiotics, is through covalent enzymatic modification of the drug, waning their bactericidal effect. Tobramycin and gentamicin are two medically important aminoglycosides targeted by several different resistance factors, including aminoglycoside 2″-nucleotidyltransferase [ANT(2″)], the primary cause of aminoglycoside resistance in North America. We describe here two crystal structures of ANT(2″), each in complex with AMPCPP, Mn(2+), and either tobramycin or gentamicin. Together these structures outline ANT(2″)'s specificity for clinically used substrates. Importantly, these structures complete our structural knowledge for the set of enzymes that most frequently confer clinically observed resistance to tobramycin and gentamicin. Comparison of tobramycin and gentamicin binding to enzymes in this resistome, as well as to the intended target, the bacterial ribosome, reveals surprising diversity in observed drug-target interactions. Analysis of the diverse binding modes informs that there are limited opportunities for developing aminoglycoside analogs capable of evading resistance.

  18. Nordihydroguaiaretic acid enhances the activities of aminoglycosides against methicillin- sensitive and resistant Staphylococcus aureus in vitro and in vivo.

    Science.gov (United States)

    Cunningham-Oakes, Edward; Soren, Odel; Moussa, Caroline; Rathor, Getika; Liu, Yingjun; Coates, Anthony; Hu, Yanmin

    2015-01-01

    Infections caused by methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) are prevalent. MRSA infections are difficult to treat and there are no new classes of antibiotics produced to the market to treat infections caused by the resistant bacteria. Therefore, using antibiotic enhancers to rescue existing classes of antibiotics is an attractive strategy. Nordihydroguaiaretic acid (NDGA) is an antioxidant compound found in extracts from plant Larrea Tridentata. It exhibits antimicrobial activity and may target bacterial cell membrane. Combination efficacies of NDGA with many classes of antibiotics were examined by chequerboard method against 200 clinical isolates of MRSA and MSSA. NDGA in combination with gentamicin, neomycin, and tobramycin was examined by time-kill assays. The synergistic combinations of NDGA and aminoglycosides were tested in vivo using a murine skin infection model. Calculations of the fractional inhibitory concentration index (FICI) showed that NDGA when combined with gentamicin, neomycin, or tobramycin displayed synergistic activities in more than 97% of MSSA and MRSA, respectively. Time kill analysis demonstrated that NDGA significantly augmented the activities of these aminoglycosides against MRSA and MSSA in vitro and in murine skin infection model. The enhanced activity of NDGA resides on its ability to damage bacterial cell membrane leading to accumulation of the antibiotics inside bacterial cells. We demonstrated that NDGA strongly revived the therapeutic potencies of aminoglycosides in vitro and in vivo. This combinational strategy could contribute major clinical implications to treat antibiotic resistant bacterial infections.

  19. A SAM-dependent methyltransferase cotranscribed with arsenate reductase alters resistance to peptidyl transferase center-binding antibiotics in Azospirillum brasilense Sp7.

    Science.gov (United States)

    Singh, Sudhir; Singh, Chhaya; Tripathi, Anil Kumar

    2014-05-01

    The genome of Azospirillum brasilense harbors a gene encoding S-adenosylmethionine-dependent methyltransferase, which is located downstream of an arsenate reductase gene. Both genes are cotranscribed and translationally coupled. When they were cloned and expressed individually in an arsenate-sensitive strain of Escherichia coli, arsenate reductase conferred tolerance to arsenate; however, methyltransferase failed to do so. Sequence analysis revealed that methyltransferase was more closely related to a PrmB-type N5-glutamine methyltransferase than to the arsenate detoxifying methyltransferase ArsM. Insertional inactivation of prmB gene in A. brasilense resulted in an increased sensitivity to chloramphenicol and resistance to tiamulin and clindamycin, which are known to bind at the peptidyl transferase center (PTC) in the ribosome. These observations suggested that the inability of prmB:km mutant to methylate L3 protein might alter hydrophobicity in the antibiotic-binding pocket of the PTC, which might affect the binding of chloramphenicol, clindamycin, and tiamulin differentially. This is the first report showing the role of PrmB-type N5-glutamine methyltransferases in conferring resistance to tiamulin and clindamycin in any bacterium.

  20. Loss of the histone methyltransferase EZH2 induces resistance to multiple drugs in acute myeloid leukemia

    DEFF Research Database (Denmark)

    Göllner, Stefanie; Oellerich, Thomas; Agrawal-Singh, Shuchi

    2017-01-01

    In acute myeloid leukemia (AML), therapy resistance frequently occurs, leading to high mortality among patients. However, the mechanisms that render leukemic cells drug resistant remain largely undefined. Here, we identified loss of the histone methyltransferase EZH2 and subsequent reduction...

  1. Structure of the phosphotransferase domain of the bifunctional aminoglycoside-resistance enzyme AAC(6′)-Ie-APH(2′′)-Ia

    Science.gov (United States)

    Smith, Clyde A.; Toth, Marta; Bhattacharya, Monolekha; Frase, Hilary; Vakulenko, Sergei B.

    2014-01-01

    The bifunctional acetyltransferase(6′)-Ie-phosphotransfer­ase(2′′)-Ia [AAC(6′)-Ie-APH(2′′)-Ia] is the most important aminoglycoside-resistance enzyme in Gram-positive bacteria, conferring resistance to almost all known aminoglycoside antibiotics in clinical use. Owing to its importance, this enzyme has been the focus of intensive research since its isolation in the mid-1980s but, despite much effort, structural details of AAC(6′)-Ie-APH(2′′)-Ia have remained elusive. The structure of the Mg2GDP complex of the APH(2′′)-Ia domain of the bifunctional enzyme has now been determined at 2.3 Å resolution. The structure of APH(2′′)-Ia is reminiscent of the structures of other aminoglycoside phosphotransferases, having a two-domain architecture with the nucleotide-binding site located at the junction of the two domains. Unlike the previously characterized APH(2′′)-IIa and APH(2′′)-IVa enzymes, which are capable of utilizing both ATP and GTP as the phosphate donors, APH(2′′)-Ia uses GTP exclusively in the phosphorylation of the aminoglycoside antibiotics, and in this regard closely resembles the GTP-dependent APH(2′′)-IIIa enzyme. In APH(2′′)-Ia this GTP selectivity is governed by the presence of a ‘gatekeeper’ residue, Tyr100, the side chain of which projects into the active site and effectively blocks access to the adenine-binding template. Mutation of this tyrosine residue to a less bulky phenylalanine provides better access for ATP to the NTP-binding template and converts APH(2′′)-Ia into a dual-specificity enzyme. PMID:24914967

  2. Detection and characterization of multidrug-resistant enterobacteria bearing aminoglycoside-modifying gene in a university hospital at Rio de Janeiro, Brazil, along three decades.

    Science.gov (United States)

    Dias-Gonçalves, Verônica; Bohrer-Lengruber, Françoise; Oliveira-Fonseca, Bianca; Santos-Pereira, Renata Meirelles; Barbosa de Melo, Luis Dione; Gazos-Lopes, Ulisses; Ribeiro-Bello, Alexandre; Adler-Pereira, José Augusto

    2015-01-01

    Multidrug-resistant Enterobacteriaceae, particularly those resistant to gentamicin, have become one of the most important causes of nosocomial infections. We sought to investigate the presence of genes conferring resistance to aminoglycosides, specially to gentamicin, in Klebsiella pneumoniae and Escherichia coli multidrug-resistant strains isolated from different clinical materials among patients hospitalized in a university hospital in Rio de Janeiro, Brazil. Ten colonization strains and 20 infection strains were evaluated during three decades (1980 to 2010) using selective media containing 8 µg/ml of gentamicin. Thirty strains were tested for antimicrobial susceptibility. Twenty two strains were subjected to plasmid DNA extraction and 12 to hybridization assays using as probe a 1.9 kb plasmid DNA fragment from one of the K. pneumoniae strains isolated from faecal samples. This fragment was sequenced and assigned to the GQ422439 GenBank record. PCR was also performed using oligonucleotides designed for aminoglycoside-modifying enzymes. An accC2 acetylase, besides transposons and insertion sequences, were evidenced. Twenty-four (80%) of the isolates were positive for the aacC2 gene in agreement with antibiotic susceptibility testing profiles, indicating the persistent presence of this gene throughout the three decades. We detected high molecular weight plasmids in 54,5% of the strains. Of the tested strains, 91% showed positive signal in the hybridization assays. A gene codifying for one specific aminoglycoside-modifying enzyme was detected all throughout the three decades. Our data back the adoption of preventive measures, such as a more conscious use of antimicrobial agents in hospital environments, which can contribute to control the dissemination of microorganisms harboring resistance gene plasmids.

  3. Sulfonamide-Based Inhibitors of Aminoglycoside Acetyltransferase Eis Abolish Resistance to Kanamycin in Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Garzan, Atefeh; Willby, Melisa J.; Green, Keith D.; Gajadeera, Chathurada S.; Hou, Caixia; Tsodikov, Oleg V.; Posey, James E.; Garneau-Tsodikova, Sylvie

    2016-12-08

    A two-drug combination therapy where one drug targets an offending cell and the other targets a resistance mechanism to the first drug is a time-tested, yet underexploited approach to combat or prevent drug resistance. By high-throughput screening, we identified a sulfonamide scaffold that served as a pharmacophore to generate inhibitors of Mycobacterium tuberculosis acetyltransferase Eis, whose upregulation causes resistance to the aminoglycoside (AG) antibiotic kanamycin A (KAN) in Mycobacterium tuberculosis. Rational systematic derivatization of this scaffold to maximize Eis inhibition and abolish the Eis-mediated KAN resistance of M. tuberculosis yielded several highly potent agents. A crystal structure of Eis in complex with one of the most potent inhibitors revealed that the inhibitor bound Eis in the AG-binding pocket held by a conformationally malleable region of Eis (residues 28–37) bearing key hydrophobic residues. These Eis inhibitors are promising leads for preclinical development of innovative AG combination therapies against resistant TB.

  4. Evaluating the Frequency of aac(6')-IIa, ant(2″)-I, intl1, and intl2 Genes in Aminoglycosides Resistant Klebsiella pneumoniae Isolates Obtained from Hospitalized Patients in Yazd, Iran.

    Science.gov (United States)

    Mokhtari, Hesam; Eslami, Gilda; Zandi, Hengameh; Dehghan-Banadkouki, Amin; Vakili, Mahmood

    2018-01-01

    Klebsiella pneumoniae (K. pneumoniae) is an opportunistic pathogen that could be resistant to many antimicrobial agents. Resistance genes can be carried among gram-negative bacteria by integrons. Enzymatic inactivation is the most important mechanism of resistance to aminoglycosides. In this study, the frequencies of two important resistance gene aac(6')-II a and ant(2″)-I, and genes coding integrase I and II, in K. pneumoniae isolates resistant to aminoglycosides were evaluated. In this cross-sectional study, an attempt was made to assess the antibiotic susceptibility of 130 K. pneumoniae isolates obtained from different samples of patients hospitalized in training hospitals of Yazd evaluated by disk diffusion method. The frequencies of aac(6')-II a, ant(2″)-I, intl1 , and intl2 genes were determined by PCR method. Data were analyzed by chi-square method using SPSS software (Ver. 16). our results showed that resistance to gentamicin, tobramycin, kanamycin, and amikacin were 34.6, 33.8, 43.8, and 14.6%, respectively. The frequencies of aac (6')-II a, ant(2″)-I, intl1 , and intl2 genes were 44.6, 27.7, 90, and 0%, respectively. This study showed there are high frequencies of genes coding aminoglycosides resistance in K. pneumoniae isolates. Hence, it is very important to monitor and inhibit the spread of antibiotic resistance genes.

  5. Effect of mutations in the A site of 16 S rRNA on aminoglycoside antibiotic-ribosome interaction

    DEFF Research Database (Denmark)

    Recht, M I; Douthwaite, S; Dahlquist, K D

    1999-01-01

    antibiotics, which also interact with this region of rRNA. Mutations of certain nucleotides in rRNA reduce aminoglycoside binding affinity, as previously demonstrated using a model RNA oligonucleotide system. Here, predictions from the oligonucleotide system were tested in the ribosome by mutation...... for the aminoglycoside paromomycin, whereas no discernible reduction in affinity was observed with 1406 mutant ribosomes. These data are consistent with prior NMR structural determination of aminoglycoside interaction with the decoding region, and further our understanding of how aminoglycoside resistance can...

  6. Molecular detection of aminoglycoside-modifying enzyme genes in Acinetobacter baumannii clinical isolates.

    Science.gov (United States)

    Heidary, Mohsen; Salimi Chirani, Alireza; Khoshnood, Saeed; Eslami, Gita; Atyabi, Seyyed Mohammad; Nazem, Habibollah; Fazilati, Mohammad; Hashemi, Ali; Soleimani, Saleh

    2017-06-01

    Acinetobacter baumannii is a major opportunistic pathogen in healthcare settings worldwide. In Iran, there are only few reports on the prevalence of aminoglycoside resistance genes among A. baumannii isolates. The aim of this study was to investigate the existence of aminoglycoside-modifying enzyme (AME) genes from A. baumannii strains collected at a university teaching hospital in Iran. One hundred A. baumannii strains were collected between 2014 and 2015 from hospitalized patients at Loghman Hakim Hospital, Tehran, Iran. Antimicrobial susceptibility was determined by disk diffusion method according to the Clinical and Laboratory Standards Institute recommendations. The DNA was extracted using a kit obtained from Bioneer Co. (Korea) and was used as a template for polymerase chain reaction. The most active antimicrobial agent against these strains was colistin. The rate of extended-spectrum cephalosporin resistance was 97%. The aadA1, aadB, aac(6')-Ib, and aac(3)-IIa genes were found in 85%, 77%, 72%, and 68% of A. baumannii isolates, respectively. This study showed a high prevalence rate of AME genes in A. baumannii. This prevalence rate has explained that further aminoglycoside resistance genes may have role in the resistance of clinical isolates of A. baumannii. Therefore, control and treatment of serious infections caused by this opportunistic pathogen should be given more consideration.

  7. Structure of AadA from Salmonella enterica: a monomeric aminoglycoside (3′′)(9) adenyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yang [Uppsala University, Biomedical Center, Box 596, SE-751 24 Uppsala (Sweden); Näsvall, Joakim [Uppsala University, Biomedical Center, Box 582, SE-751 23 Uppsala (Sweden); Wu, Shiying [Uppsala University, Biomedical Center, Box 596, SE-751 24 Uppsala (Sweden); Andersson, Dan I. [Uppsala University, Biomedical Center, Box 582, SE-751 23 Uppsala (Sweden); Selmer, Maria, E-mail: maria.selmer@icm.uu.se [Uppsala University, Biomedical Center, Box 596, SE-751 24 Uppsala (Sweden)

    2015-10-31

    The crystal structure of the aminoglycoside-adenylating enzyme AadA is reported together with functional experiments providing insights into its oligomeric state, ligand binding and catalysis. Aminoglycoside resistance is commonly conferred by enzymatic modification of drugs by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). Here, the first crystal structure of an ANT(3′′)(9) adenyltransferase, AadA from Salmonella enterica, is presented. AadA catalyses the magnesium-dependent transfer of adenosine monophosphate from ATP to the two chemically dissimilar drugs streptomycin and spectinomycin. The structure was solved using selenium SAD phasing and refined to 2.5 Å resolution. AadA consists of a nucleotidyltransferase domain and an α-helical bundle domain. AadA crystallizes as a monomer and is a monomer in solution as confirmed by small-angle X-ray scattering, in contrast to structurally similar homodimeric adenylating enzymes such as kanamycin nucleotidyltransferase. Isothermal titration calorimetry experiments show that ATP binding has to occur before binding of the aminoglycoside substrate, and structure analysis suggests that ATP binding repositions the two domains for aminoglycoside binding in the interdomain cleft. Candidate residues for ligand binding and catalysis were subjected to site-directed mutagenesis. In vivo resistance and in vitro binding assays support the role of Glu87 as the catalytic base in adenylation, while Arg192 and Lys205 are shown to be critical for ATP binding.

  8. Evolution of the Pseudomonas aeruginosa Aminoglycoside Mutational Resistome In Vitro and in the Cystic Fibrosis Setting.

    Science.gov (United States)

    López-Causapé, Carla; Rubio, Rosa; Cabot, Gabriel; Oliver, Antonio

    2018-04-01

    Inhaled administration of high doses of aminoglycosides is a key maintenance treatment of Pseudomonas aeruginosa chronic respiratory infections in cystic fibrosis (CF). We analyzed the dynamics and mechanisms of stepwise high-level tobramycin resistance development in vitro and compared the results with those of isogenic pairs of susceptible and resistant clinical isolates. Resistance development correlated with fusA1 mutations in vitro and in vivo. pmrB mutations, conferring polymyxin resistance, were also frequently selected in vitro In contrast, mutational overexpression of MexXY, a hallmark of aminoglycoside resistance in CF, was not observed in in vitro evolution experiments. Copyright © 2018 American Society for Microbiology.

  9. In vitro activity of aminoglycosides against clinical isolates of Acinetobacter baumannii complex and other nonfermentative Gram-negative bacilli causing healthcare-associated bloodstream infections in Taiwan.

    Science.gov (United States)

    Liu, Jyh-You; Wang, Fu-Der; Ho, Mao-Wang; Lee, Chen-Hsiang; Liu, Jien-Wei; Wang, Jann-Tay; Sheng, Wang-Huei; Hseuh, Po-Ren; Chang, Shan-Chwen

    2016-12-01

    Aminoglycosides possess in vitro activity against aerobic and facultative Gram-negative bacilli. However, nationwide surveillance on susceptibility data of Acinetobacter baumannii complex and Pseudomonas aeruginosa to aminoglycosides was limited, and aminoglycoside resistance has emerged in the past decade. We study the in vitro susceptibility of A. baumannii complex and other nonfermentative Gram-negative bacilli (NFGNB) to aminoglycosides. A total of 378 NFGNB blood isolates causing healthcare-associated bloodstream infections during 2008 and 2013 at four medical centers in Taiwan were tested for their susceptibilities to four aminoglycosides using the agar dilution method (gentamicin, amikacin, tobramycin, and isepamicin) and disc diffusion method (isepamicin). A. baumannii was highly resistant to all four aminoglycosides (range of susceptibility, 0-4%), whereas >80% of Acinetobacter nosocomialis and Acinetobacter pittii blood isolates were susceptible to amikacin (susceptibility: 96% and 91%, respectively), tobramycin (susceptibility: 92% and 80%, respectively), and isepamicin (susceptibility: 96% and 80%, respectively). All aminoglycosides except gentamicin possessed good in vitro activity (>94%) against P. aeruginosa. Amikacin has the best in vitro activity against P. aeruginosa (susceptibility, 98%), followed by A. nosocomialis (96%), and A. pittii (91%), whereas tobramycin and isepamicin were less potent against A. pittii (both 80%). Aminoglycoside resistances were prevalent in Stenotrophomonas maltophilia and Burkholderia cepacia complex blood isolates in Taiwan. Genospecies among the A. baumannii complex had heterogeneous susceptibility profiles to aminoglycosides. Aminoglycosides, except gentamicin, remained good in vitro antimicrobial activity against P. aeruginosa. Further in vivo clinical data and continuous resistance monitoring are warranted for clinical practice guidance. Copyright © 2015. Published by Elsevier B.V.

  10. Crystallization and preliminary crystallographic analysis of nosiheptide-resistance methyltransferase from Streptomyces actuosus in complex with SAM

    International Nuclear Information System (INIS)

    Yang, Huirong; Wang, Ping; Dong, Zhenghong; Li, Xueyuan; Gong, Rui; Yang, Ying; Li, Ze; Xu, Youwei; Xu, Yanhui

    2010-01-01

    The expression, purification and crystallization of nosiheptide-resistance methyltransferase (NSR) from Streptomyces actuosus is described. Nosiheptide-resistance methyltransferase (NSR) methylates 23S rRNA at the nucleotide adenosine 1067 in Escherichia coli and thus contributes to resistance against nosiheptide, a sulfur-containing peptide antibiotic. Here, the expression, purification and crystallization of NSR from Streptomyces actuosus are reported. Diffracting crystals were grown by the hanging-drop vapour-diffusion method in reservoir solution consisting of 0.35 M ammonium chloride, 24%(w/v) PEG 3350, 0.1 M MES pH 5.7 at 293 K. Native data have been collected from the apo enzyme and a SAM complex, as well as apo SeMet SAD data. The diffraction patterns of the apo form of NSR, of NSR complexed with SAM and of SeMet-labelled NSR crystals extended to 1.90, 1.95 and 2.25 Å resolution, respectively, using synchrotron radiation. All crystals belonged to space group P2 1 , with approximate unit-cell parameters a = 64.6, b = 69.6, c = 64.9 Å, β = 117.8°

  11. Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria

    DEFF Research Database (Denmark)

    Karminska, K. H.; Purta, E.; Hansen, L .H.

    2010-01-01

    The Cfr methyltransferase confers combined resistance to five classes of antibiotics that bind to the peptidyl tranferase center of bacterial ribosomes by catalyzing methylation of the C-8 position of 23S rRNA nucleotide A2503. The same nucleotide is targeted by the housekeeping methyltransferase...

  12. Collateral sensitivity between aminoglycosides and beta-lactam antibiotics depends on active proton pumps.

    Science.gov (United States)

    Azimi, Leila; Rastegar Lari, Abdolaziz

    2017-11-01

    Selection inversion is the hypothesis for antibiotic resistant inhabitation in bacteria and collateral sensitivity is one of the proposed phenomena for achievement of this hypothesis. The presence of collateral sensitivity associated with the proton motivation pump between the aminoglycosides and beta-lactam group of antibiotics is one of the examples of collateral sensitivity in some studies. The aim of this study was to demonstrate that collateral sensitivity between aminoglycosides and beta-lactam antibiotics associated with proton motivation pump may not be true in all cases. In this study, 100 Pseudomonas aeruginosa were surveyed. Gentamicin and imipenem-resistant strains were confirmed by disc diffusion method and MIC. Active proton motivation pumps were screened by pumps inhibitor. Semi-quantitative Real-Time PCR assay was used to confirm gene overexpression. Seventy-six and 79 out of 100 strains were resistant to gentamicin and imipenem, respectively. Seventy-five strains were resistant to both gentamicin and imipenem. The results of proton pump inhibitor test showed the involvement of active proton motivation pump in 22 of 75 imipenem- and gentamicin-resistant strains. According to Real - Time PCR assay, mexX efflux gene was overexpressed in the majority of isolates tested. The collateral sensitivity effect cannot explain the involvement of active proton motivation pumps in both imipenem and gentamicin-resistant strains simultaneously. Active and/or inactive proton pump in gentamicin-sensitive and/or resistant strains cannot be a suitable example for explanation of collateral sensitivity between aminoglycosides and beta-lactam antibiotics. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Identification of Genes Coding Aminoglycoside Modifying Enzymes in E. coli of UTI Patients in India

    Directory of Open Access Journals (Sweden)

    Abdul Rouf Mir

    2016-01-01

    Full Text Available This study is to probe the pattern of antibiotic resistance against aminoglycosides and its mechanism in E. coli obtained from patients from Chennai, India. Isolation and identification of pathogens were done on MacConkey agar. Antimicrobial sensitivity testing was done by disc diffusion test. The identification of genes encoding aminoglycoside modifying enzymes was done by Polymerase Chain Reaction (PCR. Out of 98 isolates, 71 (72.45% isolates were identified as E. coli and the remaining 27 (27.55% as other bacteria. Disc diffusion method results showed a resistance level of 72.15% for streptomycin, 73.4% for gentamicin, 63.26% for neomycin, 57.14% for tobramycin, 47.9% for netilmicin, and 8.16% for amikacin in E. coli. PCR screening showed the presence of four genes, namely, rrs, aacC2, aacA-aphD, and aphA3, in their plasmid DNA. The results point towards the novel mechanism of drug resistance in E. coli from UTI patients in India as they confirm the presence of genes encoding enzymes that cause resistance to aminoglycoside drugs. This could be an alarm for drug prescription to UTI patients.

  14. Identification of Genes Coding Aminoglycoside Modifying Enzymes in E. coli of UTI Patients in India.

    Science.gov (United States)

    Mir, Abdul Rouf; Bashir, Yasir; Dar, Firdous Ahmad; Sekhar, M

    This study is to probe the pattern of antibiotic resistance against aminoglycosides and its mechanism in E. coli obtained from patients from Chennai, India. Isolation and identification of pathogens were done on MacConkey agar. Antimicrobial sensitivity testing was done by disc diffusion test. The identification of genes encoding aminoglycoside modifying enzymes was done by Polymerase Chain Reaction (PCR). Out of 98 isolates, 71 (72.45%) isolates were identified as E. coli and the remaining 27 (27.55%) as other bacteria. Disc diffusion method results showed a resistance level of 72.15% for streptomycin, 73.4% for gentamicin, 63.26% for neomycin, 57.14% for tobramycin, 47.9% for netilmicin, and 8.16% for amikacin in E. coli. PCR screening showed the presence of four genes, namely, rrs, aacC2, aacA-aphD, and aphA3, in their plasmid DNA. The results point towards the novel mechanism of drug resistance in E. coli from UTI patients in India as they confirm the presence of genes encoding enzymes that cause resistance to aminoglycoside drugs. This could be an alarm for drug prescription to UTI patients.

  15. The Cfr rRNA methyltransferase confers resistance to Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics

    DEFF Research Database (Denmark)

    Long, K. S.; Poehlsgaard, Jacob; Kehrenberg, C.

    2006-01-01

    to overlapping sites at the peptidyl transferase center that abut nucleotide A2503, is perturbed upon Cfr-mediated methylation. Decreased drug binding to Cfr-methylated ribosomes has been confirmed by footprinting analysis. No other rRNA methyltransferase is known to confer resistance to five chemically distinct...

  16. Occurrence of aminoglycoside-modifying enzymes among isolates of Escherichia coli exhibiting high levels of aminoglycoside resistance isolated from Korean cattle farms.

    Science.gov (United States)

    Belaynehe, Kuastros Mekonnen; Shin, Seung Won; Hong-Tae, Park; Yoo, Han Sang

    2017-08-01

    This study investigated 247 Escherichia coli isolates collected from four cattle farms to characterize aminoglycoside-modifying enzyme (AME) genes, their plasmid replicons and transferability. Out of 247 isolates a high number of isolates (total 202; 81.78%) were found to be resistant to various antibiotics by disc diffusion. Of the 247 strains, 139 (56.3%) were resistant to streptomycin, and other antibiotic resistances followed as tetracycline (12.15%), ampicillin (7%), chloramphenicol (5.7%) and trimethoprim-sulfamethoxazole (0.8%). Among 247 isolates B1 was the predominant phylogenetic group identified comprising 151 isolates (61.1%), followed by groups A (27.9%), D (7%) and B2 (4%). Out of 139 isolates investigated for AME, 130 (93.5%) isolates carried at least one AME gene. aph3″-1a and aph3″-1b (46%) were the principal genes detected, followed by aac3-IVa (34.5%). ant2″-1a was the least detected gene (2.2%). Nine (6.5%) strains carried no AME genes. Twelve (63.2%) among 19 isolates transferred an AME gene to a recipient and aph3΄-1a was the dominant transferred gene. Transferability mainly occurred via the IncFIB replicon type (52.6%). Pulsed-field gel electrophoresis typing demonstrated a higher degree of diversity with 14 distinct cluster types. This result suggests that commensal microflora from food-producing animals has a tremendous ability to harbor and transfer AME genes, and poses a potential risk by dissemination of resistance to humans through the food chain. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  17. Impact of aminoglycoside cycling in six tertiary intensive care units: prospective longitudinal interventional study.

    Science.gov (United States)

    Francetić, Igor; Kalenić, Smilja; Huić, Mirjana; Mercep, Iveta; Makar-Ausperger, Ksenija; Likić, Robert; Erdeljić, Viktorija; Tripković, Vesna; Simić, Petra

    2008-04-01

    To determine the effect of aminoglycoside cycling in six tertiary intensive care units (ICU) on the rates of sepsis, aminoglycoside resistance patterns, antibiotic consumption, and costs. This was a prospective longitudinal interventional study that measured the effect of change from first-line gentamicin usage (February 2002-February 2003) to amikacin usage (February 2003-February 2004) on the aminoglycoside resistance patterns, number of patients with gram-negative bacteremia, consumption of antibiotics, and the cost of antimicrobial drugs in 6 tertiary care ICUs in Zagreb, Croatia. The change from first-line gentamicin to amikacin usage led to a decrease in the overall gentamicin resistance of gram-negative bacteria (GNB) from 42% to 26% (PAcinetobacter baumanni (P=0.014). Sepsis rate in ICUs was reduced from 3.6% to 2.2% (P<0.001; chi(2) test), with a decline in the number of nosocomial bloodstream infections from 55/100 patient-days to 26/100 patient-days (P=0.001, chi(2) test). Furthermore, amikacin use led to a 16% decrease in the overall antibiotic consumption and 0.1 euro/patient/d cost reduction. Exclusive use of amikacin significantly reduced the resistance of GNB isolates to gentamicin and netilmicin, the number of GNB nosocomial bacteremias, and the cost of total antibiotic usage in ICUs.

  18. Purification, crystallization and preliminary X-ray analysis of the aminoglycoside-6′-acetyltransferase AAC(6′)-Im

    International Nuclear Information System (INIS)

    Toth, Marta; Vakulenko, Sergei B.; Smith, Clyde A.

    2012-01-01

    AAC(6′)-Im is an N-acetyltransferase enzyme responsible for aminoglycoside resistance in E. faecium and E. coli isolates. Crystals of the kanamycin complex of this enzyme have been prepared and preliminary X-ray diffraction experiments have been undertaken. Bacterial resistance to the aminoglycoside antibiotics is primarily the result of enzymatic deactivation of the drugs. The aminoglycoside N-acetyltransferases (AACs) are a large family of bacterial enzymes that are responsible for coenzyme-A-facilitated acetylation of aminoglycosides. The gene encoding one of these enzymes, AAC(6′)-Im, has been cloned and the protein (comprising 178 amino-acid residues) was expressed in Escherichia coli, purified and crystallized as the kanamycin complex. Synchrotron diffraction data to approximately 2.0 Å resolution were collected from a crystal of this complex on beamline BL12-2 at SSRL (Stanford, California, USA). The crystals belonged to the hexagonal space group P6 5 , with approximate unit-cell parameters a = 107.75, c = 37.33 Å, and contained one molecule in the asymmetric unit. Structure determination is under way using molecular replacement

  19. Distribution of 16S rRNA Methylases Among Different Species of Aminoglycoside-Resistant Enterobacteriaceae in a Tertiary Care Hospital in Poland.

    Science.gov (United States)

    Piekarska, Katarzyna; Zacharczuk, Katarzyna; Wołkowicz, Tomasz; Rzeczkowska, Magdalena; Bareja, Elżbieta; Olak, Monika; Gierczyński, Rafał

    2016-01-01

    Aminoglycosides are a group of antimicrobial agents still the most commonly used in the treatment of life-threatening bacterial infections in human and animals. The emergence and spread of 16S rRNA methylases, which confer high-level resistance to the majority of clinically relevant aminoglycosides, constitute a major public health concern. Our goal was to evaluate the distribution of 16S rRNA methylases among different species of Enterobacteriaceae during a five month-long survey in a tertiary hospital in Warszawa, Poland. In the survey, a total of 1770 non-duplicate clinical isolates were collected from all hospital wards in a tertiary hospital in Warszawa, Poland. The survey was conducted between 19 April and 19 September 2010. The ability to produce 16S rRNA methylase was examined by determining MICs for gentamicin, kanamycin, amikacin by means of the agar dilution method. The isolates resistant to high concentration of aminoglycosides were PCR tested for genes: armA, rmtA, rmtB and rmtC. PCR products were subjected to DNA sequencing by the Sanger method. The genetic similarity of the ArmA-producing isolates was analysed by pulsed-filed gel electrophoresis (PFGE). ArmA was the only 16S rRNA methylase detected in 20 of 1770 tested isolates. The overall prevalence rate of ArmA was 1.13%. In K. pneumoniae (n = 742), P. mirabilis (n = 130), and E. cloacae (n = 253) collected in the survey, the prevalence of ArmA was 0.4%, 0.8% and 5.9%, respectively. The PFGE revealed both horizontal and clonal spread of the armA gene in the hospital. The prevalence of 16S rRNA methylase ArmA reported in this study is significantly higher than observed in other countries in Europe.

  20. Efficient transformation and regeneration of transgenic cassava using the neomycin phosphotransferase gene as aminoglycoside resistance marker gene.

    Science.gov (United States)

    Niklaus, Michael; Gruissem, Wilhelm; Vanderschuren, Hervé

    2011-01-01

    Cassava is one of the most important crops in the tropics. Its industrial use for starch and biofuel production is also increasing its importance for agricultural production in tropical countries. In the last decade cassava biotechnology has emerged as a valuable alternative to the breeding constraints of this highly heterozygous crop for improved trait development of cassava germplasm. Cassava transformation remains difficult and time-consuming because of limitations in selecting transgenic tissues and regeneration of transgenic plantlets. We have recently reported an efficient and robust cassava transformation protocol using the hygromycin phosphotransferase II (hptII) gene as selection marker and the aminoglycoside hygromycin at optimal concentrations to maximize the regeneration of transgenic plantlets. In the present work, we expanded the transformation protocol to the use of the neomycin phosphotransferase II (nptII) gene as selection marker. Several aminoglycosides compatible with the use of nptII were tested and optimal concentrations for cassava transformation were determined. Given its efficiency equivalent to hptII as selection marker with the described protocol, the use of nptII opens new possibilities to engineer transgenic cassava lines with multiple T-DNA insertions and to produce transgenic cassava with a resistance marker gene that is already deregulated in several commercial transgenic crops.

  1. Parallel Evolution of High-Level Aminoglycoside Resistance in Escherichia coli Under Low and High Mutation Supply Rates

    Directory of Open Access Journals (Sweden)

    Claudia Ibacache-Quiroga

    2018-03-01

    Full Text Available Antibiotic resistance is a major concern in public health worldwide, thus there is much interest in characterizing the mutational pathways through which susceptible bacteria evolve resistance. Here we use experimental evolution to explore the mutational pathways toward aminoglycoside resistance, using gentamicin as a model, under low and high mutation supply rates. Our results show that both normo and hypermutable strains of Escherichia coli are able to develop resistance to drug dosages > 1,000-fold higher than the minimal inhibitory concentration for their ancestors. Interestingly, such level of resistance was often associated with changes in susceptibility to other antibiotics, most prominently with increased resistance to fosfomycin. Whole-genome sequencing revealed that all resistant derivatives presented diverse mutations in five common genetic elements: fhuA, fusA and the atpIBEFHAGDC, cyoABCDE, and potABCD operons. Despite the large number of mutations acquired, hypermutable strains did not pay, apparently, fitness cost. In contrast to recent studies, we found that the mutation supply rate mainly affected the speed (tempo but not the pattern (mode of evolution: both backgrounds acquired the mutations in the same order, although the hypermutator strain did it faster. This observation is compatible with the adaptive landscape for high-level gentamicin resistance being relatively smooth, with few local maxima; which might be a common feature among antibiotics for which resistance involves multiple loci.

  2. New plasmid-mediated aminoglycoside 6'-N-acetyltransferase, AAC(6')-Ian, and ESBL, TLA-3, from a Serratia marcescens clinical isolate.

    Science.gov (United States)

    Jin, Wanchun; Wachino, Jun-Ichi; Kimura, Kouji; Yamada, Keiko; Arakawa, Yoshichika

    2015-05-01

    Enterobacteriaceae clinical isolates showing amikacin resistance (MIC 64 to >256 mg/L) in the absence of 16S rRNA methyltransferase (MTase) genes were found. The aim of this study was to clarify the molecular mechanisms underlying amikacin resistance in Enterobacteriaceae clinical isolates that do not produce 16S rRNA MTases. PCR was performed to detect already-known amikacin resistance determinants. Cloning experiments and sequence analyses were performed to characterize unknown amikacin resistance determinants. Transfer of amikacin resistance determinants was performed by conjugation and transformation. The complete nucleotide sequence of the plasmids was determined by next-generation sequencing technology. Amikacin resistance enzymes were purified with a column chromatography system. The enzymatic function of the purified protein was investigated by thin-layer chromatography (TLC) and HPLC. Among the 14 isolates, 9 were found to carry already-known amikacin resistance determinants such as aac(6')-Ia and aac(6')-Ib. Genetic analyses revealed the presence of a new amikacin acetyltransferase gene, named aac(6')-Ian, located on a 169 829 bp transferable plasmid (p11663) of the Serratia marcescens strain NUBL-11663, one of the five strains negative for known aac(6') genes by PCR. Plasmid p11663 also carried a novel ESBL gene, named blaTLA-3. HPLC and TLC analyses demonstrated that AAC(6')-Ian catalysed the transfer of an acetyl group from acetyl coenzyme A onto an amine at the 6'-position of various aminoglycosides. We identified aac(6')-Ian as a novel amikacin resistance determinant together with a new ESBL gene, blaTLA-3, on a transferable plasmid of a S. marcescens clinical isolate. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  3. Pediatric fecal microbiota harbor diverse and novel antibiotic resistance genes.

    Directory of Open Access Journals (Sweden)

    Aimée M Moore

    Full Text Available Emerging antibiotic resistance threatens human health. Gut microbes are an epidemiologically important reservoir of resistance genes (resistome, yet prior studies indicate that the true diversity of gut-associated resistomes has been underestimated. To deeply characterize the pediatric gut-associated resistome, we created metagenomic recombinant libraries in an Escherichia coli host using fecal DNA from 22 healthy infants and children (most without recent antibiotic exposure, and performed functional selections for resistance to 18 antibiotics from eight drug classes. Resistance-conferring DNA fragments were sequenced (Illumina HiSeq 2000, and reads assembled and annotated with the PARFuMS computational pipeline. Resistance to 14 of the 18 antibiotics was found in stools of infants and children. Recovered genes included chloramphenicol acetyltransferases, drug-resistant dihydrofolate reductases, rRNA methyltransferases, transcriptional regulators, multidrug efflux pumps, and every major class of beta-lactamase, aminoglycoside-modifying enzyme, and tetracycline resistance protein. Many resistance-conferring sequences were mobilizable; some had low identity to any known organism, emphasizing cryptic organisms as potentially important resistance reservoirs. We functionally confirmed three novel resistance genes, including a 16S rRNA methylase conferring aminoglycoside resistance, and two tetracycline-resistance proteins nearly identical to a bifidobacterial MFS transporter (B. longum s. longum JDM301. We provide the first report to our knowledge of resistance to folate-synthesis inhibitors conferred by a predicted Nudix hydrolase (part of the folate synthesis pathway. This functional metagenomic survey of gut-associated resistomes, the largest of its kind to date, demonstrates that fecal resistomes of healthy children are far more diverse than previously suspected, that clinically relevant resistance genes are present even without recent selective

  4. Clinical pharmacokinetics of aminoglycosides in the neonate: a review.

    Science.gov (United States)

    Pacifici, Gian Maria

    2009-04-01

    Sepsis is common in neonates and is a major cause of morbidity and mortality. Sixty percent of preterm neonates receive at least one antibiotic, and 43% of the antibiotics administered to these neonates are aminoglycosides. The clearance (Cl), serum half-life (t(1/2)), and volume of distribution (Vd) of aminoglycosides change during the neonatal life, and the pharmacokinetics of aminoglycosides need to be studied in neonates in order to optimise therapy with these drugs. The aim of this work is to review the published data on the pharmacokinetics of aminoglycosides in order to provide a critical analysis of the literature that can be a useful tool in the hands of physicians. The bibliographic search was performed electronically using PubMed, as the search engine, through July 11th, 2008. Firstly, a Medline search was performed with the keywords "pharmacokinetics of aminoglycosides in neonates" with the limit of "human". Other Medline searches were performed with the keywords "pharmacokinetics of ... in neonates" followed by the name of the aminoglycosides: amikacin, gentamicin, netilmicin and tobramycin. In addition, the book Neofax: A Manual of Drugs Used in Neonatal Care by Young and Mangum (Thomson Healthcare, 2007) was consulted. The aminoglycosides are mainly eliminated by the kidney, and their elimination rates are reduced at birth. As a consequence Cl is reduced and t(1/2) is prolonged in the neonate as compared to more mature infants. The high body-water content of the neonate results in a large Vd of aminoglycosides as these drugs are fairly water soluble. Postnatal development is an important factor in the maturation of the neonate, and as postnatal age proceeds, Cl of aminoglycosides increases. The maturation of the kidney governs the pharmacokinetics of aminoglycosides in the infant. Cl and t(1/2) are influenced by development, and this must be taken into consideration when planning a dosage regimen with aminoglycosides in the neonate. Aminoglycosides

  5. Extracellular DNA Shields against Aminoglycosides in Pseudomonas aeruginosa Biofilms

    DEFF Research Database (Denmark)

    Chiang, Wen-Chi; Nilsson, Martin; Jensen, Peter Østrup

    2013-01-01

    Within recent years, it has been established that extracellular DNA is a key constituent of the matrix of microbial biofilms. In addition, it has recently been demonstrated that DNA binds positively charged antimicrobials such as aminoglycosides and antimicrobial peptides. In the present study, we...... provide evidence that extracellular DNA shields against aminoglycosides in Pseudomonas aeruginosa biofilms. We show that exogenously supplemented DNA integrates into P. aeruginosa biofilms and increases their tolerance toward aminoglycosides. We provide evidence that biofilms formed by a DNA release......-deficient P. aeruginosa quorum-sensing mutant are more susceptible to aminoglycoside treatment than wild-type biofilms but become rescued from the detrimental action of aminoglycosides upon supplementation with exogenous DNA. Furthermore, we demonstrate that exposure to lysed polymorphonuclear leukocytes...

  6. Therapeutic drug monitoring of aminoglycosides in neonates

    NARCIS (Netherlands)

    Touw, Daniël J; Westerman, Elsbeth M; Sprij, Arwen J

    2009-01-01

    The efficacy and toxicity of aminoglycosides show a strong direct positive relationship with blood drug concentrations, therefore, therapy with aminoglycosides in adults is usually guided by therapeutic drug monitoring. Dosing regimens in adults have evolved from multiple daily dosing to

  7. A gene encoding maize caffeoyl-CoA O-methyltransferase confers quantitative resistance to multiple pathogens.

    Science.gov (United States)

    Yang, Qin; He, Yijian; Kabahuma, Mercy; Chaya, Timothy; Kelly, Amy; Borrego, Eli; Bian, Yang; El Kasmi, Farid; Yang, Li; Teixeira, Paulo; Kolkman, Judith; Nelson, Rebecca; Kolomiets, Michael; L Dangl, Jeffery; Wisser, Randall; Caplan, Jeffrey; Li, Xu; Lauter, Nick; Balint-Kurti, Peter

    2017-09-01

    Alleles that confer multiple disease resistance (MDR) are valuable in crop improvement, although the molecular mechanisms underlying their functions remain largely unknown. A quantitative trait locus, qMdr 9.02 , associated with resistance to three important foliar maize diseases-southern leaf blight, gray leaf spot and northern leaf blight-has been identified on maize chromosome 9. Through fine-mapping, association analysis, expression analysis, insertional mutagenesis and transgenic validation, we demonstrate that ZmCCoAOMT2, which encodes a caffeoyl-CoA O-methyltransferase associated with the phenylpropanoid pathway and lignin production, is the gene within qMdr 9.02 conferring quantitative resistance to both southern leaf blight and gray leaf spot. We suggest that resistance might be caused by allelic variation at the level of both gene expression and amino acid sequence, thus resulting in differences in levels of lignin and other metabolites of the phenylpropanoid pathway and regulation of programmed cell death.

  8. Recognition elements in rRNA for the tylosin resistance methyltransferase RlmA(II)

    DEFF Research Database (Denmark)

    Lebars, Isabelle; Husson, Clotilde; Yoshizawa, Satoko

    2007-01-01

    The methyltransferase RlmA(II) (formerly TlrB) is found in many Gram-positive bacteria, and methylates the N-1 position of nucleotide G748 within the loop of hairpin 35 in 23S rRNA. Methylation of the rRNA by RlmA(II) confers resistance to tylosin and other mycinosylated 16-membered ring macrolide......RNA substrate indicated that multiple contacts occur between RlmA(II) and nucleotides in stem-loops 33, 34 and 35. RlmA(II) appears to recognize its rRNA target through specific surface shape complementarity at the junction formed by these three helices. This means of recognition is highly similar...

  9. Dosing strategy based on prevailing aminoglycoside minimum inhibitory concentration in India: Evidence and issues

    Directory of Open Access Journals (Sweden)

    Balaji Veeraraghavan

    2017-01-01

    Full Text Available Aminoglycosides are important agents used for treating drug-resistant infections. The current dosing regimen of aminoglycosides does not achieve sufficient serum level concentration for the infected bacterial pathogen interpreted as susceptible based on laboratory testing. Minimum inhibitory concentration was determined for nearly 2000 isolates of Enterobacteriaceae and Pseudomonas aeruginosa by broth microdilution method. Results were interpreted based on CLSI and EUCAST interpretative criteria and the inconsistencies in the susceptibility profile were noted. This study provides insights into the inconsistencies existing in the laboratory interpretation and the corresponding clinical success rates. This urges the need for revising clinical breakpoints for amikacin, to resolve under dosing leading to clinical failure.

  10. Structural characterization of the novel aminoglycoside phosphotransferase AphVIII from Streptomyces rimosus with enzymatic activity modulated by phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Boyko, Konstantin M., E-mail: kmb@inbi.ras.ru [Bach Institute of Biochemistry, Federal Research Centre of Biotechnology of the Russian Academy of Sciences, Leninsky Prospekt. 33, Bld. 2, 119071, Moscow (Russian Federation); National Research Center “Kurchatov Institute”, Kurchatov Complex of NBICS-technologies, Akad. Kurchatova sqr., 1, Moscow, 123182 (Russian Federation); Gorbacheva, Marina A.; Korzhenevskiy, Dmitry A. [National Research Center “Kurchatov Institute”, Kurchatov Complex of NBICS-technologies, Akad. Kurchatova sqr., 1, Moscow, 123182 (Russian Federation); Alekseeva, Maria G.; Mavletova, Dilara A.; Zakharevich, Natalia V.; Elizarov, Sergey M.; Rudakova, Natalia N.; Danilenko, Valery N. [Vavilov Institute of General Genetics, Russian Academy of Sciences, Gubkina str. 3, Moscow, 119333 (Russian Federation); Popov, Vladimir O. [Bach Institute of Biochemistry, Federal Research Centre of Biotechnology of the Russian Academy of Sciences, Leninsky Prospekt. 33, Bld. 2, 119071, Moscow (Russian Federation); National Research Center “Kurchatov Institute”, Kurchatov Complex of NBICS-technologies, Akad. Kurchatova sqr., 1, Moscow, 123182 (Russian Federation)

    2016-09-02

    Aminoglycoside phosphotransferases represent a broad class of enzymes that promote bacterial resistance to aminoglycoside antibiotics via the phosphorylation of hydroxyl groups in the latter. Here we report the spatial structure of the 3′-aminoglycoside phosphotransferase of novel VIII class (AphVIII) solved by X-ray diffraction method with a resolution of 2.15 Å. Deep analysis of APHVIII structure and its comparison with known structures of aminoglycoside phosphotransferases of various types reveals that AphVIII has a typical two-domain fold and, however, possesses some unique characteristics that distinguish the enzyme from its known homologues. The most important difference is the presence of the activation loop with unique Ser146 residue. We demonstrate that in the apo-state of the enzyme the activation loop does not interact with other parts of the enzyme and seems to adopt catalytically competent state only after substrate binding. - Highlights: • 3D structure of the novel aminoglycoside phosphotransferase AphVIII was obtained. • AphVIII activation loop is clearly identified in the electron density. • AphVIII has some unique structural features in its substrate C-ring binding pocket.

  11. Crystallization and preliminary crystallographic analysis of an aminoglycoside kinase from Legionella pneumophila

    International Nuclear Information System (INIS)

    Lemke, Christopher T.; Hwang, Jiyoung; Xiong, Bing; Cianciotto, Nicholas P.; Berghuis, Albert M.

    2005-01-01

    Two crystal forms of the antibiotic resistance enzyme APH(9)-Ia from L. pneumophila are reported. 9-Aminoglycoside phosphotransferase type Ia [APH(9)-Ia] is a resistance factor in Legionella pneuemophila, the causative agent of legionnaires’ disease. It is responsible for providing intrinsic resistance to the antibiotic spectinomycin. APH(9)-Ia phosphorylates one of the hydroxyl moieties of spectinomycin in an ATP-dependent manner, abolishing the antibiotic properties of this drug. Here, the crystallization and preliminary X-ray studies of this enzyme in two crystal forms is reported. One of the these crystal forms provides diffraction data to a resolution of 1.7 Å

  12. Sublethal Triclosan Exposure Decreases Susceptibility to Gentamicin and Other Aminoglycosides in Listeria monocytogenes

    DEFF Research Database (Denmark)

    Christensen, Ellen Gerd; Gram, Lone; Kastbjerg, Vicky Gaedt

    2011-01-01

    (containing quaternary ammonium compound) in four consecutive cultures did not alter the frequency of antibiotic-tolerant isolates, as determined by plating on 2x the MIC for a range of antibiotics. Exposure of eight strains of L. monocytogenes to 1 and 4 µg/ml triclosan did not alter triclosan sensitivity...... resistance remained at a high level also after five subcultures without triclosan or gentamicin. Aminoglycoside resistance can be caused by mutations in the target site, the 16S rRNA gene. However, such mutations were not detected in the N53-1-resistant isolates. A combination of gentamicin and ampicillin...

  13. Modulation of RNA function by aminoglycoside antibiotics.

    Science.gov (United States)

    Schroeder, R; Waldsich, C; Wank, H

    2000-01-04

    One of the most important families of antibiotics are the aminoglycosides, including drugs such as neomycin B, paromomycin, gentamicin and streptomycin. With the discovery of the catalytic potential of RNA, these antibiotics became very popular due to their RNA-binding capacity. They serve for the analysis of RNA function as well as for the study of RNA as a potential therapeutic target. Improvements in RNA structure determination recently provided first insights into the decoding site of the ribosome at high resolution and how aminoglycosides might induce misreading of the genetic code. In addition to inhibiting prokaryotic translation, aminoglycosides inhibit several catalytic RNAs such as self-splicing group I introns, RNase P and small ribozymes in vitro. Furthermore, these antibiotics interfere with human immunodeficiency virus (HIV) replication by disrupting essential RNA-protein contacts. Most exciting is the potential of many RNA-binding antibiotics to stimulate RNA activities, conceiving small-molecule partners for the hypothesis of an ancient RNA world. SELEX (systematic evolution of ligands by exponential enrichment) has been used in this evolutionary game leading to small synthetic RNAs, whose NMR structures gave valuable information on how aminoglycosides interact with RNA, which could possibly be used in applied science.

  14. Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistance in bacteria

    DEFF Research Database (Denmark)

    Giessing, Anders; Jensen, Søren Skov; Rasmussen, Anette

    2009-01-01

    The Cfr methyltransferase confers combined resistance to five different classes of antibiotics that bind to the peptidyl transferase center of bacterial ribosomes. The Cfr-mediated modification has previously been shown to occur on nucleotide A2503 of 23S rRNA and has a mass corresponding......,8-dimethyladenosine. The mutation of single conserved cysteine residues in the radical SAM motif CxxxCxxC of Cfr abolishes its activity, lending support to the notion that the Cfr modification reaction occurs via a radical-based mechanism. Antibiotic susceptibility data confirm that the antibiotic resistance...

  15. O6-Methylguanine DNA Methyltransferase Status Does Not Predict Response or Resistance to Alkylating Agents in Well-Differentiated Pancreatic Neuroendocrine Tumors.

    Science.gov (United States)

    Raj, Nitya; Klimstra, David S; Horvat, Natally; Zhang, Liying; Chou, Joanne F; Capanu, Marinela; Basturk, Olca; Do, Richard Kinh Gian; Allen, Peter J; Reidy-Lagunes, Diane

    2017-07-01

    Alkylating agents have activity in well-differentiated pancreatic neuroendocrine tumors (WD panNETs). In glioblastoma multiforme, decreased activity of O-methylguanine DNA methyltransferase (MGMT) predicts response; in panNETs, MGMT relevance is unknown. We identified patients with WD panNETs treated with alkylating agents, determined best overall response by Response Evaluation Criteria In Solid Tumors (RECIST) 1.1, and performed MGMT activity testing. Fifty-six patients were identified; 26 (46%) of the 56 patients experienced partial response, 24 (43%) of 56 experienced stable disease, and 6 (11%) of 56 experienced progression of disease. O-methylguanine DNA methyltransferase status was available for 36 tumors. For tumors with partial response, 10 (67%) of 15 were MGMT deficient, and 5 (33%) of 15 were MGMT intact. For tumors with stable disease, 7 (47%) of 15 were MGMT deficient, and 8 (53%) of 15 were MGMT intact. For tumors with progression of disease, 3 (50%) of 6 were MGMT deficient, and 3 (50%) of 6 were MGMT intact. We observed response and resistance to alkylating agents in MGMT-deficient and MGMT-intact tumors. O-methylguanine DNA methyltransferase status should not guide alkylating agent therapy in WD panNETs.

  16. The fish pathogen Yersinia ruckeri produces holomycin and uses an RNA methyltransferase for self-resistance.

    Science.gov (United States)

    Qin, Zhiwei; Baker, Alexander Thomas; Raab, Andrea; Huang, Sheng; Wang, Tiehui; Yu, Yi; Jaspars, Marcel; Secombes, Christopher J; Deng, Hai

    2013-05-24

    Holomycin and its derivatives belong to a class of broad-spectrum antibacterial natural products containing a rare dithiolopyrrolone heterobicyclic scaffold. The antibacterial mechanism of dithiolopyrrolone compounds has been attributed to the inhibition of bacterial RNA polymerase activities, although the exact mode of action has not been established in vitro. Some dithiopyrrolone derivatives display potent anticancer activities. Recently the biosynthetic gene cluster of holomycin has been identified and characterized in Streptomyces clavuligerus. Here we report that the fish pathogen Yersinia ruckeri is a holomycin producer, as evidenced through genome mining, chemical isolation, and structural elucidation as well as genetic manipulation. We also identified a unique regulatory gene hom15 at one end of the gene cluster encoding a cold-shock-like protein that likely regulates the production of holomycin in low cultivation temperatures. Inactivation of hom15 resulted in a significant loss of holomycin production. Finally, gene disruption of an RNA methyltransferase gene hom12 resulted in the sensitivity of the mutant toward holomycin. A complementation experiment of hom12 restored the resistance against holomycin. Although the wild-type Escherichia coli BL21(DE3) Gold is susceptible to holomycin, the mutant harboring hom12 showed tolerance toward holomycin. High resolution liquid chromatography (LC)-ESI/MS analysis of digested RNA fragments demonstrated that the wild-type Y. ruckeri and E. coli harboring hom12 contain a methylated RNA fragment, whereas the mutated Y. ruckeri and the wild-type E. coli only contain normal non-methylated RNA fragments. Taken together, our results strongly suggest that this putative RNA methyltransferase Hom12 is the self-resistance protein that methylates the RNA of Y. ruckeri to reduce the cytotoxic effect of holomycin during holomycin production.

  17. Identification of genes involved in low aminoglycoside-induced SOS response in Vibrio cholerae: a role for transcription stalling and Mfd helicase.

    Science.gov (United States)

    Baharoglu, Zeynep; Babosan, Anamaria; Mazel, Didier

    2014-02-01

    Sub-inhibitory concentrations (sub-MIC) of antibiotics play a very important role in selection and development of resistances. Unlike Escherichia coli, Vibrio cholerae induces its SOS response in presence of sub-MIC aminoglycosides. A role for oxidized guanine residues was observed, but the mechanisms of this induction remained unclear. To select for V. cholerae mutants that do not induce low aminoglycoside-mediated SOS induction, we developed a genetic screen that renders induction of SOS lethal. We identified genes involved in this pathway using two strategies, inactivation by transposition and gene overexpression. Interestingly, we obtained mutants inactivated for the expression of proteins known to destabilize the RNA polymerase complex. Reconstruction of the corresponding mutants confirmed their specific involvement in induction of SOS by low aminoglycoside concentrations. We propose that DNA lesions formed on aminoglycoside treatment are repaired through the formation of single-stranded DNA intermediates, inducing SOS. Inactivation of functions that dislodge RNA polymerase leads to prolonged stalling on these lesions, which hampers SOS induction and repair and reduces viability under antibiotic stress. The importance of these mechanisms is illustrated by a reduction of aminoglycoside sub-MIC. Our results point to a central role for transcription blocking at DNA lesions in SOS induction, so far underestimated.

  18. Efflux Pump-mediated Drug Resistance in Burkholderia

    Directory of Open Access Journals (Sweden)

    Nicole L Podnecky

    2015-04-01

    Full Text Available Several members of the genus Burkholderia are prominent pathogens. Infections caused by these bacteria are difficult to treat because of significant antibiotic resistance. Virtually all Burkholderia species are also resistant to polymyxin, prohibiting use of drugs like colistin that are available for treatment of infections caused by most other drug resistant Gram-negative bacteria. Despite clinical significance and antibiotic resistance of Burkholderia species, characterization of efflux pumps lags behind other non-enteric Gram-negative pathogens such as Acinetobacter baumannii and Pseudomonas aeruginosa. Although efflux pumps have been described in several Burkholderia species, they have been best studied in B. cenocepacia and B. pseudomallei. As in other non-enteric Gram-negatives, efflux pumps of the resistance nodulation cell division (RND family are the clinically most significant efflux systems in these two species. Several efflux pumps were described in B. cenocepacia, which when expressed confer resistance to clinically significant antibiotics, including aminoglycosides, chloramphenicol, fluoroquinolones, and tetracyclines. Three RND pumps have been characterized in B. pseudomallei, two of which confer either intrinsic or acquired resistance to aminoglycosides, macrolides, chloramphenicol, fluoroquinolones, tetracyclines, trimethoprim, and in some instances trimethoprim+sulfamethoxazole. Several strains of the host-adapted B. mallei, a clone of B. pseudomallei, lack AmrAB-OprA and are therefore aminoglycoside and macrolide susceptible. B. thailandensis is closely related to B. pseudomallei, but non-pathogenic to humans. Its pump repertoire and ensuing drug resistance profile parallels that of B. pseudomallei. An efflux pump in B. vietnamiensis plays a significant role in acquired aminoglycoside resistance. Summarily, efflux pumps are significant players in Burkholderia drug resistance.

  19. Outbreak of Serratia marcescens Coproducing ArmA and CTX-M-15 Mediated High Levels of Resistance to Aminoglycoside and Extended-Spectrum Beta-Lactamases, Algeria.

    Science.gov (United States)

    Batah, Rima; Loucif, Lotfi; Olaitan, Abiola Olumuyiwa; Boutefnouchet, Nafissa; Allag, Hamoudi; Rolain, Jean-Marc

    2015-08-01

    Serratia marcescens is one of the most important pathogens responsible for nosocomial infections worldwide. Here, we have investigated the molecular support of antibiotic resistance and genetic relationships in a series of 54 S. marcescens clinical isolates collected from Eastern Algeria between December 2011 and July 2013. The 54 isolates were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Antibiotic susceptibility testing was performed by disc diffusion and E-test methods. Antibiotic resistance genes were detected by polymerase chain reaction (PCR). The genetic transfer of antibiotic resistance was performed by conjugation using azide-resistant Escherichia coli J53 as the recipient strain, and plasmid analysis was done by PCR-based replicon typing. The relatedness of our isolates was determined by phylogenetic analysis based on partial sequences of four protein-encoding genes (gyrB, rpoB, infB, and atpD) and then compared to MALDI-TOF MS clustering. Thirty-five out of 54 isolates yielded an extended-spectrum β-lactamase (ESBL) phenotype and carried bla(CTX-M-15) (n=32), bla(TEM-1) (n=26), bla(TEM-71) (n=1), bla(SHV-1a) (n=1), and bla(PER-2) (n=12). Among these isolates, we identified a cluster of 15 isolates from a urology unit that coharbored ESBL and the 16S rRNA methyltransferase armA. Conjugation was successful for five selected strains, demonstrating the transferability of a conjugative plasmid of incompatibility group incL/M type. Phylogenetic analysis along with MALDI-TOF clustering likely suggested an outbreak of such isolates in the urology unit. In this study, we report for the first time the co-occurrence of armA methyltransferase with ESBL in S. marcescens clinical isolates in Eastern Algeria.

  20. Options for treating carbapenem-resistant Enterobacteriaceae.

    Science.gov (United States)

    Rafailidis, Petros I; Falagas, Matthew E

    2014-12-01

    To address the therapeutic management of carbapenem-resistant Enterobacteriaceae on the basis of literature of the last 12 months. Retrospective and prospective (nonrandomized noncontrolled) studies provide data regarding the management of infections due to carbapenem-resistant Enterobacteriaceae. The combination of a carbapenem with colistin or high-dose tigecycline or aminoglycoside or even triple carbapenem-containing combinations if the minimum inhibitory concentration (MIC) range of carbapenem (meropenem and imipenem) resistance is 8 mg/l or less seems to have an advantage over monotherapy with either colistin or tigecycline or fosfomycin. For Enterobacteriaceae with MIC for carbapenems over 8 mg/l, combination regimens involve colistin, tigecycline usually administered in a double dose than that suggested by its manufacturer, fosfomycin and aminoglycosides in various combinations. Suggestions based on the limited literature cannot be made safely. Combination regimens involving carbapenems for Enterobacteriaceae with MICs 8 mg/l or less for carbapenems (in dual combination with colistin or high-dose tigecycline or aminoglycoside or even triple combinations) seem to confer some therapeutic advantage over monotherapy. For Enterobacteriaceae with higher than the above-mentioned MICs, a combination of two or even three antibiotics among colistin, high-dose tigecycline, aminoglycoside and fosfomycin seems to confer decreased mortality.

  1. In vitro bactericidal activity of aminoglycosides, including the next-generation drug plazomicin, against Brucella spp.

    Science.gov (United States)

    Plazomicin is a next-generation aminoglycoside with a potentially improved safety profile compared to other aminoglycosides. This study assessed plazomicin MICs and MBCs in four Brucella spp. reference strains. Like other aminoglycosides and aminocyclitols, plazomicin MBC values equaled MIC values ...

  2. Overexpression of the mitochondrial methyltransferase TFB1M in the mouse does not impact mitoribosomal methylation status or hearing

    DEFF Research Database (Denmark)

    Lee, Seungmin; Rose, Simon; Metodiev, Metodi D

    2015-01-01

    maternally inherited traits. The pathophysiology induced by mtDNA mutations has traditionally been attributed to deficient oxidative phosphorylation, which causes energy crisis with functional impairment of multiple cellular processes. In contrast, it was recently reported that signaling induced......Mitochondrial dysfunction is a well-established cause of sensorineural deafness, but the pathophysiological events are poorly understood. Non-syndromic deafness and predisposition to aminoglycoside-induced deafness can be caused by specific mutations in the 12S rRNA gene of mtDNA and are thus...... by 'hypermethylation' of two conserved adenosines of 12S rRNA in the mitoribosome is of key pathophysiological importance in sensorineural deafness. In support for this concept, it was reported that overexpression of the essential mitochondrial methyltransferase TFB1M in the mouse was sufficient to induce...

  3. Clinical Pharmacokinetics of Penicillins, Cephalosporins and Aminoglycosides in the Neonate: A Review

    Directory of Open Access Journals (Sweden)

    Gian Maria Pacifici

    2010-08-01

    Full Text Available Bacterial infections are common in the neonates and are a major cause of morbidity and mortality. Sixty percent of preterm infants admitted to neonatal intensive care units received at least one antibiotic during the first week of life. Penicillins, aminoglycosides and cephalosporins comprised 53, 43 and 16%, respectively. Kinetic parameters such as the half-life (t1/2, clearance (Cl, and volume of distribution (Vd change with development, so the kinetics of penicillins, cephalosporins and aminoglycosides need to be studied in order to optimise therapy with these drugs. The aim of this study is to review the pharmacokinetics of penicillins, cephalosporins and aminoglycosides in the neonate in a single article in order to provide a critical analysis of the literature and thus provide a useful tool in the hands of physicians. The bibliographic search was performed electronically using PubMed, as the search engine, until February 2nd, 2010. Medline search terms were as follows: pharmacokinetics AND (penicillins OR cephalosporins OR aminoglycosides AND infant, newborn, limiting to humans. Penicillins, cephalosporins and aminoglycosides are fairly water soluble and are mainly eliminated by the kidneys. The maturation of the kidneys governs the pharmacokinetics of penicillins, cephalosporins and aminoglycosides in the neonate. The renal excretory function is reduced in preterms compared to term infants and Cl of these drugs is reduced in premature infants. Gestational and postnatal ages are important factors in the maturation of the neonate and, as these ages proceed, Cl of penicillins, cephalosporins and aminoglycosides increases. Cl and t1/2 are influenced by development and this must be taken into consideration when planning a dosage regimen with these drugs. More pharmacokinetic studies are required to ensure that the dose recommended for the treatment of sepsis in the neonate is evidence based.

  4. Local Mechanisms for Loud Sound-Enhanced Aminoglycoside Entry into Outer Hair Cells

    Directory of Open Access Journals (Sweden)

    Hongzhe eLi

    2015-04-01

    Full Text Available Loud sound exposure exacerbates aminoglycoside ototoxicity, increasing the risk of permanent hearing loss and degrading the quality of life in affected individuals. We previously reported that loud sound exposure induces temporary threshold shifts (TTS and enhances uptake of aminoglycosides, like gentamicin, by cochlear outer hair cells (OHCs. Here, we explore mechanisms by which loud sound exposure and TTS could increase aminoglycoside uptake by OHCs that may underlie this form of ototoxic synergy.Mice were exposed to loud sound levels to induce TTS, and received fluorescently-tagged gentamicin (GTTR for 30 minutes prior to fixation. The degree of TTS was assessed by comparing auditory brainstem responses before and after loud sound exposure. The number of tip links, which gate the GTTR-permeant mechanoelectrical transducer (MET channels, was determined in OHC bundles, with or without exposure to loud sound, using scanning electron microscopy.We found wide-band noise (WBN levels that induce TTS also enhance OHC uptake of GTTR compared to OHCs in control cochleae. In cochlear regions with TTS, the increase in OHC uptake of GTTR was significantly greater than in adjacent pillar cells. In control mice, we identified stereociliary tip links at ~50% of potential positions in OHC bundles. However, the number of OHC tip links was significantly reduced in mice that received WBN at levels capable of inducing TTS.These data suggest that GTTR uptake by OHCs during TTS occurs by increased permeation of surviving, mechanically-gated MET channels, and/or non-MET aminoglycoside-permeant channels activated following loud sound exposure. Loss of tip links would hyperpolarize hair cells and potentially increase drug uptake via aminoglycoside-permeant channels expressed by hair cells. The effect of TTS on aminoglycoside-permeant channel kinetics will shed new light on the mechanisms of loud sound-enhanced aminoglycoside uptake, and consequently on ototoxic

  5. Aminoglycoside antibiotics and autism: a speculative hypothesis

    Directory of Open Access Journals (Sweden)

    Manev Hari

    2001-10-01

    Full Text Available Abstract Background Recently, it has been suspected that there is a relationship between therapy with some antibiotics and the onset of autism; but even more curious, some children benefited transiently from a subsequent treatment with a different antibiotic. Here, we speculate how aminoglycoside antibiotics might be associated with autism. Presentation We hypothesize that aminoglycoside antibiotics could a trigger the autism syndrome in susceptible infants by causing the stop codon readthrough, i.e., a misreading of the genetic code of a hypothetical critical gene, and/or b improve autism symptoms by correcting the premature stop codon mutation in a hypothetical polymorphic gene linked to autism. Testing Investigate, retrospectively, whether a link exists between aminoglycoside use (which is not extensive in children and the onset of autism symptoms (hypothesis "a", or between amino glycoside use and improvement of these symptoms (hypothesis "b". Whereas a prospective study to test hypothesis "a" is not ethically justifiable, a study could be designed to test hypothesis "b". Implications It should be stressed that at this stage no direct evidence supports our speculative hypothesis and that its main purpose is to initiate development of new ideas that, eventually, would improve our understanding of the pathobiology of autism.

  6. Aminoglycosides in septic shock: an overview, with specific consideration given to their nephrotoxic risk.

    Science.gov (United States)

    Boyer, Alexandre; Gruson, Didier; Bouchet, Stéphane; Clouzeau, Benjamin; Hoang-Nam, Bui; Vargas, Frédéric; Gilles, Hilbert; Molimard, Mathieu; Rogues, Anne-Marie; Moore, Nicholas

    2013-04-01

    Aminoglycoside nephrotoxicity has been reported in patients with sepsis, and several risk factors have been described. Once-daily dosing and shorter treatment have reduced nephrotoxicity risk, and simplified aminoglycoside monitoring. This review focuses on nephrotoxicity associated with aminoglycosides in the subset of patients with septic shock or severe sepsis. These patients are radically different from those with less severe sepsis. They may have, for instance, renal impairment due to the shock per se, sepsis-related acute kidney injury, frequent association with pre-existing risk factors for renal failure such as diabetes, dehydration and other nephrotoxic treatments. In this category of patients, these risk factors might modify substantially the benefit-risk ratio of aminoglycosides. In addition, aminoglycoside administration in critically ill patients with sepsis is complicated by an extreme inter- and intra-individual variability in drug pharmacokinetic/pharmacodynamic characteristics: the volume of distribution (Vd) is frequently increased while the elimination constant can be either increased or decreased. Consequently, and although its effect on nephrotoxicity has not been explored, a different administration schedule, i.e. a high-dose once daily (HDOD), and several therapeutic drug monitoring (TDM) options have been proposed in these patients. This review describes the historical perspective of these different options, including those applying to subsets of patients in which aminoglycoside administration is even more complex (obese intensive care unit [ICU] patients, patients needing continuous or discontinuous renal replacement therapy [CRRT/DRRT]). A simple linear dose adjustment according to aminoglycoside serum concentration can be classified as low-intensity TDM. Nomograms have also been proposed, based on the maximum (peak) plasma concentration (Cmax) objectives, weight and creatinine clearance. The Sawchuk and Zaske method (based on the

  7. Inhibition by Commercial Aminoglycosides of Human Connexin Hemichannels Expressed in Bacteria

    Directory of Open Access Journals (Sweden)

    Mariana C. Fiori

    2017-11-01

    Full Text Available In addition to gap junctional channels that mediate cell-to-cell communication, connexins form hemichannels that are present at the plasma membrane. Since hemichannels are permeable to small hydrophilic compounds, including metabolites and signaling molecules, their abnormal opening can cause or contribute to cell damage in disorders such as cardiac infarct, stroke, deafness, skin diseases, and cataracts. Therefore, hemichannels are potential pharmacological targets. A few aminoglycosides, well-known broad-spectrum antibiotics, have been shown to inhibit hemichannels. Here, we tested several commercially available aminoglycosides for inhibition of human connexin hemichannels using a cell-based bacterial growth complementation assay that we developed recently. We found that kanamycin A, kanamycin B, geneticin, neomycin, and paromomycin are effective inhibitors of hemichannels formed by connexins 26, 43, and 46 (Cx26, Cx43, and Cx46. Because of the >70 years of clinical experience with aminoglycosides and the fact that several of the aminoglycosides tested here have been used in humans, they are promising starting points for the development of effective connexin hemichannel inhibitors.

  8. Functional characterization of KanP, a methyltransferase from the kanamycin biosynthetic gene cluster of Streptomyces kanamyceticus.

    Science.gov (United States)

    Nepal, Keshav Kumar; Yoo, Jin Cheol; Sohng, Jae Kyung

    2010-09-20

    KanP, a putative methyltransferase, is located in the kanamycin biosynthetic gene cluster of Streptomyces kanamyceticus ATCC12853. Amino acid sequence analysis of KanP revealed the presence of S-adenosyl-L-methionine binding motifs, which are present in other O-methyltransferases. The kanP gene was expressed in Escherichia coli BL21 (DE3) to generate the E. coli KANP recombinant strain. The conversion of external quercetin to methylated quercetin in the culture extract of E. coli KANP proved the function of kanP as S-adenosyl-L-methionine-dependent methyltransferase. This is the first report concerning the identification of an O-methyltransferase gene from the kanamycin gene cluster. The resistant activity assay and RT-PCR analysis demonstrated the leeway for obtaining methylated kanamycin derivatives from the wild-type strain of kanamycin producer. 2009 Elsevier GmbH. All rights reserved.

  9. Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae: Roles in ribosome biogenesis and translation.

    Science.gov (United States)

    Al-Hadid, Qais; White, Jonelle; Clarke, Steven

    2016-02-12

    A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed -1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Plant isoflavone and isoflavanone O-methyltransferase genes

    Science.gov (United States)

    Broeckling, Bettina E.; Liu, Chang-Jun; Dixon, Richard A.

    2014-08-19

    The invention provides enzymes that encode O-methyltransferases (OMTs) from Medicago truncatula that allow modification to plant (iso)flavonoid biosynthetic pathways. In certain aspects of the invention, the genes encoding these enzymes are provided. The invention therefore allows the modification of plants for isoflavonoid content. Transgenic plants comprising such enzymes are also provided, as well as methods for improving disease resistance in plants. Methods for producing food and nutraceuticals, and the resulting compositions, are also provided.

  11. Bacterial Enzymes and Antibiotic Resistance- Oral Presentation

    Energy Technology Data Exchange (ETDEWEB)

    Maltz, Lauren [SLAC National Accelerator Lab., Menlo Park, CA (United States)

    2015-08-25

    By using protein crystallography and X-ray diffraction, structures of bacterial enzymes were solved to gain a better understanding of how enzymatic modification acts as an antibacterial resistance mechanism. Aminoglycoside phosphotransferases (APHs) are one of three aminoglycoside modifying enzymes that confer resistance to the aminoglycoside antibiotics via enzymatic modification, rendering many drugs obsolete. Specifically, the APH(2”) family vary in their substrate specificities and also in their preference for the phosphate donor (ADP versus GDP). By solving the structures of members of the APH(2”) family of enzymes, we can see how domain movements are important to their substrate specificity. Our structure of the ternary complex of APH(2”)-IIIa with GDP and kanamycin, when compared to the known structures of APH(2”)-IVa, reveals that there are real physical differences between these two enzymes, a structural finding that explains why the two enzymes differ in their preferences for certain aminoglycosides. Another important group of bacterial resistance enzymes are the Class D β-lactamases. Oxacillinase carbapenemases (OXAs) are part of this enzyme class and have begun to confer resistance to ‘last resort’ drugs, most notably carbapenems. Our structure of OXA-143 shows that the conformational flexibility of a conserved hydrophobic residue in the active site (Val130) serves to control the entry of a transient water molecule responsible for a key step in the enzyme’s mechanism. Our results provide insight into the structural mechanisms of these two different enzymes.

  12. Accuracy of genetic code translation and its orthogonal corruption by aminoglycosides and Mg2+ ions.

    Science.gov (United States)

    Zhang, Jingji; Pavlov, Michael Y; Ehrenberg, Måns

    2018-02-16

    We studied the effects of aminoglycosides and changing Mg2+ ion concentration on the accuracy of initial codon selection by aminoacyl-tRNA in ternary complex with elongation factor Tu and GTP (T3) on mRNA programmed ribosomes. Aminoglycosides decrease the accuracy by changing the equilibrium constants of 'monitoring bases' A1492, A1493 and G530 in 16S rRNA in favor of their 'activated' state by large, aminoglycoside-specific factors, which are the same for cognate and near-cognate codons. Increasing Mg2+ concentration decreases the accuracy by slowing dissociation of T3 from its initial codon- and aminoglycoside-independent binding state on the ribosome. The distinct accuracy-corrupting mechanisms for aminoglycosides and Mg2+ ions prompted us to re-interpret previous biochemical experiments and functional implications of existing high resolution ribosome structures. We estimate the upper thermodynamic limit to the accuracy, the 'intrinsic selectivity' of the ribosome. We conclude that aminoglycosides do not alter the intrinsic selectivity but reduce the fraction of it that is expressed as the accuracy of initial selection. We suggest that induced fit increases the accuracy and speed of codon reading at unaltered intrinsic selectivity of the ribosome.

  13. Deciphering the details of RNA aminoglycoside interactions: from atomistic models to biotechnological applications

    Energy Technology Data Exchange (ETDEWEB)

    Ilgu, Muslum [Iowa State Univ., Ames, IA (United States)

    2012-01-01

    A detailed study was done of the neomycin-B RNA aptamer for determining its selectivity and binding ability to both neomycin– and kanamycin-class aminoglycosides. A novel method to increase drug concentrations in cells for more efficiently killing is described. To test the method, a bacterial model system was adopted and several small RNA molecules interacting with aminoglycosides were cloned downstream of T7 RNA polymerase promoter in an expression vector. Then, the growth analysis of E. coli expressing aptamers was observed for 12-hour period. Our analysis indicated that aptamers helped to increase the intracellular concentration of aminoglycosides thereby increasing their efficacy.

  14. Antibiotic resistant enterococci—Tales of a drug resistance gene trafficker

    DEFF Research Database (Denmark)

    Werner, Guido; Coque, Teresa M.; Franz, Charles M.A.P.

    2013-01-01

    Enterococci have been recognized as important hospital-acquired pathogens in recent years, and isolates of E. faecalis and E. faecium are the third- to fourth-most prevalent nosocomial pathogen worldwide. Acquired resistances, especially against penicilin/ampicillin, aminoglycosides (high-level) ...

  15. Radioimmunoassay and radioenzymatic assay of a new aminoglycoside antibiotic, netilmicin

    International Nuclear Information System (INIS)

    Broughton, A.; Strong, J.E.; Pickering, L.K.; Knight, J.; Bodey, G.P.

    1978-01-01

    A radioimmunoassay and a radioenzymatic assay for netilmicin, a new aminoglycoside, were developed in our laboratories to assist in the study of the pharmacology of the drug and establish values for use in its monitoring. The assays are sensitive, precise, and rapid, giving results that correlate (r = 0.90) with each other and with those of a microbiological assay in which Klebsiella pneumoniae is used as the test organism. Preliminary pharmacological studies show the drug to have a biological half-life of 135 min, which is comparable to that for other aminoglycosides

  16. Domain V of 23S rRNA contains all the structural elements necessary for recognition by the ErmE methyltransferase

    DEFF Research Database (Denmark)

    Vester, B; Douthwaite, S

    1994-01-01

    investigated what structural elements in 23S rRNA are required for specific recognition by the ErmE methyltransferase. The ermE gene was cloned into R1 plasmid derivatives, providing a means of inducible expression in Escherichia coli. Expression of the methyltransferase in vivo confers resistance......, and the enzyme efficiently modifies 23S rRNA in vitro. Removal of most of the 23S rRNA structure, so that only domain V (nucleotides 2000 to 2624) remains, does not affect the efficiency of modification by the methyltransferase. In addition, modification still occurs after the rRNA tertiary structure has been...

  17. Aminoglucósidos: mirada actual desde su historia Aminoglycosides: a present look based on their history

    Directory of Open Access Journals (Sweden)

    Miriam Aliño Santiago

    2007-06-01

    Full Text Available Se refiere la historia, mecanismos de acción y eficacia de los aminoglucósidos en los pacientes pediátricos, así como las limitaciones de su utilidad por el surgimiento de resistencias bacterianas originadas por empleo abusivo. Se presenta la estrategia de administración de monodosis, como alternativa frente al método tradicional de dosis fraccionadas, y también las complicaciones más frecuentes y graves de los aminoglucósidos y su sinergismo con otras familias de antimicrobianos. Y se citan investigaciones realizadas en el país en materia de terapia antibiótica.We referred to history, mechanisms of action and efficacy of aminoglycosides in pediatric patients as well as limitations in their use because of the emergence of bacterial resistance caused by overuse. The one-dose administration strategy as an alternative to the traditional methods of fractioned doses, the most frequent and serious complictions of aminoglycosides and their sinergism with other antimicrobial families were presented. We quoted research studies on antibiotic therapy made in the country.

  18. Detection and Quantification of Ribosome Inhibition by Aminoglycoside Antibiotics in Living Bacteria Using an Orthogonal Ribosome-Controlled Fluorescent Reporter.

    Science.gov (United States)

    Huang, Shijie; Zhu, Xuechen; Melançon, Charles E

    2016-01-15

    The ribosome is the quintessential antibacterial drug target, with many structurally and mechanistically distinct classes of antibacterial agents acting by inhibiting ribosome function. Detecting and quantifying ribosome inhibition by small molecules and investigating their binding modes and mechanisms of action are critical to antibacterial drug discovery and development efforts. To develop a ribosome inhibition assay that is operationally simple, yet provides direct information on the drug target and the mechanism of action, we have developed engineered E. coli strains harboring an orthogonal ribosome-controlled green fluorescent protein (GFP) reporter that produce fluorescent signal when the orthogonal ribosome is inhibited. As a proof of concept, we demonstrate that these strains, when coexpressing homogeneous populations of aminoglycoside resistant ribosomes, act as sensitive and quantitative detectors of ribosome inhibition by a set of 12 structurally diverse aminoglycoside antibiotics. We suggest that this strategy can be extended to quantifying ribosome inhibition by other drug classes.

  19. Pyruvate cycle increases aminoglycoside efficacy and provides respiratory energy in bacteria.

    Science.gov (United States)

    Su, Yu-Bin; Peng, Bo; Li, Hui; Cheng, Zhi-Xue; Zhang, Tian-Tuo; Zhu, Jia-Xin; Li, Dan; Li, Min-Yi; Ye, Jin-Zhou; Du, Chao-Chao; Zhang, Song; Zhao, Xian-Liang; Yang, Man-Jun; Peng, Xuan-Xian

    2018-02-13

    The emergence and ongoing spread of multidrug-resistant bacteria puts humans and other species at risk for potentially lethal infections. Thus, novel antibiotics or alternative approaches are needed to target drug-resistant bacteria, and metabolic modulation has been documented to improve antibiotic efficacy, but the relevant metabolic mechanisms require more studies. Here, we show that glutamate potentiates aminoglycoside antibiotics, resulting in improved elimination of antibiotic-resistant pathogens. When exploring the metabolic flux of glutamate, it was found that the enzymes that link the phosphoenolpyruvate (PEP)-pyruvate-AcCoA pathway to the TCA cycle were key players in this increased efficacy. Together, the PEP-pyruvate-AcCoA pathway and TCA cycle can be considered the pyruvate cycle (P cycle). Our results show that inhibition or gene depletion of the enzymes in the P cycle shut down the TCA cycle even in the presence of excess carbon sources, and that the P cycle operates routinely as a general mechanism for energy production and regulation in Escherichia coli and Edwardsiella tarda These findings address metabolic mechanisms of metabolite-induced potentiation and fundamental questions about bacterial biochemistry and energy metabolism.

  20. A versatile non-radioactive assay for DNA methyltransferase activity and DNA binding

    Science.gov (United States)

    Frauer, Carina; Leonhardt, Heinrich

    2009-01-01

    We present a simple, non-radioactive assay for DNA methyltransferase activity and DNA binding. As most proteins are studied as GFP fusions in living cells, we used a GFP binding nanobody coupled to agarose beads (GFP nanotrap) for rapid one-step purification. Immobilized GFP fusion proteins were subsequently incubated with different fluorescently labeled DNA substrates. The absolute amounts and molar ratios of GFP fusion proteins and bound DNA substrates were determined by fluorescence spectroscopy. In addition to specific DNA binding of GFP fusion proteins, the enzymatic activity of DNA methyltransferases can also be determined by using suicide DNA substrates. These substrates contain the mechanism-based inhibitor 5-aza-dC and lead to irreversible covalent complex formation. We obtained covalent complexes with mammalian DNA methyltransferase 1 (Dnmt1), which were resistant to competition with non-labeled canonical DNA substrates, allowing differentiation between methyltransferase activity and DNA binding. By comparison, the Dnmt1C1229W catalytic site mutant showed DNA-binding activity, but no irreversible covalent complex formation. With this assay, we could also confirm the preference of Dnmt1 for hemimethylated CpG sequences. The rapid optical read-out in a multi-well format and the possibility to test several different substrates in direct competition allow rapid characterization of sequence-specific binding and enzymatic activity. PMID:19129216

  1. Purification, crystallization and preliminary X-ray analysis of aminoglycoside-2′′-phosphotransferase-Ic [APH(2′′)-Ic] from Enterococcus gallinarum

    International Nuclear Information System (INIS)

    Byrnes, Laura J.; Badarau, Adriana; Vakulenko, Sergei B.; Smith, Clyde A.

    2008-01-01

    APH(2′′)-Ic is an enzyme that is responsible for high-level gentamicin resistance in E. gallinarum isolates. Crystals of the wild-type enzyme and three mutants have been prepared and a complete X-ray diffraction data set was collected to 2.15 Å resolution from an F108L crystal. Bacterial resistance to aminoglycoside antibiotics is primarily the result of deactivation of the drugs. Three families of enzymes are responsible for this activity, with one such family being the aminoglycoside phosphotransferases (APHs). The gene encoding one of these enzymes, aminoglycoside-2′′-phosphotransferase-Ic [APH(2′′)-Ic] from Enterococcus gallinarum, has been cloned and the wild-type protein (comprising 308 amino-acid residues) and three mutants that showed elevated minimum inhibitory concentrations towards gentamicin (F108L, H258L and a double mutant F108L/H258L) were expressed in Escherichia coli and subsequently purified. All APH(2′′)-Ic variants were crystallized in the presence of 14–20%(w/v) PEG 4000, 0.25 M MgCl 2 , 0.1 M Tris–HCl pH 8.5 and 1 mM Mg 2 GTP. The crystals belong to the monoclinic space group C2, with one molecule in the asymmetric unit. The approximate unit-cell parameters are a = 82.4, b = 54.2, c = 77.0 Å, β = 108.8°. X-ray diffraction data were collected to approximately 2.15 Å resolution from an F108L crystal at beamline BL9-2 at SSRL, Stanford, California, USA

  2. Aminoglycoside-derived amphiphilic nanoparticles for molecular delivery.

    Science.gov (United States)

    Miryala, Bhavani; Godeshala, Sudhakar; Grandhi, Taraka Sai Pavan; Christensen, Matthew D; Tian, Yanqing; Rege, Kaushal

    2016-10-01

    The development of effective drug carriers can lead to improved outcomes in a variety of disease conditions. Aminoglycosides have been used as antibacterial therapeutics, and are attractive as monomers for the development of polymeric materials in various applications. Here, we describe the development of novel aminoglycoside-derived amphiphilic nanoparticles for drug delivery, with an eye towards ablation of cancer cells. The aminoglycoside paromomycin was first cross-linked with resorcinol diglycidyl ether leading to the formation of a poly (amino ether), PAE. PAE molecules were further derivatized with methoxy-terminated poly(ethylene glycol) or mPEG resulting in the formation of mPEG-PAE polymer, which self-assembled to form nanoparticles. Formation of the mPEG-PAE amphiphile was characterized using (1)H NMR, (13)C NMR, gel permeation chromatography (GPC) and FTIR spectroscopy. Self-assembly of the polymer into nanoparticles was characterized using dynamic light scattering, zeta potential analyses, atomic force microscopy (AFM) and the pyrene fluorescence assay. mPEG-PAE nanoparticles were able to carry significant amounts of doxorubicin (DOX), presumably by means of hydrophobic interactions between the drug and the core. Cell-based studies indicated that mPEG-PAE nanoparticles, loaded with doxorubicin, were able to induce significant loss in viabilities of PC3 human prostate cancer, MDA-MB-231 human breast cancer, and MB49 murine bladder cancer cells; empty nanoparticles resulted in negligible losses of cell viability under the conditions investigated. Taken together, our results indicate that the mPEG-PAE nanoparticle platform is attractive for drug delivery in different applications, including cancer. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Purification, crystallization and preliminary X-ray analysis of Enterococcus faecium aminoglycoside-2′′-phosphotransferase-Ib [APH(2′′)-Ib

    International Nuclear Information System (INIS)

    Walanj, Rupa; Young, Paul; Baker, Heather M.; Baker, Edward N.; Metcalf, Peter; Chow, Joseph W.; Lerner, Stephen; Vakulenko, Sergei; Smith, Clyde A.

    2005-01-01

    APH(2′′)-Ib is an enzyme responsible for high-level gentamicin resistance in E. faecium isolates. Native crystals of this enzyme have been prepared and preliminary X-ray diffraction experiments have been undertaken. Bacterial resistance to the aminoglycoside antibiotics is primarily the result of deactivation of the drugs. Three families of enzymes are responsible for this activity, with one such family being the aminoglycoside phosphotransferases (APHs). The gene encoding one of these enzymes, APH(2′′)-Ib, has been cloned and the protein (comprising 299 amino-acid residues) expressed in Escherichia coli, purified and crystallized in the presence of 16%(w/v) PEG 3350 and gentamicin. The crystals belong to the monoclinic space group P2 1 , with approximate unit-cell parameters a = 79.7, b = 58.8, c = 81.4 Å, β = 98.4°, and preliminary X-ray diffraction analysis is consistent with the presence of two molecules in the asymmetric unit. Synchrotron diffraction data to approximately 2.65 Å resolution were collected from a native APH(2′′)-Ib crystal at beamline BL9-2 at SSRL (Stanford, CA, USA). Selenium-substituted crystals have also been produced and structure determination is proceeding

  4. Loss of Slc4a1b chloride/bicarbonate exchanger function protects mechanosensory hair cells from aminoglycoside damage in the zebrafish mutant persephone.

    Directory of Open Access Journals (Sweden)

    Dale W Hailey

    Full Text Available Mechanosensory hair cell death is a leading cause of hearing and balance disorders in the human population. Hair cells are remarkably sensitive to environmental insults such as excessive noise and exposure to some otherwise therapeutic drugs. However, individual responses to damaging agents can vary, in part due to genetic differences. We previously carried out a forward genetic screen using the zebrafish lateral line system to identify mutations that alter the response of larval hair cells to the antibiotic neomycin, one of a class of aminoglycoside compounds that cause hair cell death in humans. The persephone mutation confers resistance to aminoglycosides. 5 dpf homozygous persephone mutants are indistinguishable from wild-type siblings, but differ in their retention of lateral line hair cells upon exposure to neomycin. The mutation in persephone maps to the chloride/bicarbonate exchanger slc4a1b and introduces a single Ser-to-Phe substitution in zSlc4a1b. This mutation prevents delivery of the exchanger to the cell surface and abolishes the ability of the protein to import chloride across the plasma membrane. Loss of function of zSlc4a1b reduces hair cell death caused by exposure to the aminoglycosides neomycin, kanamycin, and gentamicin, and the chemotherapeutic drug cisplatin. Pharmacological block of anion transport with the disulfonic stilbene derivatives DIDS and SITS, or exposure to exogenous bicarbonate, also protects hair cells against damage. Both persephone mutant and DIDS-treated wild-type larvae show reduced uptake of labeled aminoglycosides. persephone mutants also show reduced FM1-43 uptake, indicating a potential impact on mechanotransduction-coupled activity in the mutant. We propose that tight regulation of the ionic environment of sensory hair cells, mediated by zSlc4a1b activity, is critical for their sensitivity to aminoglycoside antibiotics.

  5. Combined Effect of the Cfr Methyltransferase and Ribosomal Protein L3 Mutations on Resistance to Ribosome-Targeting Antibiotics.

    Science.gov (United States)

    Pakula, Kevin K; Hansen, Lykke H; Vester, Birte

    2017-09-01

    Several groups of antibiotics inhibit bacterial growth by binding to bacterial ribosomes. Mutations in ribosomal protein L3 have been associated with resistance to linezolid and tiamulin, which both bind at the peptidyl transferase center in the ribosome. Resistance to these and other antibiotics also occurs through methylation of 23S rRNA at position A2503 by the methyltransferase Cfr. The mutations in L3 and the cfr gene have been found together in clinical isolates, raising the question of whether they have a combined effect on antibiotic resistance or growth. We transformed a plasmid-borne cfr gene into a uL3-depleted Escherichia coli strain containing either wild-type L3 or L3 with one of seven mutations, G147R, Q148F, N149S, N149D, N149R, Q150L, or T151P, expressed from plasmid-carried rplC genes. The L3 mutations are well tolerated, with small to moderate growth rate decreases. The presence of Cfr has a very minor influence on the growth rate. The resistance of the transformants to linezolid, tiamulin, florfenicol, and Synercid (a combination of quinupristin and dalfopristin [Q-D]) was measured by MIC assays. The resistance from Cfr was, in all cases, stronger than the effects of the L3 mutations, but various effects were obtained with the combinations of Cfr and L3 mutations ranging from a synergistic to an antagonistic effect. Linezolid and tiamulin susceptibility varied greatly among the L3 mutations, while no significant effects on florfenicol and Q-D susceptibility were seen. This study underscores the complex interplay between various resistance mechanisms and cross-resistance, even from antibiotics with overlapping binding sites. Copyright © 2017 American Society for Microbiology.

  6. Aminoglycoside Concentrations Required for Synergy with Carbapenems against Pseudomonas aeruginosa Determined via Mechanistic Studies and Modeling.

    Science.gov (United States)

    Yadav, Rajbharan; Bulitta, Jürgen B; Schneider, Elena K; Shin, Beom Soo; Velkov, Tony; Nation, Roger L; Landersdorfer, Cornelia B

    2017-12-01

    This study aimed to systematically identify the aminoglycoside concentrations required for synergy with a carbapenem and characterize the permeabilizing effect of aminoglycosides on the outer membrane of Pseudomonas aeruginosa Monotherapies and combinations of four aminoglycosides and three carbapenems were studied for activity against P. aeruginosa strain AH298-GFP in 48-h static-concentration time-kill studies (SCTK) (inoculum: 10 7.6 CFU/ml). The outer membrane-permeabilizing effect of tobramycin alone and in combination with imipenem was characterized via electron microscopy, confocal imaging, and the nitrocefin assay. A mechanism-based model (MBM) was developed to simultaneously describe the time course of bacterial killing and prevention of regrowth by imipenem combined with each of the four aminoglycosides. Notably, 0.25 mg/liter of tobramycin, which was inactive in monotherapy, achieved synergy (i.e., ≥2-log 10 more killing than the most active monotherapy at 24 h) combined with imipenem. Electron micrographs, confocal image analyses, and the nitrocefin uptake data showed distinct outer membrane damage by tobramycin, which was more extensive for the combination with imipenem. The MBM indicated that aminoglycosides enhanced the imipenem target site concentration up to 4.27-fold. Tobramycin was the most potent aminoglycoside to permeabilize the outer membrane; tobramycin (0.216 mg/liter), gentamicin (0.739 mg/liter), amikacin (1.70 mg/liter), or streptomycin (5.19 mg/liter) was required for half-maximal permeabilization. In summary, our SCTK, mechanistic studies and MBM indicated that tobramycin was highly synergistic and displayed the maximum outer membrane disruption potential among the tested aminoglycosides. These findings support the optimization of highly promising antibiotic combination dosage regimens for critically ill patients. Copyright © 2017 American Society for Microbiology.

  7. Synthesis of Netilmicin and Apramycin Derivatives for the Treatment of Multidrug-Resistant Infectious Diseases

    Science.gov (United States)

    Sonousi, Amr

    The ever-growing bacterial resistance to existing antibiotics is alarming to humanity. Many researchers decided to revisit aminoglycosides with renewed emphasis on chemical modification as they have long been used as highly potent antibiotics for treating severe bacterial infections. The bactericidal effect of aminoglycosides is mainly due to protein synthesis inhibition by binding to the A-site of the bacterial ribosomes. However, the high potency and the broad spectrum of aminoglycosides has been outweighed by their side effects, especially ototoxicity, and by the resistance of pathogens. The goal of this research was the modification of existing aminoglycosides to develop derivatives which are less toxic and that evade resistance. The chapters in the thesis discuss the chemical synthesis as well as the biological evaluation of the newly synthesized analogs. This study has focused on the modification of aminoglycosides netilmicin and apramycin. Chapter one introduces the MDR bacterial infection problem and its influence. Chapter one also introduces the aminoglycosides elaborating their history, classifications, and their mechanism of action. The resistance mechanisms against aminoglycosides and their adverse effects, as well as the ways to prevent them are briefly explained. Chapter two discusses modifications of netilmicin at the 4'-position conducted with a view to reducing the ototoxicity but not the antibiotic activity, as was previously done in the 4,5-series with paromomycin. The antibacterial activity and antiribosomal activity of the six netilmicin derivatives synthesized were determined. The 4'-position is more sensitive to modification in 4,6-series than in the 4,5-series to the extent that such modifications are ineffective. Chapter two also highlights the use of phenyl triazenes as selective protecting groups for secondary amines in the presence of primary amines. Several polyamine substrates were selectively protected as phenyl triazenes, and primary

  8. Simple measurement of isepamicin, a new aminoglycoside antibiotic, in guinea pig and human plasma, using high-performance liquid chromatography with ultraviolet detection

    International Nuclear Information System (INIS)

    Dionisotti, S.; Bamonte, F.; Scaglione, F.; Ongini, E.

    1991-01-01

    Isepamicin, the 1-N-(S-alpha-hydroxy-beta-aminopropionyl) derivative of gentamicin B, is a new aminoglycoside antibiotic, which not only has most of the properties of amikacin but also is effective against several amikacin-resistant strains of bacteria. The drug was assayed in guinea-pig and human plasma with a high-performance liquid chromatographic procedure using precolumn derivatization with 1-fluoro-2,4-dinitrobenzene and ultraviolet detection. Linearity was established over the range 0.5-40 micrograms/ml using 50 microliters of plasma. Accuracy has a mean relative error of less than 3% and precision a mean coefficient of variation of 5%. Isepamicin was determined without interference from plasma constituents or other drugs commonly prescribed during aminoglycoside therapy. This procedure correlates well with radioimmunoassay and can be used either in experimental studies or therapeutic monitoring of plasma levels

  9. Methyltransferase Erm(37) Slips on rRNA to Confer Atypical Resistance in Mycobacterium tuberculosis

    Czech Academy of Sciences Publication Activity Database

    Madsen, Ch. T.; Jakobsen, L.; Buriánková, Karolína; Doucet-Populaire, F.; Perdonet, J. L.; Douthwaite, S.

    2005-01-01

    Roč. 280, č. 47 (2005), s. 38942-38947 ISSN 0021-9258 R&D Projects: GA ČR GA310/03/0292 Institutional research plan: CEZ:AV0Z50200510 Keywords : methyltransferase erm * mycobacterium tuberculosis * rRNA Subject RIV: EE - Microbiology, Virology Impact factor: 5.854, year: 2005

  10. Overexpression of a soybean salicylic acid methyltransferase gene confers resistance to soybean cyst nematode

    Science.gov (United States)

    Salicylic acid plays a critical role in activating plant defence responses after pathogen attack. Salicylic acid methyltransferase (SAMT) modulates the level of salicylic acid by converting salicylic acid to methyl salicylate. Here, we report that a SAMT gene from soybean (GmSAMT1) plays a role in s...

  11. Activity of some aminoglycoside antibiotics against true fungi, Phytophthora and Pythium species.

    Science.gov (United States)

    Lee, H B; Kim, Y; Kim, J C; Choi, G J; Park, S-H; Kim, C-J; Jung, H S

    2005-01-01

    To investigate the in vitro antifungal and antioomycete activities of some aminoglycosides against true fungi and Phytophthora and Pythium species and to evaluate the potential of the antibiotics against Phytophthora late blight on plants. Antifungal and antioomycete activities of aminoglycoside antibiotics (neomycin, paromomycin, ribostamycin and streptomycin) and a paromomycin-producing strain (Streptomyces sp. AMG-P1) against Phytophthora and Pythium species and 10 common fungi were measured in potato dextrose broth (PDB) and on seedlings in pots. Paromomycin was the most active against Phytophthora and Pythium species with a minimal inhibitory concentration of 1-10 microg ml(-1) in PDB, but displayed low to moderate activities towards other common fungi at the same concentration. Paromomycin also showed potent in vivo activity against red pepper and tomato late blight diseases with 80 and 99% control value, respectively, at 100 microg ml(-1). In addition, culture broth of Streptomyces sp. AMG-P1 as a paromomycin producer exhibited high in vivo activity against late blight at 500 microg freeze-dried weight per millilitre. Among tested aminoglycoside antibiotics, paromomycin was the most active against oomycetes both in vitro and in vivo. Data from this study show that aminoglycoside antibiotics have in vitro and in vivo activities against oomycetes, suggesting that Streptomyces sp. AMG-P1 may be used as a biocontrol agent against oomycete diseases.

  12. Heparin interferes with the radioenzymatic and homogeneous enzyme immunoassays for aminoglycosides

    International Nuclear Information System (INIS)

    Krogstad, D.J.; Granich, G.G.; Murray, P.R.; Pfaller, M.A.; Valdes, R.

    1981-01-01

    Heparin interferes with measurement of aminoglycosides in serum by biological, radioenzymatic, and homogeneous enzyme immunoassay techniques, but not with radioimmunoassay. At concentrations greater than or equal to 10 5 and greater than or equal to 3 X 10 6 USP units/L, respectively, it interferes with the radioenzymatic assay by inhibiting the gentamicin 3-acetyltransferase and kanamycin 6'-acetyltransferase enzymes used in the assay. It interferes with the homogeneous enzyme immunoassays for gentamicin and tobramycin (at concentrations greater than or equal to 10 5 and greater than or equal to10 4 USP units/L, respectively), but not with the commercially available homogeneous enzyme immunoassays for other drugs. Heparin interference with the homogeneous enzyme immunoassay for aminoglycosides requires both the heparin polyanion and glucose-6-phosphate dehydrogenase bound to a cationic aminoglycoside. This interference can be reproduced with dextran sulfate (but not dextran), and does not occur with free enzyme (glucose-6-phosphate dehydrogenase) alone. Heparin interference with these two assays and at concentrations that may be present in intravenous infusions or in seriously underfilled blood-collection tubes is described

  13. Dissemination of antibiotic resistance in methicillin-resistant Staphylococcus aureus and vancomycin-resistant S aureus strains isolated from hospital effluents.

    Science.gov (United States)

    Mandal, Santi M; Ghosh, Ananta K; Pati, Bikas R

    2015-12-01

    Vancomycin-resistant Staphylococcus aureus (VRSA) and methicillin-resistant S aureus (MRSA) strains were examined in hospital effluents. Most S aureus strains are resistant to methicillin (MRSA), followed by tetracycline. Approximately 15% of MRSA strains are also resistant to vancomycin (VRSA). All VRSA strains developed a VanR/VanS-regulated 2-component system of VanA-type resistance in their genome. Results indicate that there is a possibility of developing resistance to aminoglycosides by VRSA strains in the near future. Copyright © 2015 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

  14. A Novel 6'-N-Aminoglycoside Acetyltransferase, AAC(6')-Ial, from a Clinical Isolate of Serratia marcescens.

    Science.gov (United States)

    Tada, Tatsuya; Miyoshi-Akiyama, Tohru; Shimada, Kayo; Dahal, Rajan K; Mishra, Shyam K; Ohara, Hiroshi; Kirikae, Teruo; Pokhrel, Bharat M

    2016-03-01

    Serratia marcescens IOMTU115 has a novel 6'-N-aminoglycoside acetyltransferase-encoding gene, aac(6')-Ial. The encoded protein AAC(6')-Ial has 146 amino acids, with 91.8% identity to the amino acid sequence of AAC(6')-Ic in S. marcescens SM16 and 97.3% identity to the amino acid sequence of AAC(6')-Iap in S. marcescens WW4. The minimum inhibitory concentrations of aminoglycosides for Escherichia coli expressing AAC(6')-Ial were similar to those for E. coli expressing AAC(6')-Ic or AAC(6')-Iap. Thin-layer chromatography showed that AAC(6')-Ial, AAC(6')-Ic, or AAC(6')-Iap acetylated all the aminoglycosides tested, except for apramycin, gentamicin, and lividomycin. Kinetics assays revealed that AAC(6')-Ial is a functional acetyltransferase against aminoglycosides. The aac(6')-Ial gene was located on chromosomal DNA.

  15. Mosaic Structure of a Multiple-Drug-Resistant, Conjugative Plasmid from Campylobacter jejuni

    National Research Council Canada - National Science Library

    Nirdnoy, Warawadee; Mason, Carl J; Guerry, Patricia

    2005-01-01

    ..., where it apparently integrated into the chromosome and expressed high-level resistance to multiple aminoglycoside antibiotics. This work provides new information about both the nature of drug resistance in C...

  16. Purification, crystallization and preliminary X-ray analysis of Enterococcus faecium aminoglycoside-2′′-phosphotransferase-Ib [APH(2′′)-Ib

    Energy Technology Data Exchange (ETDEWEB)

    Walanj, Rupa; Young, Paul; Baker, Heather M.; Baker, Edward N.; Metcalf, Peter [Laboratory of Structural Biology, School of Biological Sciences, University of Auckland, Auckland (New Zealand); Chow, Joseph W.; Lerner, Stephen [Division of Infectious Diseases, Wayne State University School of Medicine and VA Medical Center, Detroit, Michigan 48201 (United States); Vakulenko, Sergei [Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556 (United States); Smith, Clyde A., E-mail: csmith@slac.stanford.edu [Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CA 94025 (United States); Laboratory of Structural Biology, School of Biological Sciences, University of Auckland, Auckland (New Zealand)

    2005-04-01

    APH(2′′)-Ib is an enzyme responsible for high-level gentamicin resistance in E. faecium isolates. Native crystals of this enzyme have been prepared and preliminary X-ray diffraction experiments have been undertaken. Bacterial resistance to the aminoglycoside antibiotics is primarily the result of deactivation of the drugs. Three families of enzymes are responsible for this activity, with one such family being the aminoglycoside phosphotransferases (APHs). The gene encoding one of these enzymes, APH(2′′)-Ib, has been cloned and the protein (comprising 299 amino-acid residues) expressed in Escherichia coli, purified and crystallized in the presence of 16%(w/v) PEG 3350 and gentamicin. The crystals belong to the monoclinic space group P2{sub 1}, with approximate unit-cell parameters a = 79.7, b = 58.8, c = 81.4 Å, β = 98.4°, and preliminary X-ray diffraction analysis is consistent with the presence of two molecules in the asymmetric unit. Synchrotron diffraction data to approximately 2.65 Å resolution were collected from a native APH(2′′)-Ib crystal at beamline BL9-2 at SSRL (Stanford, CA, USA). Selenium-substituted crystals have also been produced and structure determination is proceeding.

  17. Coexistence of blaOXA-23 with armA in quinolone-resistant Acinetobacter baumannii from a Chinese university hospital.

    Science.gov (United States)

    Shen, Min; Luan, Guangxin; Wang, Yanhong; Chang, Yaowen; Zhang, Chi; Yang, Jingni; Deng, Shanshan; Ling, Baodong; Jia, Xu

    2016-03-01

    A total of 101 Acinetobacter baumannii isolates were collected to determine the mechanisms of quinolone resistance and investigate the occurrence of carbapenem and high-level aminoglycoside resistance genes among quinolone-resistant strains. Among 77 quinolone-resistant A. baumannii harbored mutations of gyrA and parC, 41 isolates, which belonged to European clone II, had resistance to aminoglycosides and carbapenems due to the expression of armA and acquisition of blaOXA-23. Most of sequence type belonged to clonal complex 92. These results suggested hospital dissemination of multidrug-resistant A. baumannii carrying blaOXA-23, armA, and mutations of quinolone resistance-determining regions in western China. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Methyltransferase That Modifies Guanine 966 of the 16 S rRNA: FUNCTIONAL IDENTIFICATION AND TERTIARY STRUCTURE*

    Science.gov (United States)

    Lesnyak, Dmitry V.; Osipiuk, Jerzy; Skarina, Tatiana; Sergiev, Petr V.; Bogdanov, Alexey A.; Edwards, Aled; Savchenko, Alexei; Joachimiak, Andrzej; Dontsova, Olga A.

    2010-01-01

    N2-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m2G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m2G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m2G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05 Å. The structure closely resembles RsmC rRNA methyltransferase, specific for m2G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m2G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed. PMID:17189261

  19. Methyltransferase that modifies guanine 966 of the 16 S rRNA: functional identification and tertiary structure.

    Science.gov (United States)

    Lesnyak, Dmitry V; Osipiuk, Jerzy; Skarina, Tatiana; Sergiev, Petr V; Bogdanov, Alexey A; Edwards, Aled; Savchenko, Alexei; Joachimiak, Andrzej; Dontsova, Olga A

    2007-02-23

    N(2)-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m(2)G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m(2)G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m(2)G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05A(.) The structure closely resembles RsmC rRNA methyltransferase, specific for m(2)G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m(2)G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed.

  20. Roles of DNA methyltransferases in Arabidopsis development ...

    African Journals Online (AJOL)

    Mutations that cause severe loss of DNA methylation often leads to abnormal development. In the present review, we summarized recent findings of the three major DNA methyltransferases mutants playing vital role in development of Arabidopsis thaliana. Keywords: DNA methylation, epigenetics, methyltransferase, mutant ...

  1. Natural bizbenzoquinoline derivatives protect zebrafish lateral line sensory hair cells from aminoglycoside toxicity

    Directory of Open Access Journals (Sweden)

    Matthew eKruger

    2016-03-01

    Full Text Available Moderate to severe hearing loss affects 360 million people worldwide and most often results from damage to sensory hair cells. Hair cell damage can result from aging, genetic mutations, excess noise exposure, and certain medications including aminoglycoside antibiotics. Aminoglycosides are effective at treating infections associated with cystic fibrosis and other life-threatening conditions such as sepsis, but cause hearing loss in 20-30% of patients. It is therefore imperative to develop new therapies to combat hearing loss and allow safe use of these potent antibiotics. We approach this drug discovery question using the larval zebrafish lateral line because zebrafish hair cells are structurally and functionally similar to mammalian inner ear hair cells and respond similarly to toxins. We screened a library of 502 natural compounds in order to identify novel hair cell protectants. Our screen identified four bisbenzylisoquinoline derivatives: berbamine, E6 berbamine, hernandezine, and isotetrandrine, each of which robustly protected hair cells from aminoglycoside-induced damage. Using fluorescence microscopy and electrophysiology, we demonstrated that the natural compounds confer protection by reducing antibiotic uptake into hair cells and showed that hair cells remain functional during and after incubation in E6 berbamine. We also determined that these natural compounds do not reduce antibiotic efficacy. Together, these natural compounds represent a novel source of possible otoprotective drugs that may offer therapeutic options for patients receiving aminoglycoside treatment.

  2. Structural Studies of Bacterial Enzymes and their Relation to Antibiotic Resistance Mechanisms - Final Paper

    Energy Technology Data Exchange (ETDEWEB)

    Maltz, Lauren [SLAC National Accelerator Lab., Menlo Park, CA (United States)

    2015-08-27

    By using protein crystallography and X-ray diffraction, structures of bacterial enzymes were solved to gain a better understanding of how enzymatic modification acts as an antibacterial resistance mechanism. Aminoglycoside phosphotransferases (APHs) are one of three aminoglycoside modifying enzymes that confer resistance to the aminoglycoside antibiotics via enzymatic modification, rendering many drugs obsolete. Specifically, the APH(2”) family vary in their substrate specificities and also in their preference for the phosphate donor (ADP versus GDP). By solving the structures of members of the APH(2”) family of enzymes, we can see how domain movements are important to their substrate specificity. Our structure of the ternary complex of APH(2”)-IIIa with GDP and kanamycin, when compared to the known structures of APH(2”)-IVa, reveals that there are real physical differences between these two enzymes, a structural finding that explains why the two enzymes differ in their preferences for certain aminoglycosides. Another important group of bacterial resistance enzymes are the Class D β- lactamases. Oxacillinase carbapenemases (OXAs) are part of this enzyme class and have begun to confer resistance to ‘last resort’ drugs, most notably carbapenems. Our structure of OXA-143 shows that the conformational flexibility of a conserved hydrophobic residue in the active site (Val130) serves to control the entry of a transient water molecule responsible for a key step in the enzyme’s mechanism. Our results provide insight into the structural mechanisms of these two different enzymes

  3. Production of Se-methylselenocysteine in transgenic plants expressing selenocysteine methyltransferase

    Directory of Open Access Journals (Sweden)

    Harris Hugh

    2004-01-01

    Full Text Available Abstract Background It has become increasingly evident that dietary Se plays a significant role in reducing the incidence of lung, colorectal and prostate cancer in humans. Different forms of Se vary in their chemopreventative efficacy, with Se-methylselenocysteine being one of the most potent. Interestingly, the Se accumulating plant Astragalus bisulcatus (Two-grooved poison vetch contains up to 0.6% of its shoot dry weight as Se-methylselenocysteine. The ability of this Se accumulator to biosynthesize Se-methylselenocysteine provides a critical metabolic shunt that prevents selenocysteine and selenomethionine from entering the protein biosynthetic machinery. Such a metabolic shunt has been proposed to be vital for Se tolerance in A. bisulcatus. Utilization of this mechanism in other plants may provide a possible avenue for the genetic engineering of Se tolerance in plants ideally suited for the phytoremediation of Se contaminated land. Here, we describe the overexpression of a selenocysteine methyltransferase from A. bisulcatus to engineer Se-methylselenocysteine metabolism in the Se non-accumulator Arabidopsis thaliana (Thale cress. Results By over producing the A. bisulcatus enzyme selenocysteine methyltransferase in A. thaliana, we have introduced a novel biosynthetic ability that allows the non-accumulator to accumulate Se-methylselenocysteine and γ-glutamylmethylselenocysteine in shoots. The biosynthesis of Se-methylselenocysteine in A. thaliana also confers significantly increased selenite tolerance and foliar Se accumulation. Conclusion These results demonstrate the feasibility of developing transgenic plant-based production of Se-methylselenocysteine, as well as bioengineering selenite resistance in plants. Selenite resistance is the first step in engineering plants that are resistant to selenate, the predominant form of Se in the environment.

  4. Low Prevalence of Carbapenem-Resistant Bacteria in River Water: Resistance Is Mostly Related to Intrinsic Mechanisms.

    Science.gov (United States)

    Tacão, Marta; Correia, António; Henriques, Isabel S

    2015-10-01

    Carbapenems are last-resort antibiotics to handle serious infections caused by multiresistant bacteria. The incidence of resistance to these antibiotics has been increasing and new resistance mechanisms have emerged. The dissemination of carbapenem resistance in the environment has been overlooked. The main goal of this research was to assess the prevalence and diversity of carbapenem-resistant bacteria in riverine ecosystems. The presence of frequently reported carbapenemase-encoding genes was inspected. The proportion of imipenem-resistant bacteria was on average 2.24 CFU/ml. Imipenem-resistant strains (n=110) were identified as Pseudomonas spp., Stenotrophomonas maltophilia, Aeromonas spp., Chromobacterium haemolyticum, Shewanella xiamenensis, and members of Enterobacteriaceae. Carbapenem-resistant bacteria were highly resistant to other beta-lactams such as quinolones, aminoglycosides, chloramphenicol, tetracyclines, and sulfamethoxazole/trimethoprim. Carbapenem resistance was mostly associated with intrinsically resistant bacteria. As intrinsic resistance mechanisms, we have identified the blaCphA gene in 77.3% of Aeromonas spp., blaL1 in all S. maltophilia, and blaOXA-48-like in all S. xiamenensis. As acquired resistance mechanisms, we have detected the blaVIM-2 gene in six Pseudomonas spp. (5.45%). Integrons with gene cassettes encoding resistance to aminoglycosides (aacA and aacC genes), trimethoprim (dfrB1b), and carbapenems (blaVIM-2) were found in Pseudomonas spp. Results suggest that carbapenem resistance dissemination in riverine ecosystems is still at an early stage. Nevertheless, monitoring these aquatic compartments for the presence of resistance genes and its host organisms is essential to outline strategies to minimize resistance dissemination.

  5. Beneficial read-through of a USH1C nonsense mutation by designed aminoglycoside NB30 in the retina.

    Science.gov (United States)

    Goldmann, Tobias; Rebibo-Sabbah, Annie; Overlack, Nora; Nudelman, Igor; Belakhov, Valery; Baasov, Timor; Ben-Yosef, Tamar; Wolfrum, Uwe; Nagel-Wolfrum, Kerstin

    2010-12-01

    The human Usher syndrome (USH) is the most frequent cause of inherited combined deaf-blindness. USH is clinically and genetically heterogeneous, assigned to three clinical types. The most severe type is USH1, characterized by profound inner ear defects and retinitis pigmentosa. Thus far, no effective treatment for the ophthalmic component of USH exists. The p.R31X nonsense mutation in USH1C leads to a disease causing premature termination of gene translation. Here, we investigated the capability of the novel synthetic aminoglycoside NB30 for the translational read-through of the USH1C-p.R31X nonsense mutation as a retinal therapy option. Read-through of p.R31X by three commercial, clinically applied aminoglycosides and the synthetic derivative NB30 was validated in vitro, in cell culture, and in retinal explants. Restoration of harmonin functions was monitored in GST pull-downs (scaffold function) and by F-actin bundling analysis in HEK293T cells. Biocompatibility of aminoglycosides was determined in retinal explants by TUNEL assays. In vitro translation and analyses of transfected HEK293T cells revealed a dose-dependent read-through by all aminoglycosides. In addition, gentamicin, paromomycin, and NB30 induced read-through of p.R31X in mouse retinal explants. The read-through of p.R31X restored harmonin protein function. In contrast to all commercial aminoglycosides NB30 showed good biocompatibility. Commercial aminoglycosides and NB30 induced significant read-through of the USH1C-p.R31X nonsense mutation. However, the observed read-through efficiency, along with its significantly reduced toxicity and good biocompatibility, indicate that the novel derivate NB30 represents a better choice than commercial aminoglycosides in a read-through therapy of USH1C and other ocular diseases.

  6. The risks of concurrent treatment with tenofovir and aminoglycosides ...

    African Journals Online (AJOL)

    The risks of concurrent treatment with tenofovir and aminoglycosides in patients with HIV-associated tuberculosis. C Kenyon, N Wearne, R Burton, G Meintjes. Abstract. The South African public sector antiretroviral treatment (ART) guidelines have recently been changed to include tenofovir in the first-line regimen.1 ...

  7. Diversity of enterococcal species and characterization of high-level aminoglycoside resistant enterococci of samples of wastewater and surface water in Tunisia.

    Science.gov (United States)

    Ben Said, Leila; Klibi, Naouel; Lozano, Carmen; Dziri, Raoudha; Ben Slama, Karim; Boudabous, Abdellatif; Torres, Carmen

    2015-10-15

    One hundred-fourteen samples of wastewater (n=64) and surface-water (n=50) were inoculated in Slanetz-Bartley agar plates supplemented or not with gentamicin (SB-Gen and SB plates, respectively) for enterococci recovery. Enterococci were obtained from 75% of tested samples in SB media (72% in wastewater; 78% in surface-water), and 85 enterococcal isolates (one/positive-sample) were obtained. Enterococcus faecium was the most prevalent species (63.5%), followed by Enterococcus faecalis (20%), Enterococcus hirae (9.4%), Enterococcus casseliflavus (4.7%), and Enterococcus gallinarum/Enterococcus durans (2.4%). Antibiotic resistance detected among these enterococci was as follows [percentage/detected gene (number isolates)]: kanamycin [29%/aph(3')-IIIa (n=22)], streptomycin [8%/ant(6)-Ia (n=4)], erythromycin [44%/erm(B) (n=34)], tetracycline [18%/tet(M) (n=6)/tet(M)-tet(L) (n=9)], chloramphenicol [2%/cat(A) (n=1)], ciprofloxacin [7%] and trimethoprim-sulfamethoxazole [94%]. High-level-gentamicin resistant (HLR-G) enterococci were recovered from 15 samples in SB-Gen or SB plates [12/64 samples of wastewater (19%) and 3/50 samples of surface-water (6%)]; HLR-G isolates were identified as E. faecium (n=7), E. faecalis (n=6), and E. casseliflavus (n=2). These HLR-G enterococci carried the aac(6')-Ie-aph(2")-Ia and erm(B) genes, in addition to aph(3')-IIIa (n=10), ant(6)-Ia (n=9), tet(M) (n=13), tet(L) (n=8) and cat(A) genes (n=2). Three HLR-G enterococci carried the esp virulence gene. Sequence-types detected among HLR-G enterococci were as follows: E. faecalis (ST480, ST314, ST202, ST55, and the new ones ST531 and ST532) and E. faecium (ST327, ST12, ST296, and the new ones ST985 and ST986). Thirty-two different PFGE patterns were detected among 36 high-level-aminoglycoside-resistant enterococci recovered in water samples. Diverse genetic lineages of HLR-G enterococci were detected in wastewater and surface-water in Tunisia. Water can represent an important source for the

  8. Small Molecule Inhibitors That Selectively Block Dengue Virus Methyltransferase*

    Science.gov (United States)

    Lim, Siew Pheng; Sonntag, Louis Sebastian; Noble, Christian; Nilar, Shahul H.; Ng, Ru Hui; Zou, Gang; Monaghan, Paul; Chung, Ka Yan; Dong, Hongping; Liu, Boping; Bodenreider, Christophe; Lee, Gladys; Ding, Mei; Chan, Wai Ling; Wang, Gang; Jian, Yap Li; Chao, Alexander Theodore; Lescar, Julien; Yin, Zheng; Vedananda, T. R.; Keller, Thomas H.; Shi, Pei-Yong

    2011-01-01

    Crystal structure analysis of Flavivirus methyltransferases uncovered a flavivirus-conserved cavity located next to the binding site for its cofactor, S-adenosyl-methionine (SAM). Chemical derivatization of S-adenosyl-homocysteine (SAH), the product inhibitor of the methylation reaction, with substituents that extend into the identified cavity, generated inhibitors that showed improved and selective activity against dengue virus methyltransferase (MTase), but not related human enzymes. Crystal structure of dengue virus MTase with a bound SAH derivative revealed that its N6-substituent bound in this cavity and induced conformation changes in residues lining the pocket. These findings demonstrate that one of the major hurdles for the development of methyltransferase-based therapeutics, namely selectivity for disease-related methyltransferases, can be overcome. PMID:21147775

  9. Antimicrobial sensitivity and frequency of DRUG resistance among bacterial strains isolated from cancer patients

    International Nuclear Information System (INIS)

    Faiz, M.; Bashir, T.

    2004-01-01

    Blood stream infections (bacteremia) is potentially life threatening. Concomitant with a change in the incidence and epidemiology of infecting organisms, there has been an increase in resistance to many antibiotic compounds. The widespread emergence of resistance among bacterial pathogens has an impact on our ability to treat patients effectively. The changing spectrum of microbial pathogens and widespread emergence of microbial resistance to antibiotic drugs has emphasized the need to monitor the prevalence of resistance in these strains. In the present study frequency of isolation of clinically significant bacteria and their susceptibility and resistance pattern against a wide range of antimicrobial drugs from positive blood cultures collected during 2001-2003 was studied. A total of 102 consecutive isolates were found with 63% gram positive and 44% gram negative strains. The dominating pathogens were Staphylococcus aureus (51%), Streptococci (31%), Pseudomonas (40%), Proteus (13%), Klebsiella (13%). The isolated strains were tested against a wide range of antibiotics belonging to cephalosporins, aminoglycosides and quinolone derivative group by disk diffusion method. It has been observed that isolated strains among gram positive and negative strains showed different level of resistance against aminoglycosides and cephalosporin group of antibiotics with gram positives showing highest number and frequency of resistance against aminoglycosides (40-50%) and cephalosporins.(35-45%) whereas cephalosporins were found to be more effective against gram negatives with low frequency of resistant strains. Cabapenem and quinolone derivative drugs were found to be most effective among other groups in both gram positive and negative strains with 23-41% strains found sensitive to these two drugs. The frequency of sensitive strains against aminoglycoside and cephalosporin in gram negative and gram positive strains were found to be decreasing yearwise with a trend towards an

  10. Andrographolide: A potent antituberculosis compound that targets Aminoglycoside 2'-N-acetyltransferase in Mycobacterium tuberculosis.

    Science.gov (United States)

    Prabu, Amudha; Hassan, Sameer; Prabuseenivasan; Shainaba, A S; Hanna, L E; Kumar, Vanaja

    2015-09-01

    Tuberculosis (TB) still remains a major challenging infectious disease. The increased rate of emergence of multi-drug resistant and extensively-drug resistant strains of the organism has further complicated the situation, resulting in an urgent need for new anti-TB drugs. Antimycobacterial activity of Andrographis paniculata was evaluated using a rapid LRP assay and the probable targets were identified by docking analysis. The methanolic extract of A. paniculata showed maximum antimycobacterial activity at 250μg/ml against all the tested strains of M. tuberculosis (H37Rv, MDR, and drug sensitive). Based on bioassay guided fractionation, andrographolide was identified as the potent molecule. With the docking analysis, both ICDH (Isocitrate Dehydrogenase) and AAC (Aminoglycoside 2'-N-acetyltransferase) were predicted as targets of andrographolide in M. tuberculosis. Molecular simulation revealed that, ICDH showed low binding affinity to andrographolide. However, for AAC, the andrographolide was observed to be well within the active site after 10ns of molecular simulation. This suggests that ACC (PDB ID 1M4I) could be the probable target for andrographolide. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Potentiation of aminoglycoside antibiotic activity using the body fat from the snake Boa constrictor

    Directory of Open Access Journals (Sweden)

    Felipe S. Ferreira

    2011-05-01

    Full Text Available Boa constrictor is widely used in traditional communities in many different folk remedies and products derived from it are sold in public markets throughout northeastern Brazil and as its body fat has many different therapeutic indications as a folk remedy. The present work evaluates the antibacterial activity of the body fat from the snake Boa constrictor when employed either alone or in combination with antibiotics and discusses the ecological implications of the use of this traditional remedy. Oil (OBC was extracted from body fat located in the ventral region of B. constrictor using hexane as a solvent. The antibacterial activity of OBC was tested against standard as well as multi-resistant lines, either alone and in combination with antibiotics. OBC did not demonstrate any relevant antibacterial activity against standard or multidrug-resistant bacterial strains. OBC showed synergistic activity when combined with the aminoglycoside antibiotics. Our results indicate that the body fat of Boa constrictor does not possess bactericidal activity, from the clinical point of view, but when combined with an antibiotic, the fat demonstrated a significant synergistic activity.

  12. Potentiation of aminoglycoside antibiotic activity using the body fat from the snake Boa constrictor

    Directory of Open Access Journals (Sweden)

    Felipe S. Ferreira

    2011-06-01

    Full Text Available Boa constrictor is widely used in traditional communities in many different folk remedies and products derived from it are sold in public markets throughout northeastern Brazil and as its body fat has many different therapeutic indications as a folk remedy. The present work evaluates the antibacterial activity of the body fat from the snake Boa constrictor when employed either alone or in combination with antibiotics and discusses the ecological implications of the use of this traditional remedy. Oil (OBC was extracted from body fat located in the ventral region of B. constrictor using hexane as a solvent. The antibacterial activity of OBC was tested against standard as well as multi-resistant lines, either alone and in combination with antibiotics. OBC did not demonstrate any relevant antibacterial activity against standard or multidrug-resistant bacterial strains. OBC showed synergistic activity when combined with the aminoglycoside antibiotics. Our results indicate that the body fat of Boa constrictor does not possess bactericidal activity, from the clinical point of view, but when combined with an antibiotic, the fat demonstrated a significant synergistic activity.

  13. Determination of aminoglycoside antibiotics using complex compounds of chromotropic acid bisazoderivatives with rare earth ions

    International Nuclear Information System (INIS)

    Alykov, N.M.

    1981-01-01

    Studies of complex formation of bisazo derivatives of chromotropic acid with rare earth ions and aminoglycoside antibiotics have made it possible to choose carboxyarsenazo, orthanyl R and carboxynitrazo as highly sensitive reagents for determining aminoglycoside antibiotics. Conditions have been found for the formation of precipitates of different-ligand complexes containing rare earth ions, bisazo derivatives of chromotropic acid and aminogylcoside antibiotics. A procedure has been worked out of determining the antibiotics in biological samples with carboxyarsenazo [ru

  14. Burkholderia pseudomallei isolates from Sarawak, Malaysian Borneo, are predominantly susceptible to aminoglycosides and macrolides.

    Science.gov (United States)

    Podin, Yuwana; Sarovich, Derek S; Price, Erin P; Kaestli, Mirjam; Mayo, Mark; Hii, KingChing; Ngian, Hieung; Wong, SeeChang; Wong, IngTien; Wong, JinShyan; Mohan, Anand; Ooi, MongHow; Fam, TemLom; Wong, Jack; Tuanyok, Apichai; Keim, Paul; Giffard, Philip M; Currie, Bart J

    2014-01-01

    Melioidosis is a potentially fatal disease caused by the saprophytic bacterium Burkholderia pseudomallei. Resistance to gentamicin is generally a hallmark of B. pseudomallei, and gentamicin is a selective agent in media used for diagnosis of melioidosis. In this study, we determined the prevalence and mechanism of gentamicin susceptibility found in B. pseudomallei isolates from Sarawak, Malaysian Borneo. We performed multilocus sequence typing and antibiotic susceptibility testing on 44 B. pseudomallei clinical isolates from melioidosis patients in Sarawak district hospitals. Whole-genome sequencing was used to identify the mechanism of gentamicin susceptibility. A novel allelic-specific PCR was designed to differentiate gentamicin-sensitive isolates from wild-type B. pseudomallei. A reversion assay was performed to confirm the involvement of this mechanism in gentamicin susceptibility. A substantial proportion (86%) of B. pseudomallei clinical isolates in Sarawak, Malaysian Borneo, were found to be susceptible to the aminoglycoside gentamicin, a rare occurrence in other regions where B. pseudomallei is endemic. Gentamicin sensitivity was restricted to genetically related strains belonging to sequence type 881 or its single-locus variant, sequence type 997. Whole-genome sequencing identified a novel nonsynonymous mutation within amrB, encoding an essential component of the AmrAB-OprA multidrug efflux pump. We confirmed the role of this mutation in conferring aminoglycoside and macrolide sensitivity by reversion of this mutation to the wild-type sequence. Our study demonstrates that alternative B. pseudomallei selective media without gentamicin are needed for accurate melioidosis laboratory diagnosis in Sarawak. This finding may also have implications for environmental sampling of other locations to test for B. pseudomallei endemicity.

  15. Stimulation of diesel degradation and biosurfactant production by aminoglycosides in a novel oil-degrading bacterium Pseudomonas luteola PRO23

    Directory of Open Access Journals (Sweden)

    Atanasković Iva M.

    2016-01-01

    Full Text Available Bioremediation is promising technology for dealing with oil hydrocarbons contamination. In this research growth kinetics and oil biodegradation efficiency of Pseudomonas luteola PRO23, isolated from crude oil-contaminated soil samples, were investigated under different concentrations (5, 10 and 20 g/L of light and heavy crude oil. More efficient biodegradation and more rapid adaptation and cell growth were obtained in conditions with light oil. The 5 to 10 g/L upgrade of light oil concentration stimulated the microbial growth and the biodegradation efficiency. Further upgrade of light oil concentration and the upgrade of heavy oil concentration both inhibited the microbial growth, as well as biodegradation process. Aminoglycosides stimulated biosurfactant production in P. luteola in the range of sub-inhibitory concentrations (0.3125, 0.625 μg/mL. Aminoglycosides also induced biofilm formation. The production of biosurfactants was the most intense during lag phase and continues until stationary phase. Aminoglycosides also induced changes in P. luteola growth kinetics. In the presence of aminoglycosides this strain degraded 82% of diesel for 96 h. These results indicated that Pseudomonas luteola PRO23 potentially can be used in bioremediation of crude oil-contaminated environments and that aminoglycosides could stimulate this process. [Projekat Ministarstva nauke Republike Srbije, br. TR31080

  16. A low-barrier hydrogen bond mediates antibiotic resistance in a noncanonical catalytic triad

    Science.gov (United States)

    2018-01-01

    One group of enzymes that confer resistance to aminoglycoside antibiotics through covalent modification belongs to the GCN5-related N-acetyltransferase (GNAT) superfamily. We show how a unique GNAT subfamily member uses a previously unidentified noncanonical catalytic triad, consisting of a glutamic acid, a histidine, and the antibiotic substrate itself, which acts as a nucleophile and attacks the acetyl donor molecule. Neutron diffraction studies allow for unambiguous identification of a low-barrier hydrogen bond, predicted in canonical catalytic triads to increase basicity of the histidine. This work highlights the role of this unique catalytic triad in mediating antibiotic resistance while providing new insights into the design of the next generation of aminoglycosides. PMID:29632894

  17. Structural Chemistry of Human RNA Methyltransferases.

    Science.gov (United States)

    Schapira, Matthieu

    2016-03-18

    RNA methyltransferases (RNMTs) play important roles in RNA stability, splicing, and epigenetic mechanisms. They constitute a promising target class that is underexplored by the medicinal chemistry community. Information of relevance to drug design can be extracted from the rich structural coverage of human RNMTs. In this work, the structural chemistry of this protein family is analyzed in depth. Unlike most methyltransferases, RNMTs generally feature a substrate-binding site that is largely open on the cofactor-binding pocket, favoring the design of bisubstrate inhibitors. Substrate purine or pyrimidines are often sandwiched between hydrophobic walls that can accommodate planar ring systems. When the substrate base is laying on a shallow surface, a 5' flanking base is sometimes anchored in a druggable cavity. The cofactor-binding site is structurally more diverse than in protein methyltransferases and more druggable in SPOUT than in Rossman-fold enzymes. Finally, conformational plasticity observed both at the substrate and cofactor binding sites may be a challenge for structure-based drug design. The landscape drawn here may inform ongoing efforts toward the discovery of the first human RNMT inhibitors.

  18. Heavy metals in liquid pig manure in light of bacterial antimicrobial resistance

    Energy Technology Data Exchange (ETDEWEB)

    Hoelzel, Christina S., E-mail: Christina.Hoelzel@wzw.tum.de [Chair of Animal Hygiene, Technische Universitaet Muenchen, Weihenstephaner Berg 3, 85354 Freising (Germany); Mueller, Christa [Institute for Agroecology, Organic Farming and Soil Protection, Bavarian State Research Center for Agriculture (LfL), Lange Point 12, 85354 Freising (Germany); Harms, Katrin S. [Chair of Animal Hygiene, Technische Universitaet Muenchen, Weihenstephaner Berg 3, 85354 Freising (Germany); Mikolajewski, Sabine [Department for Quality Assurance and Analytics, Bavarian State Research Center for Agriculture (LfL), Lange Point 4, 85354 Freising (Germany); Schaefer, Stefanie; Schwaiger, Karin; Bauer, Johann [Chair of Animal Hygiene, Technische Universitaet Muenchen, Weihenstephaner Berg 3, 85354 Freising (Germany)

    2012-02-15

    Heavy metals are regularly found in liquid pig manure, and might interact with bacterial antimicrobial resistance. Concentrations of heavy metals were determined by atomic spectroscopic methods in 305 pig manure samples and were connected to the phenotypic resistance of Escherichia coli (n=613) against 29 antimicrobial drugs. Concentrations of heavy metals (/kg dry matter) were 0.08-5.30 mg cadmium, 1.1-32.0 mg chrome, 22.4-3387.6 mg copper, <2.0-26.7 mg lead, <0.01-0.11 mg mercury, 3.1-97.3 mg nickel and 93.0-8239.0 mg zinc. Associated with the detection of copper and zinc, resistance rates against {beta}-lactams were significantly elevated. By contrast, the presence of mercury was significantly associated with low antimicrobial resistance rates of Escherichia coli against {beta}-lactams, aminoglycosides and other antibiotics. Effects of subinhibitory concentrations of mercury on bacterial resistance against penicillins, cephalosporins, aminoglycosides and doxycycline were also demonstrated in a laboratory trial. Antimicrobial resistance in the porcine microflora might be increased by copper and zinc. By contrast, the occurrence of mercury in the environment might, due to co-toxicity, act counter-selective against antimicrobial resistant strains.

  19. Chemical Probes of Histone Lysine Methyltransferases

    Science.gov (United States)

    2015-01-01

    Growing evidence suggests that histone methyltransferases (HMTs, also known as protein methyltransferases (PMTs)) play an important role in diverse biological processes and human diseases by regulating gene expression and the chromatin state. Therefore, HMTs have been increasingly recognized by the biomedical community as a class of potential therapeutic targets. High quality chemical probes of HMTs, as tools for deciphering their physiological functions and roles in human diseases and testing therapeutic hypotheses, are critical for advancing this promising field. In this review, we focus on the discovery, characterization, and biological applications of chemical probes for HMTs. PMID:25423077

  20. Determination of aminoglycoside antibiotics using an on-chip microfluidic device with chemiluminescence detection

    International Nuclear Information System (INIS)

    Sierra-Rodero, M.; Fernandez-Romero, J.M.; Gomez-Hens, A.

    2012-01-01

    We describe an on-chip microflow injection (μFI) approach for the determination of aminoglycoside antibiotics using chemiluminescence (CL) detection. The method is based on the inhibition of the Cu(II)-catalyzed CL reaction of luminol and hydrogen peroxide by the aminoglycosides due to the formation of a complex between the antibiotic and Cu(II). The main features of the method include small sample volumes and a fast response. Syringe pumps were used to insert the sample and the reagents into the microfluidic device. CL was collected using a fiber optic bundle connected to a luminescence detector. All instrumental, hydrodynamic and chemical variables involved in the system were optimized using neomycin as the aminoglycoside model. Inhibition is proportional to the concentration of the antibiotics. The dynamic ranges of the calibration graphs obtained for neomycin, streptomycin and amikacin are 0.3-3.3, 0.9-13.7, and 0.8-8.5 μmol L -1 , and the detection limits are 0.09, 0.28 and 0.24 μmol L -1 , respectively. The precision of the methods, expressed as relative standard deviation, is in the range from 0.8 to 5.0 %. The method was successfully applied to the determination of neomycin in water samples, with recoveries ranging from 80 to 120 %. (author)

  1. Readthrough of stop codons by use of aminoglycosides in cells from xeroderma pigmentosum group C patients.

    Science.gov (United States)

    Kuschal, Christiane; Khan, Sikandar G; Enk, Benedikt; DiGiovanna, John J; Kraemer, Kenneth H

    2015-04-01

    Readthrough of premature termination (stop) codons (PTC) is a new approach to treatment of genetic diseases. We recently reported that readthrough of PTC in cells from some xeroderma pigmentosum complementation group C (XP-C) patients could be achieved with the aminoglycosides geneticin or gentamicin. We found that the response depended on several factors including the PTC sequence, its location within the gene and the aminoglycoside used. Here, we extended these studies to investigate the effects of other aminoglycosides that are already on the market. We reasoned that topical treatment could deliver much higher concentrations of drug to the skin, the therapeutic target, and thus increase the therapeutic effect while reducing renal or ototoxicity in comparison with systemic treatment. Our prior clinical studies indicated that only a few percent of normal XPC expression was associated with mild clinical disease. We found minimal cell toxicity in the XP-C cells with several aminoglycosides. We found increased XPC mRNA expression in PTC-containing XP-C cells with G418, paromomycin, neomycin and kanamycin and increased XPC protein expression with G418. We conclude that in selected patients with XP, topical PTC therapy can be investigated as a method of personalized medicine to alleviate their cutaneous symptoms. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

  2. Methyl transfer in glucosinolate biosynthesis mediated by indole glucosinolate O-Methyltransferase 5

    DEFF Research Database (Denmark)

    Pfalz, Marina; Mukhaimar, Maisara; Perreau, François

    2016-01-01

    in position 1 (1-IG modification) or 4 (4-IG modification). Products of the 4-IG modification pathway mediate plant-enemy interactions and are particularly important for Arabidopsis innate immunity. While CYP81Fs encoding cytochrome P450 monooxygenases and IGMTs encoding indole glucosinolate O...... with moderate similarity to previously characterized IGMTs, encodes the methyltransferase that is responsible for the conversion of 1OHI3M to 1MOI3M. Disruption of IGMT5 function increases resistance against the root-knot nematode Meloidogyne javanica and suggests a potential role for the 1-IG modification...

  3. Coenzyme Q10 protects hair cells against aminoglycoside.

    Directory of Open Access Journals (Sweden)

    Kazuma Sugahara

    Full Text Available It is well known that the production of free radicals is associated with sensory cell death induced by an aminoglycoside. Many researchers have reported that antioxidant reagents protect sensory cells in the inner ear, and coenzyme Q10 (CoQ10 is an antioxidant that is consumed as a health food in many countries. The purpose of this study was to investigate the role of CoQ10 in mammalian vestibular hair cell death induced by aminoglycoside. Cultured utricles of CBA/CaN mice were divided into three groups (control group, neomycin group, and neomycin + CoQ10 group. In the neomycin group, utricles were cultured with neomycin (1 mM to induce hair cell death. In the neomycin + CoQ10 group, utricles were cultured with neomycin and water-soluble CoQ10 (30-0.3 µM. Twenty-four hours after exposure to neomycin, the cultured tissues were fixed, and vestibular hair cells were labeled using an anti-calmodulin antibody. Significantly more hair cells survived in the neomycin + CoQ10 group than in the neomycin group. These data indicate that CoQ10 protects sensory hair cells against neomycin-induced death in the mammalian vestibular epithelium; therefore, CoQ10 may be useful as a protective drug in the inner ear.

  4. Resistance to Linezolid

    DEFF Research Database (Denmark)

    Vester, Birte; Ntokou, Eleni

    2017-01-01

    Linezolid is an antimicrobial agent that binds to the bacterial ribosome and thereby inhibits protein synthesis. Soon after its release as a clinical drug, it became clear that bacteria could become resistant to linezolid. The resistance mechanisms are mainly causing alteration of the drug target...... site, but probably efflux might also play a role. The resistance is still rare in surveillance studies, but outbreaks of resistant clones from hospitals have been observed. So far the main mechanisms of resistance are occurrence of mutations in ribosomal genes or obtaining plasmids with a gene coding...... for a methyltransferase providing resistance. The most obvious way to avoid resistance may be development of derivatives of linezolid overcoming the known resistance mechanisms....

  5. Is the addition of aminoglycosides to beta-lactams in cancer patients with febrile neutropenia needed?

    Science.gov (United States)

    Contreras, Valeria; Sepúlveda, Sebastián; Heredia, Ana

    2016-02-24

    It is still controversial if the combined use of beta-lactam antibiotics and aminoglycosides has advantages over broad-spectrum beta-lactam monotherapy for the empirical treatment of cancer patients with febrile neutropenia. Searching in Epistemonikos database, which is maintained by screening 30 databases, we identified three systematic reviews including 14 pertinent randomized trials. We combined the evidence using meta-analysis and generated a summary of findings table following the GRADE approach. We concluded the combination of beta-lactam antibiotics and aminoglycosides probably does not lead to a reduced mortality in febrile neutropenic cancer patients and it might increase nephrotoxicity.

  6. An integrated epigenetic and genetic analysis of DNA methyltransferase genes (DNMTs) in tumor resistant and susceptible chicken lines

    Science.gov (United States)

    Both epigenetic alterations and genetic variations play essential roles in tumorigenesis. The epigenetic modification of DNA methylation is catalyzed and maintained by the DNA methyltransferases (DNMT3a, DNMT3b and DNMT1). DNA mutations and DNA methylation profiles of DNMTs themselves and their rela...

  7. Antibiotic resistant Escherichia coli in southeastern Australian pig herds and implications for surveillance.

    Science.gov (United States)

    van Breda, L K; Dhungyel, O P; Ward, M P

    2018-02-01

    To investigate public health implications of antibiotics to control post-weaning scours, we surveyed 22 commercial pig herds in southeastern Australia. Fifty faecal samples per herd were collected from pre- and post-weaned piglets. Presumptive Escherichia coli isolates were confirmed by MALDI-TOF MS. Isolates (n = 325) were screened for susceptibility to 19 veterinary antibiotics using MIC broth microdilution. All 325 E. coli isolates underwent further testing against 27 antibiotics used in human medicine and were screened for ETEC adhesin and enterotoxin genes (F4 (K88), F5 (K99), F6 (987P), F18, F41, STa, STb, Stx2e and LT) by multiplex PCR. Isolates identified as phenotypically resistant to third-generation cephalosporin (3GC) and aminoglycoside antibiotics were screened by multiplex PCR/reverse line blot to detect common β-lactam and aminoglycosides resistance genes, confirmed by sequencing. Twenty (6.1%) of the E. coli isolates were resistant to 3GC antibiotics and 24 (7.4%) to the aminoglycoside antibiotic gentamicin. Genetic analysis revealed six different extended spectrum β-lactamase (ESBL) genes (blaCTX-M-1, -14, -15, -27, blaSHV-12 and blaCMY-2-like genes), four of which have not been previously reported in Australian pigs. Critically, the prevalence of 3GC resistance was higher in non-pathogenic (non-ETEC) isolates and those from clinically normal (non-diarrhoeal) samples. This highlights the importance of non-ETECE. coli as reservoirs of antimicrobial resistance genes in piglet pens. Antimicrobial resistance surveillance in pig production focused on diagnostic specimens from clinically-affected animals might be potentially misleading. We recommend that surveillance for emerging antimicrobial resistance such as to 3GC antibiotics should include clinically healthy pigs. © 2017 Blackwell Verlag GmbH.

  8. Unusual Complication of Multidrug Resistant Tuberculosis

    Directory of Open Access Journals (Sweden)

    Prerna Sharma

    2017-01-01

    Full Text Available Introduction. Capreomycin is a second-line drug often used for multidrug-resistant tuberculosis which can result in nephrotoxic effects similar to other aminoglycosides. We describe a case of capreomycin induced Bartter-like syndrome with hypocalcemic tetany. Case Report. 23-year-old female patient presented with carpopedal spasms and tingling sensations in hands. Patient was being treated with capreomycin for two months for tuberculosis. On further investigation, hypocalcemia, hyponatremia, hypomagnesemia, hypokalemia, and hypochloremic metabolic alkalosis were noted. Vitamin D and serum PTH levels were within normal limits. Hypercalciuria was confirmed by urine calcium/creatinine ratio. Calcium, potassium, and magnesium supplementation was given and capreomycin was discontinued. Electrolytes normalized in two days after cessation of capreomycin with no further abnormalities on repeat investigations. Discussion. Aminoglycosides can result in renal tubular dysfunction leading to Fanconi syndrome, Bartter syndrome, and distal tubular acidosis. Impaired mitochondrial function in the tubular cells has been hypothesized as the possible cause of these tubulopathies. Acquired Bartter-like syndrome phenotypically resembles autosomal dominant type 5 Bartter syndrome. Treatment consists of correction of electrolyte abnormalities, indomethacin, and potassium-sparing diuretics. Prompt diagnosis and treatment of severe dyselectrolytemia are warranted in patients on aminoglycoside therapy.

  9. Functional Characterization of Bacteria Isolated from Ancient Arctic Soil Exposes Diverse Resistance Mechanisms to Modern Antibiotics

    Science.gov (United States)

    Perron, Gabriel G.; Whyte, Lyle; Turnbaugh, Peter J.; Goordial, Jacqueline; Hanage, William P.; Dantas, Gautam; Desai, Michael M.

    2015-01-01

    Using functional metagenomics to study the resistomes of bacterial communities isolated from different layers of the Canadian high Arctic permafrost, we show that microbial communities harbored diverse resistance mechanisms at least 5,000 years ago. Among bacteria sampled from the ancient layers of a permafrost core, we isolated eight genes conferring clinical levels of resistance against aminoglycoside, β-lactam and tetracycline antibiotics that are naturally produced by microorganisms. Among these resistance genes, four also conferred resistance against amikacin, a modern semi-synthetic antibiotic that does not naturally occur in microorganisms. In bacteria sampled from the overlaying active layer, we isolated ten different genes conferring resistance to all six antibiotics tested in this study, including aminoglycoside, β-lactam and tetracycline variants that are naturally produced by microorganisms as well as semi-synthetic variants produced in the laboratory. On average, we found that resistance genes found in permafrost bacteria conferred lower levels of resistance against clinically relevant antibiotics than resistance genes sampled from the active layer. Our results demonstrate that antibiotic resistance genes were functionally diverse prior to the anthropogenic use of antibiotics, contributing to the evolution of natural reservoirs of resistance genes. PMID:25807523

  10. Acid detergent lignin, lodging resistance index, and expression of the caffeic acid O-methyltransferase gene in brown midrib-12 sudangrass.

    Science.gov (United States)

    Li, Yuan; Liu, Guibo; Li, Jun; You, Yongliang; Zhao, Haiming; Liang, Huan; Mao, Peisheng

    2015-09-01

    Understanding the relationship between acid detergent lignin (ADL) and lodging resistance index (LRI) is essential for breeding new varieties of brown midrib (bmr) sudangrass (Sorghum sudanense (Piper) Stapf.). In this study, bmr-12 near isogenic lines and their wild-types obtained by back cross breeding were used to compare relevant forage yield and quality traits, and to analyze expression of the caffeic acid O-methyltransferase (COMT) gene using quantitative real time-PCR. The research showed that the mean ADL content of bmr-12 mutants (20.94 g kg(-1)) was significantly (P bmr-12 mutants (0.29) was significantly (P bmr-12 materials (r = -0.44, P > 0.05). Sequence comparison of the COMT gene revealed two point mutations present in bmr-12 but not in the wild-type, the second mutation changed amino acid 129 of the protein from Gln (CAG) to a stop codon (UAG). The relative expression level of COMT gene was significantly reduced, which likely led to the decreased ADL content observed in the bmr-12 mutant.

  11. Clinical evaluation and mitochondrial DNA sequence analysis in two Chinese families with aminoglycoside-induced and non-syndromic hearing loss

    International Nuclear Information System (INIS)

    Zhao Lidong; Wang Qiuju; Qian Yaping; Li Ronghua; Cao Juayng; Hart, Laura Christine; Zhai Suoqiang; Han Dongyi; Young Wieyen; Guan Minxin

    2005-01-01

    We report here the clinical, genetic, and molecular characterization of two Chinese pedigrees with aminoglycoside-induced and non-syndromic hearing impairment. Clinical evaluation revealed the variable phenotype of hearing impairment including audiometric configuration in these subjects. Penetrances of hearing loss in BJ105 and BJ106 pedigrees are 67% and 33%, respectively. In particular, three of 10 affected matrilineal relatives of BJ105 pedigree had aminoglycoside-induced hearing loss, while seven affected matrilineal relatives in BJ105 pedigree and six affected matrilineal relatives in BJ106 pedigree did not have a history of exposure to aminoglycosides. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the identical homoplasmic A1555G mutation and distinct sets of mtDNA variants belonging to haplogroups F3 and M7b. These variants showed no evolutionary conservation, implying that mitochondrial haplotype may not play a significant role in the phenotypic expression of the A1555G mutation in these Chinese pedigrees. However, aminoglycosides and nuclear backgrounds appear to be major modifier factors for the phenotypic manifestation of the A1555G mutation in these Chinese families

  12. Heavy metals in liquid pig manure in light of bacterial antimicrobial resistance

    International Nuclear Information System (INIS)

    Hölzel, Christina S.; Müller, Christa; Harms, Katrin S.; Mikolajewski, Sabine; Schäfer, Stefanie; Schwaiger, Karin; Bauer, Johann

    2012-01-01

    Heavy metals are regularly found in liquid pig manure, and might interact with bacterial antimicrobial resistance. Concentrations of heavy metals were determined by atomic spectroscopic methods in 305 pig manure samples and were connected to the phenotypic resistance of Escherichia coli (n=613) against 29 antimicrobial drugs. Concentrations of heavy metals (/kg dry matter) were 0.08–5.30 mg cadmium, 1.1–32.0 mg chrome, 22.4–3387.6 mg copper, <2.0–26.7 mg lead, <0.01–0.11 mg mercury, 3.1–97.3 mg nickel and 93.0–8239.0 mg zinc. Associated with the detection of copper and zinc, resistance rates against β-lactams were significantly elevated. By contrast, the presence of mercury was significantly associated with low antimicrobial resistance rates of Escherichia coli against β-lactams, aminoglycosides and other antibiotics. Effects of subinhibitory concentrations of mercury on bacterial resistance against penicillins, cephalosporins, aminoglycosides and doxycycline were also demonstrated in a laboratory trial. Antimicrobial resistance in the porcine microflora might be increased by copper and zinc. By contrast, the occurrence of mercury in the environment might, due to co-toxicity, act counter-selective against antimicrobial resistant strains.

  13. Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria

    DEFF Research Database (Denmark)

    Batchelor, Miranda; Hopkins, Katie L; Liebana, Ernesto

    2008-01-01

    We describe the development of a miniaturised microarray for the detection of antimicrobial resistance genes in Gram-negative bacteria. Included on the array are genes encoding resistance to aminoglycosides, trimethoprim, sulphonamides, tetracyclines and beta-lactams, including extended-spectrum ...

  14. Potentiation of antibiotic activity of aminoglycosides by natural products from Cordia verbenacea DC.

    Science.gov (United States)

    Matias, Edinardo F F; Alves, Erivania F; Silva, Maria K N; Carvalho, Victoria R A; Medeiros, Cassio R; Santos, Francisco A V; Bitu, Vanessa C N; Souza, Celestina E S; Figueredo, Fernando G; Boligon, Aline A; Athayde, Margareth L; Costa, José G M; Coutinho, Henrique D M

    2016-06-01

    Medicinal plants are often the only therapeutic resource for many communities and ethnic groups. Cordia verbenacea DC., "Erva-baleeira," is one of the species of plants currently used to produce a phytotherapeutic product extracted from its leaves. The present study aimed to establish its chemical profile, antibacterial activity and resistance-modulating potential. The C. verbenacea extracts were prepared from fresh leaves using solvents as methanol and hexane. Ethyl Acetate was used for the preparation of the fraction. Phytochemical screening was carried out using HPLC-DAD for determination and quantification of the secondary metabolites present in the fractions. Antibacterial and resistance-modulation assays were performed to determine minimum inhibitory concentration (MIC) using a microdilution assay. The data were subjected to statistical analysis with two-way ANOVA and Bonferroni posttests. Results of phytochemical prospecting and HPLC analysis of the fractions were in agreement with the literature. The natural products presented moderate antibacterial activity when considering the clinical relevance of a MIC of 256 μg/mL against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, and 512 μg/mL against P. aeruginosa. However, when the fractions were combined with antibiotics we observed a synergic effect, as natural products enhanced the antibacterial effect of aminoglycosides, significantly decreasing the MIC of antibiotics at 12.5%-98.4%. We believe that the data obtained from phytochemical analysis and from antibacterial and resistance modulation assays of C. verbenacea extracts new can open perspectives in the search for new alternatives for the treatment of bacterial infections and stimulate the renewed use of antibiotics with reduced effectiveness due to resistance. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Whole genome sequencing-based characterization of extensively drug resistant (XDR) strains of Mycobacterium tuberculosis from Pakistan

    KAUST Repository

    Hasan, Zahra; Ali, Asho; McNerney, Ruth; Mallard, Kim; Hill-Cawthorne, Grant A.; Coll, Francesc; Nair, Mridul; Pain, Arnab; Clark, Taane G.; Hasan, Rumina

    2015-01-01

    Objectives: The global increase in drug resistance in Mycobacterium tuberculosis (MTB) strains increases the focus on improved molecular diagnostics for MTB. Extensively drug-resistant (XDR) - TB is caused by MTB strains resistant to rifampicin, isoniazid, fluoroquinolone and aminoglycoside antibiotics. Resistance to anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs), in particular MTB genes. However, there is regional variation between MTB lineages and the SNPs associated with resistance. Therefore, there is a need to identify common resistance conferring SNPs so that effective molecular-based diagnostic tests for MTB can be developed. This study investigated used whole genome sequencing (WGS) to characterize 37 XDR MTB isolates from Pakistan and investigated SNPs related to drug resistance. Methods: XDR-TB strains were selected. DNA was extracted from MTB strains, and samples underwent WGS with 76-base-paired end fragment sizes using Illumina paired end HiSeq2000 technology. Raw sequence data were mapped uniquely to H37Rv reference genome. The mappings allowed SNPs and small indels to be called using SAMtools/BCFtools. Results: This study found that in all XDR strains, rifampicin resistance was attributable to SNPs in the rpoB RDR region. Isoniazid resistance-associated mutations were primarily related to katG codon 315 followed by inhA S94A. Fluoroquinolone resistance was attributable to gyrA 91-94 codons in most strains, while one did not have SNPs in either gyrA or gyrB. Aminoglycoside resistance was mostly associated with SNPs in rrs, except in 6 strains. Ethambutol resistant strains had embB codon 306 mutations, but many strains did not have this present. The SNPs were compared with those present in commercial assays such as LiPA Hain MDRTBsl, and the sensitivity of the assays for these strains was evaluated. Conclusions: If common drug resistance associated with SNPs evaluated the concordance between phenotypic and

  16. Whole genome sequencing-based characterization of extensively drug resistant (XDR) strains of Mycobacterium tuberculosis from Pakistan

    KAUST Repository

    Hasan, Zahra

    2015-03-01

    Objectives: The global increase in drug resistance in Mycobacterium tuberculosis (MTB) strains increases the focus on improved molecular diagnostics for MTB. Extensively drug-resistant (XDR) - TB is caused by MTB strains resistant to rifampicin, isoniazid, fluoroquinolone and aminoglycoside antibiotics. Resistance to anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs), in particular MTB genes. However, there is regional variation between MTB lineages and the SNPs associated with resistance. Therefore, there is a need to identify common resistance conferring SNPs so that effective molecular-based diagnostic tests for MTB can be developed. This study investigated used whole genome sequencing (WGS) to characterize 37 XDR MTB isolates from Pakistan and investigated SNPs related to drug resistance. Methods: XDR-TB strains were selected. DNA was extracted from MTB strains, and samples underwent WGS with 76-base-paired end fragment sizes using Illumina paired end HiSeq2000 technology. Raw sequence data were mapped uniquely to H37Rv reference genome. The mappings allowed SNPs and small indels to be called using SAMtools/BCFtools. Results: This study found that in all XDR strains, rifampicin resistance was attributable to SNPs in the rpoB RDR region. Isoniazid resistance-associated mutations were primarily related to katG codon 315 followed by inhA S94A. Fluoroquinolone resistance was attributable to gyrA 91-94 codons in most strains, while one did not have SNPs in either gyrA or gyrB. Aminoglycoside resistance was mostly associated with SNPs in rrs, except in 6 strains. Ethambutol resistant strains had embB codon 306 mutations, but many strains did not have this present. The SNPs were compared with those present in commercial assays such as LiPA Hain MDRTBsl, and the sensitivity of the assays for these strains was evaluated. Conclusions: If common drug resistance associated with SNPs evaluated the concordance between phenotypic and

  17. Human C6orf211 Encodes Armt1, a Protein Carboxyl Methyltransferase that Targets PCNA and Is Linked to the DNA Damage Response

    Directory of Open Access Journals (Sweden)

    J. Jefferson P. Perry

    2015-03-01

    Full Text Available Recent evidence supports the presence of an L-glutamyl methyltransferase(s in eukaryotic cells, but this enzyme class has been defined only in certain prokaryotic species. Here, we characterize the human C6orf211 gene product as “acidic residue methyltransferase-1” (Armt1, an enzyme that specifically targets proliferating cell nuclear antigen (PCNA in breast cancer cells, predominately methylating glutamate side chains. Armt1 homologs share structural similarities with the SAM-dependent methyltransferases, and negative regulation of activity by automethylation indicates a means for cellular control. Notably, shRNA-based knockdown of Armt1 expression in two breast cancer cell lines altered survival in response to genotoxic stress. Increased sensitivity to UV, adriamycin, and MMS was observed in SK-Br-3 cells, while in contrast, increased resistance to these agents was observed in MCF7 cells. Together, these results lay the foundation for defining the mechanism by which this post-translational modification operates in the DNA damage response (DDR.

  18. Correlations between Income inequality and antimicrobial resistance.

    Science.gov (United States)

    Kirby, Andrew; Herbert, Annie

    2013-01-01

    The aim of this study is to investigate if correlations exist between income inequality and antimicrobial resistance. This study's hypothesis is that income inequality at the national level is positively correlated with antimicrobial resistance within developed countries. Income inequality data were obtained from the Standardized World Income Inequality Database. Antimicrobial resistance data were obtained from the European antimicrobial Resistance Surveillance Network and outpatient antimicrobial consumption data, measured by Defined daily Doses per 1000 inhabitants per day, from the European Surveillance of antimicrobial Consumption group. Spearman's correlation coefficient (r) defined strengths of correlations of: > 0.8 as strong, > 0.5 as moderate and > 0.2 as weak. Confidence intervals and p values were defined for all r values. Correlations were calculated for the time period 2003-10, for 15 European countries. Income inequality and antimicrobial resistance correlations which were moderate or strong, with 95% confidence intervals > 0, included the following. Enterococcus faecalis resistance to aminopenicillins, vancomycin and high level gentamicin was moderately associated with income inequality (r= ≥0.54 for all three antimicrobials). Escherichia coli resistance to aminoglycosides, aminopenicillins, third generation cephalosporins and fluoroquinolones was moderately-strongly associated with income inequality (r= ≥0.7 for all four antimicrobials). Klebsiella pneumoniae resistance to third generation cephalosporins, aminoglycosides and fluoroquinolones was moderately associated with income inequality (r= ≥0.5 for all three antimicrobials). Staphylococcus aureus methicillin resistance and income inequality were strongly associated (r=0.87). As income inequality increases in European countries so do the rates of antimicrobial resistance for bacteria including E. faecalis, E. coli, K. pneumoniae and S. aureus. Further studies are needed to confirm these

  19. Whole genome sequencing-based characterization of extensively drug resistant (XDR strains of Mycobacterium tuberculosis from Pakistan

    Directory of Open Access Journals (Sweden)

    Zahra Hasan

    2015-01-01

    Conclusions: If common drug resistance associated with SNPs evaluated the concordance between phenotypic and genotypic testing, the results would be rifampicin (100%, isoniazid (89%, fluoroquinolones (95%, aminoglycoside (81% and ethambutol (61%. This work highlights the importance of expanded targets for drug resistance detection in MTB isolates.

  20. Small Molecule Inhibitors That Selectively Block Dengue Virus Methyltransferase*

    OpenAIRE

    Lim, Siew Pheng; Sonntag, Louis Sebastian; Noble, Christian; Nilar, Shahul H.; Ng, Ru Hui; Zou, Gang; Monaghan, Paul; Chung, Ka Yan; Dong, Hongping; Liu, Boping; Bodenreider, Christophe; Lee, Gladys; Ding, Mei; Chan, Wai Ling; Wang, Gang

    2010-01-01

    Crystal structure analysis of Flavivirus methyltransferases uncovered a flavivirus-conserved cavity located next to the binding site for its cofactor, S-adenosyl-methionine (SAM). Chemical derivatization of S-adenosyl-homocysteine (SAH), the product inhibitor of the methylation reaction, with substituents that extend into the identified cavity, generated inhibitors that showed improved and selective activity against dengue virus methyltransferase (MTase), but not related human enzymes. Crysta...

  1. Risk Factors for Emergence of Resistance to Broad-Spectrum Cephalosporins among Enterobacter spp.

    Science.gov (United States)

    Kaye, Keith S.; Cosgrove, Sara; Harris, Anthony; Eliopoulos, George M.; Carmeli, Yehuda

    2001-01-01

    Among 477 patients with susceptible Enterobacter spp., 49 subsequently harbored third-generation cephalosporin-resistant Enterobacter spp. Broad-spectrum cephalosporins were independent risk factors for resistance (relative risk [OR] = 2.3, P = 0.01); quinolone therapy was protective (OR = 0.4, P = 0.03). There were trends toward decreased risk for resistance among patients receiving broad-spectrum cephalosporins and either aminoglycosides or imipenem. Of the patients receiving broad-spectrum cephalosporins, 19% developed resistance. PMID:11502540

  2. Mitochondrial 12S rRNA A827G mutation is involved in the genetic susceptibility to aminoglycoside ototoxicity

    International Nuclear Information System (INIS)

    Xing Guangqian; Chen Zhibin; Wei Qinjun; Tian Huiqin; Li Xiaolu; Zhou Aidong; Bu Xingkuan; Cao Xin

    2006-01-01

    We have analyzed the clinical and molecular characterization of a Chinese family with aminoglycoside-induced and non-syndromic hearing impairment. Clinical evaluations revealed that only those family members who had a history of exposure to aminoglycoside antibiotics subsequently developed hearing loss, suggesting mitochondrial genome involvement. Sequence analysis of the mitochondrial 12S rRNA and tRNA Ser(UCN) genes led to the identification of a homoplasmic A827G mutation in all maternal relatives, a mutation that was identified previously in a few sporadic patients and in another Chinese family with non-syndromic deafness. The pathogenicity of the A827G mutation is strongly supported by the occurrence of the same mutation in two independent families and several genetically unrelated subjects. The A827G mutation is located at the A-site of the mitochondrial 12S rRNA gene which is highly conserved in mammals. It is possible that the alteration of the tertiary or quaternary structure of this rRNA by the A827G mutation may lead to mitochondrial dysfunction, thereby playing a role in the pathogenesis of hearing loss and aminoglycoside hypersensitivity. However, incomplete penetrance of hearing impairment indicates that the A827G mutation itself is not sufficient to produce clinical phenotype but requires the involvement of modifier factors for the phenotypic expression. Indeed, aminoglycosides may contribute to the phenotypic manifestation of the A827G mutation in this family. In contrast with the congenital or early-onset hearing impairment in another Chinese family carrying the A827G mutation, three patients in this pedigree developed hearing loss only after use of aminoglycosides. This discrepancy likely reflects the difference of genetic backgrounds, either mitochondrial haplotypes or nuclear modifier genes, between two families

  3. The Sensitivity to Aminoglycosides and Heavy Metals of Isolates of ...

    African Journals Online (AJOL)

    Eighty-two clinical isolates of Pseudomonas aeruginosa strains were tested for their sensitivity to aminoglycosides by an agar diffusion method and to heavy metals by a dilution technique on tri –buffered mineral salt agar containing 10 – 100mg/L CdCl2.H20, CoCl2.6H20, ZnCl2, AgNO3 and HgCl2. All the strains tested ...

  4. Structural characterization of the mitomycin 7-O-methyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Shanteri; Chang, Aram; Goff, Randal D.; Bingman, Craig A.; Grüschow, Sabine; Sherman, David H.; Phillips, Jr., George N.; Thorson, Jon S. (Michigan); (UW)

    2014-10-02

    Mitomycins are quinone-containing antibiotics, widely used as antitumor drugs in chemotherapy. Mitomycin-7-O-methyltransferase (MmcR), a key tailoring enzyme involved in the biosynthesis of mitomycin in Streptomyces lavendulae, catalyzes the 7-O-methylation of both C9{beta}- and C9{alpha}-configured 7-hydroxymitomycins. We have determined the crystal structures of the MmcR-S-adenosylhomocysteine (SAH) binary complex and MmcR-SAH-mitomycin A (MMA) ternary complex at resolutions of 1.9 and 2.3 {angstrom}, respectively. The study revealed MmcR to adopt a common S-adenosyl-L-methionine-dependent O-methyltransferase fold and the presence of a structurally conserved active site general acid-base pair is consistent with a proton-assisted methyltransfer common to most methyltransferases. Given the importance of C7 alkylation to modulate mitomycin redox potential, this study may also present a template toward the future engineering of catalysts to generate uniquely bioactive mitomycins.

  5. Early transcriptional response to aminoglycoside antibiotic suggests alternate pathways leading to apoptosis of sensory hair cells in the mouse inner ear

    Directory of Open Access Journals (Sweden)

    Neil eSegil

    2015-05-01

    Full Text Available Aminoglycoside antibiotics are the drug of choice for treating many bacterial infections, but their administration results in hearing loss in nearly one fourth of the patients who receive them. Several biochemical pathways have been implicated in aminoglycoside antibiotic ototoxicity; however, little is known about how hair cells respond to aminoglycoside antibiotics at the transcriptome level. Here we have investigated the genome-wide response to the aminoglycoside antibiotic gentamicin. Using organotypic cultures of the perinatal organ of Corti, we performed RNA sequencing using cDNA libraries obtained from FACS-purified hair cells. Within 3 hours of gentamicin treatment, the messenger RNA level of more than three thousand genes in hair cells changed significantly. Bioinformatic analysis of these changes highlighted several known signal transduction pathways, including the JNK pathway and the NF-κB pathway, in addition to genes involved in the stress response, apoptosis, cell cycle control, and DNA damage repair. In contrast, only 698 genes, mainly involved in cell cycle and metabolite biosynthetic processes, were significantly affected in the non-hair cell population. The gene expression profiles of hair cells in response to gentamicin share a considerable similarity with those previously observed in gentamicin-induced nephrotoxicity. Our findings suggest that previously observed early responses to gentamicin in hair cells in specific signaling pathways are reflected in changes in gene expression. Additionally, the observed changes in gene expression of cell cycle regulatory genes indicate a disruption of the postmitotic state, which may suggest an alternative pathway regulating gentamicin-induced hair cell death. This work provides a more comprehensive view of aminoglycoside antibiotic ototoxicity, and thus contribute to identifying potential pathways or therapeutic targets to alleviate this important side effect of aminoglycoside

  6. Metal and antibiotic resistance of bacteria isolated from the Baltic Sea.

    Science.gov (United States)

    Moskot, Marta; Kotlarska, Ewa; Jakóbkiewicz-Banecka, Joanna; Gabig-Cimińska, Magdalena; Fari, Karolina; Wegrzyn, Grzegorz; Wróbel, Borys

    2012-09-01

    The resistance of 49 strains of bacteria isolated from surface Baltic Sea waters to 11 antibiotics was analyzed and the resistance of selected strains to three metal ions (Ni2+, Mn2+, Zn2+) was tested. Most isolates belonged to Gammaproteobacteria (78%), while Alphaproteobacteria (8%), Actinobacteria (10%), and Bacteroidetes (4%) were less abundant. Even though previous reports suggested relationships between resistance and the presence of plasmids or the ability to produce pigments, no compelling evidence for such relationships was obtained for the strains isolated in this work. In particular, strains resistant to multiple antibiotics did not carry plasmids more frequently than sensitive strains. A relation between resistance and the four aminoglycosides tested (gentamycin, kanamycin, neomycin, and streptomycin), but not to spectinomycin, was demonstrated. This observation is of interest given that spectinomycin is not always classified as an aminoglycoside because it lacks a traditional sugar moiety. Statistical analysis indicated relationships between resistance to some antibiotics (ampicillin and erythromycin, chloramphenicol and erythromycin, chloramphenicol and tetracycline, erythromycin and tetracycline), suggesting the linkage of resistance genes for antibiotics belonging to different classes. The effects of NiSO4, ZnCl2 and MnCl2 on various media suggested that the composition of Marine Broth might result in low concentrations of Mn2+ due to chemical interactions that potentially lead to precipitation.

  7. Single biosensor immunoassay for the detection of five aminoglycosides in reconstituted skimmed milk

    NARCIS (Netherlands)

    Haasnoot, W.; Cazemier, G.; Koets, M.; Amerongen, van A.

    2003-01-01

    The application of an optical biosensor (Biacore 3000), with four flow channels (Fcs), in combination with a mixture of four specific antibodies resulted in a competitive inhibition biosensor immunoassay (BIA) for the simultaneous detection of the five relevant aminoglycosides in reconstituted

  8. Effect of basic amino acids and aminoglycosides on 3H-gentamicin uptake in cortical slices of rat and human kindney

    International Nuclear Information System (INIS)

    Bennett, W.M.; Plamp, C.E.; Elliott, W.C.; Parker, R.A.; Porter, G.A.

    1982-01-01

    The uptake of 3 H-gentamicin was assessed in renal cortical slices of Fischer 344 male rats and four human cadaver kidneys not utilized for renal transplantation. In both species the uptake was maximal at 90 min and maintained a steady state therafter. The characteristics of the energy-dependent component of 3 H-gentamicin uptake were not altered by various basic amino acids, but competitive inhibition was induced by other aminoglycosides in a dose-dependent fashion. Thus aminoglycosides appear to share a transport process that is distinct from those of organic bases or other cationic substances. In addition, under the experimental conditions employed, the basolateral membranes of the tubular cell is capable of energy-dependent uptake of gentamicin. The role of this route of cellular uptake of aminoglycoside in clinical nephrotoxicity is speculative

  9. Regulation of the Two-Component Regulator CpxR on Aminoglycosides and β-lactams Resistance in Salmonella enterica Serovar Typhimurium

    Directory of Open Access Journals (Sweden)

    Hui eHuang

    2016-04-01

    RNA expression levels of the efflux pump acrD and mdtA genes, as compared to strain JS△cpxR. Our results indicate that the two-component regulator CpxR contributes to resistance of S. enterica serovar Typhimurium to aminoglycosides and β-lactams by influencing the expression level of the MDR-related genes.

  10. Role of Bacterioferritin & Ferritin in M. tuberculosis Pathogenesis and Drug Resistance: A Future Perspective by Interactomic Approach

    Directory of Open Access Journals (Sweden)

    Divakar Sharma

    2017-06-01

    Full Text Available Tuberculosis is caused by Mycobacterium tuberculosis, one of the most successful and deadliest human pathogen. Aminoglycosides resistance leads to emergence of extremely drug resistant strains of M. tuberculosis. Iron is crucial for the biological functions of the cells. Iron assimilation, storage and their utilization is not only involved in pathogenesis but also in emergence of drug resistance strains. We previously reported that iron storing proteins (bacterioferritin and ferritin were found to be overexpressed in aminoglycosides resistant isolates. In this study we performed the STRING analysis of bacterioferritin & ferritin proteins and predicted their interactive partners [ferrochelatase (hemH, Rv1877 (hypothetical protein/probable conserved integral membrane protein, uroporphyrinogen decarboxylase (hemE trigger factor (tig, transcriptional regulatory protein (MT3948, hypothetical protein (MT1928, glnA3 (glutamine synthetase, molecular chaperone GroEL (groEL1 & hsp65, and hypothetical protein (MT3947]. We suggested that interactive partners of bacterioferritin and ferritin are directly or indirectly involved in M. tuberculosis growth, homeostasis, iron assimilation, virulence, resistance, and stresses.

  11. Risk factors and outcomes of carbapenem-resistant Klebsiella pneumoniae infections

    Directory of Open Access Journals (Sweden)

    Eleonora Pistella

    2016-12-01

    Full Text Available In the nosocomial setting, antimicrobial-resistant Enterobacteriaceae are a growing challenge, and alarming trends in resistance are currently reported all over the world. Isolates of Enterobacteriaceae producing ampC β-lactamases and extended spectrum β-lactamases are endemic in many hospitals, and are frequently resistant also to other classes of antibiotics, such as fluoroquinolones and aminoglycosides. The risk of infections due to multi-drug resistant strains should be considered also for outpatients who have had recent contact with the health system. Both nosocomial and health-care associated infections should be treated with a combination of antibiotics active against multi-drug resistant Gram negative and methicillin-resistant Staphylococcus aureus. In the absence of effective antimicrobial stewardship programs, this aggressive therapeutic approach might lead to abuse of broad-spectrum antibiotics, with consequent increase in resistances. To contain the possible antibiotic overuse, several decisional strategies, often based on risk-score systems supporting the clinical decisions, have been proposed. In this context of high antibiotic selection pressure, carbapenem-resistant pathogens recently began to spread in many hospitals. Carbapenem-resistant Klebsiella pneumoniae, as well as carbapenem-resistant Acinetobacter baumannii and P. aeruginosa, represent the new major challenges to patient safety. Against these organisms the initial empiric treatment is generally ineffective. The poor clinical outcome associated with carbapenem- resistant K. pneumoniae infections is probably due to the delete in the beginning of an appropriate antibiotic treatment, rather than to the increased virulence of pathogens. Only few therapeutic options are available, including colistin, tigecycline, aminoglycosides and carbapenems in selected cases. Several combinations of these antibiotics have been used, but no ideal regimen has been currently established.

  12. Drosophila arginine methyltransferase 1 (DART1) is an ecdysone receptor co-repressor

    International Nuclear Information System (INIS)

    Kimura, Shuhei; Sawatsubashi, Shun; Ito, Saya; Kouzmenko, Alexander; Suzuki, Eriko; Zhao, Yue; Yamagata, Kaoru; Tanabe, Masahiko; Ueda, Takashi; Fujiyama, Sari; Murata, Takuya; Matsukawa, Hiroyuki; Takeyama, Ken-ichi; Yaegashi, Nobuo

    2008-01-01

    Histone arginine methylation is an epigenetic marker that regulates gene expression by defining the chromatin state. Arginine methyltransferases, therefore, serve as transcriptional co-regulators. However, unlike other transcriptional co-regulators, the physiological roles of arginine methyltransferases are poorly understood. Drosophila arginine methyltransferase 1 (DART1), the mammalian PRMT1 homologue, methylates the arginine residue of histone H4 (H4R3me2). Disruption of DART1 in Drosophila by imprecise P-element excision resulted in low viability during metamorphosis in the pupal stages. In the pupal stage, an ecdysone hormone signal is critical for developmental progression. DART1 interacted with the nuclear ecdysone receptor (EcR) in a ligand-dependent manner, and co-repressed EcR in intact flies. These findings suggest that DART1, a histone arginine methyltransferase, is a co-repressor of EcR that is indispensable for normal pupal development in the intact fly

  13. Wild-type MIC distributions for aminoglycoside and cyclic polypeptide antibiotics used for treatment of Mycobacterium tuberculosis infections.

    Science.gov (United States)

    Juréen, P; Angeby, K; Sturegård, E; Chryssanthou, E; Giske, C G; Werngren, J; Nordvall, M; Johansson, A; Kahlmeter, G; Hoffner, S; Schön, T

    2010-05-01

    The aminoglycosides and cyclic polypeptides are essential drugs in the treatment of multidrug-resistant tuberculosis, underscoring the need for accurate and reproducible drug susceptibility testing (DST). The epidemiological cutoff value (ECOFF) separating wild-type susceptible strains from non-wild-type strains is an important but rarely used tool for indicating susceptibility breakpoints against Mycobacterium tuberculosis. In this study, we established wild-type MIC distributions on Middlebrook 7H10 medium for amikacin, kanamycin, streptomycin, capreomycin, and viomycin using 90 consecutive clinical isolates and 21 resistant strains. Overall, the MIC variation between and within runs did not exceed +/-1 MIC dilution step, and validation of MIC values in Bactec 960 MGIT demonstrated good agreement. Tentative ECOFFs defining the wild type were established for all investigated drugs, including amikacin and viomycin, which currently lack susceptibility breakpoints for 7H10. Five out of seven amikacin- and kanamycin-resistant isolates were classified as susceptible to capreomycin according to the current critical concentration (10 mg/liter) but were non-wild type according to the ECOFF (4 mg/liter), suggesting that the critical concentration may be too high. All amikacin- and kanamycin-resistant isolates were clearly below the ECOFF for viomycin, and two of them were below the ECOFF for streptomycin, indicating that these two drugs may be considered for treatment of amikacin-resistant strains. Pharmacodynamic indices (peak serum concentration [Cmax]/MIC) were more favorable for amikacin and viomycin compared to kanamycin and capreomycin. In conclusion, our data emphasize the importance of establishing wild-type MIC distributions for improving the quality of drug susceptibility testing against Mycobacterium tuberculosis.

  14. Chaperonins fight aminoglycoside-induced protein misfolding and promote short-term tolerance in Escherichia coli

    DEFF Research Database (Denmark)

    Goltermann, Lise; Good, Liam; Bentin, Thomas

    2013-01-01

    For almost half of a century, we have known that aminoglycoside antibiotics corrupt ribosomes, causing translational misreading, yet it remains unclear whether or not misreading triggers protein misfolding, and possible effects of chaperone action on drug susceptibilities are poorly understood...

  15. Influence of very short patch mismatch repair on SOS inducing lesions after aminoglycoside treatment in Escherichia coli.

    Science.gov (United States)

    Baharoglu, Zeynep; Mazel, Didier

    2014-01-01

    Low concentrations of aminoglycosides induce the SOS response in Vibrio cholerae but not in Escherichia coli. In order to determine whether a specific factor present in E. coli prevents this induction, we developed a genetic screen where only SOS inducing mutants are viable. We identified the vsr gene coding for the Vsr protein of the very short patch mismatch repair (VSPR) pathway. The effect of mismatch repair (MMR) mutants was also studied. We propose that lesions formed upon aminoglycoside treatment are preferentially repaired by VSPR without SOS induction in E. coli and by MMR when VSPR is impaired. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  16. The association between measurements of antimicrobial use and resistance in the faeces microbiota of finisher batches.

    Science.gov (United States)

    Andersen, V D; DE Knegt, L V; Munk, P; Jensen, M S; Agersø, Y; Aarestrup, F M; Vigre, H

    2017-10-01

    The objectives were to present three approaches for calculating antimicrobial (AM) use in pigs that take into account the rearing period and rearing site, and to study the association between these measurements and phenotypical resistance and abundance of resistance genes in faeces samples from 10 finisher batches. The AM use was calculated relative to the rearing period of the batches as (i) 'Finisher Unit Exposure' at unit level, (ii) 'Lifetime Exposure' at batch level and (iii) 'Herd Exposure' at herd level. A significant effect on the occurrence of tetracycline resistance measured by cultivation was identified for Lifetime Exposure for the AM class: tetracycline. Furthermore, for Lifetime Exposure for the AM classes: macrolide, broad-spectrum penicillin, sulfonamide and tetracycline use as well as Herd Unit Exposure for the AM classes: aminoglycoside, lincosamide and tetracycline use, a significant effect was observed on the occurrence of genes coding for the AM resistance classes: aminoglycoside, lincosamide, macrolide, β-lactam, sulfonamide and tetracycline. No effect was observed for Finisher Unit Exposure. Overall, the study shows that Lifetime Exposure is an efficient measurement of AM use in finisher batches, and has a significant effect on the occurrence of resistance, measured either by cultivation or metagenomics.

  17. A Picrinine N-Methyltransferase Belongs to a New Family of γ-Tocopherol-Like Methyltransferases Found in Medicinal Plants That Make Biologically Active Monoterpenoid Indole Alkaloids1[OPEN

    Science.gov (United States)

    Levac, Dylan; Cázares, Paulo; Yu, Fang

    2016-01-01

    Members of the Apocynaceae plant family produce a large number of monoterpenoid indole alkaloids (MIAs) with different substitution patterns that are responsible for their various biological activities. A novel N-methyltransferase involved in the vindoline pathway in Catharanthus roseus showing distinct similarity to γ-tocopherol C-methyltransferases was used in a bioinformatic screen of transcriptomes from Vinca minor, Rauvolfia serpentina, and C. roseus to identify 10 γ-tocopherol-like N-methyltransferases from a large annotated transcriptome database of different MIA-producing plant species (www.phytometasyn.ca). The biochemical function of two members of this group cloned from V. minor (VmPiNMT) and R. serpentina (RsPiNMT) have been characterized by screening their biochemical activities against potential MIA substrates harvested from the leaf surfaces of MIA-accumulating plants. The approach was validated by identifying the MIA picrinine from leaf surfaces of Amsonia hubrichtii as a substrate of VmPiNMT and RsPiNMT. Recombinant proteins were shown to have high substrate specificity and affinity for picrinine, converting it to N-methylpicrinine (ervincine). Developmental studies with V. minor and R. serpentina showed that RsPiNMT and VmPiNMT gene expression and biochemical activities were highest in younger leaf tissues. The assembly of at least 150 known N-methylated MIAs within members of the Apocynaceae family may have occurred as a result of the evolution of the γ-tocopherol-like N-methyltransferase family from γ-tocopherol methyltransferases. PMID:26848097

  18. Monolignol 4-O-methyltransferases and uses thereof

    Science.gov (United States)

    Liu, Chang-Jun; Bhuiya, Mohammad-Wadud; Zhang, Kewei

    2014-11-18

    Modified (iso)eugenol 4-O-methyltransferase enzymes having novel capacity for methylation of monolignols and reduction of lignin polymerization in plant cell wall are disclosed. Sequences encoding the modified enzymes are disclosed.

  19. Involvement of methyltransferases enzymes during the energy

    African Journals Online (AJOL)

    Mgina

    INVOLVEMENT OF METHYLTRANSFERASES ENZYMES DURING THE. ENERGY METABOLISM OF ..... cell extract still exhibited relatively high methanogenesis with methanol (Fig ... product CH3-CoM into methane (see Fig. 1). The HS-CoM ...

  20. O6-methylguanine DNA-methyltransferase (MGMT) overexpression in melanoma cells induces resistance to nitrosoureas and temozolomide but sensitizes to mitomycin C

    International Nuclear Information System (INIS)

    Passagne, Isabelle; Evrard, Alexandre; Depeille, Philippe; Cuq, Pierre; Cupissol, Didier; Vian, Laurence

    2006-01-01

    Alkylating agents play an important role in the chemotherapy of malignant melanomas. The activity of alkylating agents depends on their capacity to form alkyl adducts with DNA, in some cases causing cross-linking of DNA strands. However, the use of these agents is limited by cellular resistance induced by the DNA repair enzyme O 6 -methylguanine DNA-methyltransferase (MGMT) which removes alkyl groups from alkylated DNA strands. To determine to what extent the expression of MGMT in melanoma cells induces resistance to alkylating agents, the human cell line CAL77 Mer- (i.e., MGMT deficient) were transfected with pcMGMT vector containing human MGMT cDNA. Several clones expressing MGMT at a high level were selected to determine their sensitivity to chemotherapeutic drugs. Melanoma-transfected cells were found to be significantly less sensitive to nitrosoureas (carmustine, fotemustine, streptozotocin) and temozolomide with an increase of IC 5 values between 3 and 14 when compared to parent cells. No difference in cell survival rates between MGMT-proficient and -deficient cells was observed for melphalan, chlorambucil, busulphan, thiotepa and cisplatin which preferentially induce N 7 guanine lesions. Surprisingly, MGMT overexpression increased the sensitivity of CAL77 cells to mitomycin C by approximately 10-fold. Treatment of clonal cell lines with buthionine-[S,R]-sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase which depletes cellular glutathione, completely reversed this unexpected increase in sensitivity to mitomycin C. This observation suggests that glutathione is involved in the sensitivity of MGMT-transfected cells to mitomycin C and may act synergistically with MGMT via an unknown mechanism

  1. O(6)-methylguanine DNA-methyltransferase (MGMT) overexpression in melanoma cells induces resistance to nitrosoureas and temozolomide but sensitizes to mitomycin C.

    Science.gov (United States)

    Passagne, Isabelle; Evrard, Alexandre; Depeille, Philippe; Cuq, Pierre; Cupissol, Didier; Vian, Laurence

    2006-03-01

    Alkylating agents play an important role in the chemotherapy of malignant melanomas. The activity of alkylating agents depends on their capacity to form alkyl adducts with DNA, in some cases causing cross-linking of DNA strands. However, the use of these agents is limited by cellular resistance induced by the DNA repair enzyme O(6)-methylguanine DNA-methyltransferase (MGMT) which removes alkyl groups from alkylated DNA strands. To determine to what extent the expression of MGMT in melanoma cells induces resistance to alkylating agents, the human cell line CAL77 Mer- (i.e., MGMT deficient) were transfected with pcMGMT vector containing human MGMT cDNA. Several clones expressing MGMT at a high level were selected to determine their sensitivity to chemotherapeutic drugs. Melanoma-transfected cells were found to be significantly less sensitive to nitrosoureas (carmustine, fotemustine, streptozotocin) and temozolomide with an increase of IC(50) values between 3 and 14 when compared to parent cells. No difference in cell survival rates between MGMT-proficient and -deficient cells was observed for melphalan, chlorambucil, busulphan, thiotepa and cisplatin which preferentially induce N(7) guanine lesions. Surprisingly, MGMT overexpression increased the sensitivity of CAL77 cells to mitomycin C by approximately 10-fold. Treatment of clonal cell lines with buthionine-[S,R]-sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase which depletes cellular glutathione, completely reversed this unexpected increase in sensitivity to mitomycin C. This observation suggests that glutathione is involved in the sensitivity of MGMT-transfected cells to mitomycin C and may act synergistically with MGMT via an unknown mechanism.

  2. Draft Genome Sequence of a Multidrug-Resistant Klebsiella quasipneumoniae subsp. similipneumoniae Isolate from a Clinical Source

    Energy Technology Data Exchange (ETDEWEB)

    Ozer, Egon A.; Morris, Andrew R.; Krapp, Fiorella; Henry, Christopher S.; Tyo, Keith E.; Lathem, Wyndham W.; Hauser, Alan R.

    2016-05-26

    We report here the draft genome sequence of a multidrug-resistant clinical isolate ofKlebsiella quasipneumoniaesubsp.similipneumoniae, KP_Z4175. This strain, isolated as part of a hospital infection-control screening program, is resistant to multiple β-lactam antibiotics, aminoglycosides, and trimethoprim-sulfamethoxazole.

  3. The effect of the circadian rhythm on the clearance of aminoglycosides in ICU patients

    NARCIS (Netherlands)

    Van Maarseveen, E.; Proost, J.; Neef, C.; Touw, D.

    Aims: Critically ill patients, admitted to an intensive care unit, are at high risk of acquiring a nosocomial infection. Aminoglycosides (AMGs) are frequently used as first line therapy in severe hospital-acquired pneumonia or sepsis. It has been shown that once daily dosing (ODD) can reduce toxic

  4. Wild-Type MIC Distributions for Aminoglycoside and Cyclic Polypeptide Antibiotics Used for Treatment of Mycobacterium tuberculosis Infections▿

    Science.gov (United States)

    Juréen, P.; Ängeby, K.; Sturegård, E.; Chryssanthou, E.; Giske, C. G.; Werngren, J.; Nordvall, M.; Johansson, A.; Kahlmeter, G.; Hoffner, S.; Schön, T.

    2010-01-01

    The aminoglycosides and cyclic polypeptides are essential drugs in the treatment of multidrug-resistant tuberculosis, underscoring the need for accurate and reproducible drug susceptibility testing (DST). The epidemiological cutoff value (ECOFF) separating wild-type susceptible strains from non-wild-type strains is an important but rarely used tool for indicating susceptibility breakpoints against Mycobacterium tuberculosis. In this study, we established wild-type MIC distributions on Middlebrook 7H10 medium for amikacin, kanamycin, streptomycin, capreomycin, and viomycin using 90 consecutive clinical isolates and 21 resistant strains. Overall, the MIC variation between and within runs did not exceed ±1 MIC dilution step, and validation of MIC values in Bactec 960 MGIT demonstrated good agreement. Tentative ECOFFs defining the wild type were established for all investigated drugs, including amikacin and viomycin, which currently lack susceptibility breakpoints for 7H10. Five out of seven amikacin- and kanamycin-resistant isolates were classified as susceptible to capreomycin according to the current critical concentration (10 mg/liter) but were non-wild type according to the ECOFF (4 mg/liter), suggesting that the critical concentration may be too high. All amikacin- and kanamycin-resistant isolates were clearly below the ECOFF for viomycin, and two of them were below the ECOFF for streptomycin, indicating that these two drugs may be considered for treatment of amikacin-resistant strains. Pharmacodynamic indices (peak serum concentration [Cmax]/MIC) were more favorable for amikacin and viomycin compared to kanamycin and capreomycin. In conclusion, our data emphasize the importance of establishing wild-type MIC distributions for improving the quality of drug susceptibility testing against Mycobacterium tuberculosis. PMID:20237102

  5. Biochemical and Structural Analysis of an Eis Family Aminoglycoside Acetyltransferase from Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Green, Keith D.; Biswas, Tapan; Chang, Changsoo; Wu, Ruiying; Chen, Wenjing; Janes, Brian K.; Chalupska, Dominika; Gornicki, Piotr; Hanna, Philip C.; Tsodikov, Oleg V.; Joachimiak, Andrzej; Garneau-Tsodikova, Sylvie

    2015-05-26

    Proteins from the enhanced intracellular survival (Eis) family are versatile acetyltransferases that acetylate amines at multiple positions of several aminoglycosides (AGs). Their upregulation confers drug resistance. Homologues of Eis are present in diverse bacteria, including many pathogens. Eis from Mycobacterium tuberculosis (Eis_Mtb) has been well characterized. In this study, we explored the AG specificity and catalytic efficiency of the Eis family protein from Bacillus anthracis (Eis_Ban). Kinetic analysis of specificity and catalytic efficiency of acetylation of six AGs indicates that Eis_Ban displays significant differences from Eis_Mtb in both substrate binding and catalytic efficiency. The number of acetylated amines was also different for several AGs, indicating a distinct regiospecificity of Eis_Ban. Furthermore, most recently identified inhibitors of Eis_Mtb did not inhibit Eis_Ban, underscoring the differences between these two enzymes. To explain these differences, we determined an Eis_Ban crystal structure. The comparison of the crystal structures of Eis_Ban and Eis_Mtb demonstrates that critical residues lining their respective substrate binding pockets differ substantially, explaining their distinct specificities. Our results suggest that acetyltransferases of the Eis family evolved divergently to garner distinct specificities while conserving catalytic efficiency, possibly to counter distinct chemical challenges. The unique specificity features of these enzymes can be utilized as tools for developing AGs with novel modifications and help guide specific AG treatments to avoid Eis-mediated resistance.

  6. Therapeutic drug monitoring by radioimmunoassay: Determination of aminoglycoside antibiotics and vancomycin in plasma

    International Nuclear Information System (INIS)

    Glaubitt, D.; Drechsler, H.J.; Knoch, K.; Siafarikas, K.

    1984-01-01

    Radioimmunoassay of aminoglycoside antibiotics (gentamicin, tobramycin, netilmicin) or vancomycin in plasma may considerably aid to assess their appropriate dosage and, if necessary, to rapidly adjust it to the assumed requirement. Thus the dosage of the antibiotic is kept large enough as to lead to the desired therapeutic result but not as high as to cause side effects. (orig.)

  7. The O-methyltransferase PMT2 mediates methylation of pinosylvin in Scots pine.

    Science.gov (United States)

    Paasela, Tanja; Lim, Kean-Jin; Pietiäinen, Milla; Teeri, Teemu H

    2017-06-01

    Heartwood extractives are important determinants of the natural durability of pine heartwood. The most important phenolic compounds affecting durability are the stilbenes pinosylvin and its monomethylether, which in addition have important functions as phytoalexins in active defense. A substantial portion of the synthesized pinosylvin is 3-methoxylated but the O-methyltransferase responsible for this modification has not been correctly identified. We studied the expression of the stilbene pathway during heartwood development as well as in response to wounding of xylem and UV-C treatment of needles. We isolated and enzymatically characterized a novel O-methyltransferase, PMT2. The methylated product was verified as pinosylvin monomethylether using ultra performance liquid chromatography-tandem mass spectrometry and high performance liquid chromatography analyses. The PMT2 enzyme was highly specific for stilbenes as substrate, in contrast to caffeoyl-CoA O-methyltransferase (CCoAOMT) and PMT1 that were multifunctional. Expression profile and multifunctional activity of CCoAOMT suggest that it might have additional roles outside lignin biosynthesis. PMT1 is not involved in the stilbene pathway and its biological function remains an open question. We isolated a new specific O-methyltransferase responsible for 3-methoxylation of pinosylvin. Expression of PMT2 closely follows stilbene biosynthesis during developmental and stress induction. We propose that PMT2 is responsible for pinosylvin methylation in Scots pine (Pinus sylvestris), instead of the previously characterized methyltransferase, PMT1. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  8. Global DNA hypermethylation-associated cancer chemotherapy resistance and its reversion with the demethylating agent hydralazine

    Directory of Open Access Journals (Sweden)

    Benitez-Bribiesca Luis

    2006-08-01

    Full Text Available Abstract Background The development of resistance to cytotoxic chemotherapy continues to be a major obstacle for successful anticancer therapy. It has been shown that cells exposed to toxic concentrations of commonly used cancer chemotherapy agents develop DNA hypermetylation. Hence, demethylating agents could play a role in overcoming drug resistance. Methods MCF-7 cells were rendered adriamycin-resistant by weekly treatment with adriamycin. Wild-type and the resulting MCF-7/Adr cells were analyzed for global DNA methylation. DNA methyltransferase activity and DNA methyltransferase (dnmt gene expression were also determined. MCF-7/Adr cells were then subjected to antisense targeting of dnmt1, -3a, and -b genes and to treatment with the DNA methylation inhibitor hydralazine to investigate whether DNA demethylation restores sensitivity to adriamycin. Results MCF-7/Adr cells exhibited the multi-drug resistant phenotype as demonstrated by adriamycin resistance, mdr1 gene over-expression, decreased intracellular accumulation of adriamycin, and cross-resistance to paclitaxel. The mdr phenotype was accompanied by global DNA hypermetylation, over-expression of dnmt genes, and increased DNA methyltransferase activity as compared with wild-type MCF-7 cells. DNA demethylation through antisense targeting of dnmts or hydralazine restored adriamycin sensitivity of MCF-7/Adr cells to a greater extent than verapamil, a known inhibitor of mdr protein, suggesting that DNA demethylation interferes with the epigenetic reprogramming that participates in the drug-resistant phenotype. Conclusion We provide evidence that DNA hypermethylation is at least partly responsible for development of the multidrug-resistant phenotype in the MCF-7/Adr model and that hydralazine, a known DNA demethylating agent, can revert the resistant phenotype.

  9. Flavivirus methyltransferase as target for virus treatment

    Czech Academy of Sciences Publication Activity Database

    Krafčíková, Petra; Chalupská, Dominika; Hercík, Kamil; Nencka, Radim; Bouřa, Evžen

    2017-01-01

    Roč. 284, Suppl 1 (2017), s. 216-217 ISSN 1742-464X. [FEBS Congress /42./ From Molecules to Cells and Back. 10.09.2017-14.09.2017, Jerusalem] Institutional support: RVO:61388963 Keywords : flavivirus methyltransferase * antivirals Subject RIV: CE - Biochemistry

  10. Surveillance for Travel and Domestically Acquired Multidrug-Resistant Human Shigella Infections-Pennsylvania, 2006-2014.

    Science.gov (United States)

    Li, Yu Lung; Tewari, Deepanker; Yealy, Courtney C; Fardig, David; M'ikanatha, Nkuchia M

    2016-01-01

    Shigellosis is a leading cause of enteric infections in the United States. We compared antimicrobial resistance in Shigella infections related to overseas travel (travel-associated) and in those acquired domestically by analyzing antimicrobial resistance patterns, geographic distributions, and pulsed-field gel electrophoresis (PFGE) patterns. We tested samples (n = 204) from a collection of isolates recovered from patients in Pennsylvania between 2006 and 2014. Isolates were grouped into travel- and non-travel-associated categories. Eighty-one (79.4%) of the Shigella isolates acquired during international travel were resistant to multiple antibiotics compared to 53 (52.1%) of the infections transmitted in domestic settings. A majority (79.4%) of isolates associated with international travel demonstrated resistance to aminoglycosides and tetracyclines, whereas 47 (46.1%) of the infections acquired domestically were resistant to tetracycline. Almost all isolates (92.2%) transmitted in domestic settings were resistant to aminoglycosides, and 5 isolates from adult male patients were resistant to azithromycin, a drug often used for empiric treatment of severe shigellosis. Twenty (19.6%) isolates associated with illnesses acquired during overseas travel in 4 countries were resistant to quinolones. One S. sonnei PFGE pattern was traced to a multidrug-resistant isolate acquired overseas that had caused a multistate outbreak of shigellosis, suggesting global dissemination of a drug-resistant species. Resistance to certain drugs-for example, tetracycline-increased in both overseas- and domestic-acquired infections during the study period. The prevalence of resistance to macrolides (azithromycin) and third-generation cephalosporins (ceftriaxone) was less than 1%; however, efforts to better monitor changes in drug resistance over time combined with increased antimicrobial stewardship are essential at the local, national, and global levels.

  11. Association between the Presence of Aminoglycoside-Modifying Enzymes and In Vitro Activity of Gentamicin, Tobramycin, Amikacin, and Plazomicin against Klebsiella pneumoniae Carbapenemase- and Extended-Spectrum-β-Lactamase-Producing Enterobacter Species.

    Science.gov (United States)

    Haidar, Ghady; Alkroud, Ammar; Cheng, Shaoji; Churilla, Travis M; Churilla, Bryce M; Shields, Ryan K; Doi, Yohei; Clancy, Cornelius J; Nguyen, M Hong

    2016-09-01

    We compared the in vitro activities of gentamicin (GEN), tobramycin (TOB), amikacin (AMK), and plazomicin (PLZ) against 13 Enterobacter isolates possessing both Klebsiella pneumoniae carbapenemase and extended-spectrum β-lactamase (KPC+/ESBL+) with activity against 8 KPC+/ESBL-, 6 KPC-/ESBL+, and 38 KPC-/ESBL- isolates. The rates of resistance to GEN and TOB were higher for KPC+/ESBL+ (100% for both) than for KPC+/ESBL- (25% and 38%, respectively), KPC-/ESBL+ (50% and 17%, respectively), and KPC-/ESBL- (0% and 3%, respectively) isolates. KPC+/ESBL+ isolates were more likely than others to possess an aminoglycoside-modifying enzyme (AME) (100% versus 38%, 67%, and 5%; P = 0.007, 0.06, and 1 AME than with ≤1 AME. The presence of at least 2/3 of KPC, SHV, and TEM predicted the presence of AMEs. PLZ MICs against all isolates were ≤4 μg/ml, regardless of KPC/ESBL pattern or the presence of AMEs. In conclusion, GEN and TOB are limited as treatment options against KPC+ and ESBL+ Enterobacter PLZ may represent a valuable addition to the antimicrobial armamentarium. A full understanding of AMEs and other aminoglycoside resistance mechanisms will allow clinicians to incorporate PLZ rationally into treatment regimens. The development of molecular assays that accurately and rapidly predict antimicrobial responses among KPC- and ESBL-producing Enterobacter spp. should be a top research priority. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  12. Floral Benzenoid Carboxyl Methyltransferases: From in Vitro to in Planta Function

    Energy Technology Data Exchange (ETDEWEB)

    Effmert,U.; Saschenbrecker, S.; Ross, J.; Negre, F.; Fraser, C.; Noel, J.; Dudareva, N.; Piechulla, B.

    2005-01-01

    Benzenoid carboxyl methyltransferases synthesize methyl esters (e.g., methyl benzoate and methyl salicylate), which are constituents of aromas and scents of many plant species and play important roles in plant communication with the surrounding environment. Within the past five years, eleven such carboxyl methyltransferases were isolated and most of them were comprehensively investigated at the biochemical, molecular and structural level. Two types of enzymes can be distinguished according to their substrate preferences: the SAMT-type enzymes isolated from Clarkia breweri, Stephanotis floribunda, Antirrhinum majus, Hoya carnosa, and Petunia hybrida, which have a higher catalytic efficiency and preference for salicylic acid, while BAMT-type enzymes from A. majus, Arabidopsis thaliana, Arabidopsis lyrata, and Nicotiana suaveolens prefer benzoic acid. The elucidation of C. breweri SAMT's three-dimensional structure allowed a detailed modelling of the active sites of the carboxyl methyltransferases and revealed that the SAM binding pocket is highly conserved among these enzymes while the methyl acceptor binding site exhibits some variability, allowing a classification into SAMT-type and BAMT-type enzymes. The analysis of expression patterns coupled with biochemical characterization showed that these carboxyl methyltransferases are involved either in floral scent biosynthesis or in plant defense responses. While the latter can be induced by biotic or abiotic stress, the genes responsible for floral scent synthesis exhibit developmental and rhythmic expression pattern. The nature of the product and efficiency of its formation in plants depend on the availability of substrates, the catalytic efficiency of the enzyme toward benzoic acid and/or salicylic acid, and the transcriptional, translational, and post-translational regulation at the enzyme level. The biochemical properties of benzenoid carboxyl methyltransferases suggest that the genes involved in plant defenses

  13. Two unusual cases of severe recalcitrant hypocalcemia due to aminoglycoside-induced hypomagnesemia

    Directory of Open Access Journals (Sweden)

    Tarun Varma

    2013-01-01

    Full Text Available Aminoglycoside (AMG-induced renal toxicity is well-known and may manifest with non-oliguric renal failure or renal tubular dysfunction like Fanconi-like syndrome, Barter syndrome-like syndrome or distal renal tubular acidosis (RTA. These phenomena have been described with Gentamycin and Amikacin though rarely with Kanamycin. We present two cases of pulmonary tuberculosis that were treated with Kanamycin and during the course of treatment, developed severe recalcitrant hypocalcemia along with hypomagnesemia.

  14. Histone methyltransferases in cancer

    DEFF Research Database (Denmark)

    Albert, Mareike; Helin, Kristian

    2009-01-01

    Cancer is perceived as a heterogeneous group of diseases that is characterized by aberrant patterns of gene expression. In the last decade, an increasing amount of data has pointed to a key role for epigenetic alterations in human cancer. In this review, we focus on a subclass of epigenetic...... regulators, namely histone methyltransferases (HMTs). Several HMTs have been linked to different types of cancer; however, in most cases we only have limited knowledge regarding the molecular mechanisms by which the HMTs contribute to disease development. We summarize the current knowledge regarding some...

  15. Assessment of antibiotic resistance genes and integrons in commensal Escherichia coli from the Indian urban waste water: Implications and significance for public health

    Directory of Open Access Journals (Sweden)

    Nambram Somendro Singh

    2017-10-01

    Full Text Available Antibiotics like β-lactams, quinolones/fluoroquinolones, aminoglycosides and tetracycline constitute the major mainstay of treatment against most infectious diseases including Escherichia coli. Indiscriminate use of antibiotics for human and animal well-being has generated an enormous evolutionary pressure on bacteria especially E.coli, which has a highly plastic/evolving genome. Though, antibiotic resistance (AR has been extensively studied in pathogenic E.coli, commensal strains have been studied less owing to lesser clinical significance. However, commensal strains pose a serious threat as reservoirs and transmitters of resistance genes to other bacteria. Therefore, the present study was undertaken to investigate the prevalence of resistance genes and integrons in commensal E.coli isolated from river Yamuna, Delhi, India, which receives plentiful urban waste water. Eighty three well-characterized E.coli strains of phylogroups A and B1 isolated from river Yamuna were investigated. Antimicrobial susceptibilities and minimal inhibitory concentrations (MICs for β-lactams, aminoglycosides, tetracycline and quinolone/fluoroquinolone were determined by disk diffusion and Etest, according to Clinical and Laboratory Standards Institute (CLSI guidelines. Production of Extended spectrum β-lactamases (ESBL and AmpC was investigated. Prevalence of antibiotic-resistance genes for β-lactams (blaTEM,blaSHV, blaCTX-M, blaOXA, blaCMY-42, aminoglycosides (rmtA, rmtB, rmtC, armA, str, aacC2, tetracycline (tetA, tetR, tetM, tetW, and plasmid-mediated quinolone resistance, PMQR (qnrA, qnrB, qnrC, qnrD, qnrS, qep, aac were assessed. Integrons and  gene-cassette arrays were characterized. Commensal E.coli strains showed a higher resistance to ampicillin (95%, less to cefazolin (45% and still lesser to tetracycline (15%. About 19% of these strains showed multidrug resistant (three or more classes of antibiotics, of which 15% also produced ESBLs. None of the

  16. [Gene cloning and bioinformatics analysis of SABATH methyltransferase in Lonicera japonica var. chinensis].

    Science.gov (United States)

    Yu, Xiao-Dan; Jiang, Chao; Huang, Lu-Qi; Qin, Shuang-Shuang; Zeng, Xiang-Mei; Chen, Ping; Yuan, Yuan

    2013-08-01

    To clone SABATH methyltransferase (rLjSABATHMT) gene in Lonicera japonica var. chinensis, and compare the gene expression and intron sequence of SABATH methyltransferase orthologous in L. japonica with L. japonica var. chinensis. It provide a basis for gene regulate the formation of L. japonica floral scents. The cDNA and genome sequences of LjSABATHMT from L. japonica var. chinensis were cloned according to the gene fragments in cDNA library. The LjSABATHMT protein was characterized by bioinformatics analysis. SABATH family phylogenetic tree were built by MEGA 5.0. The transcripted level of SABATHMT orthologous were analyzed in different organs and different flower periods of L. japonica and L. japonica var. chinensis using RT-PCR analysis. Intron sequences of SABATHMT orthologous were also analyzied. The cDNA of LjSABATHMT was 1 251 bp, had a complete coding frame with 365 amino acids. The protein had the conservative SABATHMT domain, and phylogenetic tree showed that it may be a salicylic acid/benzoic acid methyltransferase. Higher expression of SABATH methyltransferase orthologous was found in flower. The intron sequence of L. japonica and L. japonica var. chinensis had rich polymorphism, and two SNP are unique genotype of L. japonica var. chinensis. The motif elements in two orthologous genes were significant differences. The intron difference of SABATH methyltransferase orthologous could be inducing to difference of gene expression between L. japonica and L. japonica var. chinensis. These results will provide important base on regulating active compounds of L. japonica.

  17. Biochemical and Computational Analysis of the Substrate Specificities of Cfr and RlmN Methyltransferases

    DEFF Research Database (Denmark)

    Ntokou, Eleni; Hansen, Lykke Haastrup; Kongsted, Jacob

    2015-01-01

    -ray structure of RlmN. We used a trinucleotide as target sequence and assessed its positioning at the active site for methylation. The calculations are in accordance with different poses of the trinucleotide in the two enzymes indicating major evolutionary changes to shift the C2/C8 specificities. To explore......Cfr and RlmN methyltransferases both modify adenine 2503 in 23S rRNA (Escherichia coli numbering). RlmN methylates position C2 of adenine while Cfr methylates position C8, and to a lesser extent C2, conferring antibiotic resistance to peptidyl transferase inhibitors. Cfr and RlmN show high sequence...... interchangeability between Cfr and RlmN we constructed various combinations of their genes. The function of the mixed genes was investigated by RNA primer extension analysis to reveal methylation at 23S rRNA position A2503 and by MIC analysis to reveal antibiotic resistance. The catalytic site is expected...

  18. Methylated nucleosides in tRNA and tRNA methyltransferases

    Directory of Open Access Journals (Sweden)

    Hiroyuki eHori

    2014-05-01

    Full Text Available To date, more than 90 modified nucleosides have been found in tRNA and the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s. Recent studies of the biosynthetic pathways have demonstrated that the availability of methyl group donors for the methylation in tRNA is important for correct and efficient protein synthesis. In this review, I focus on the methylated nucleosides and tRNA methyltransferases. The primary functions of tRNA methylations are linked to the different steps of protein synthesis, such as the stabilization of tRNA structure, reinforcement of the codon–anticodon interaction, regulation of wobble base pairing, and prevention of frameshift errors. However, beyond these basic functions, recent studies have demonstrated that tRNA methylations are also involved in the RNA quality control system and regulation of tRNA localization in the cell. In a thermophilic eubacterium, tRNA modifications and the modification enzymes form a network that responses to temperature changes. Furthermore, several modifications are involved in genetic diseases, infections, and the immune response. Moreover, structural, biochemical, and bioinformatics studies of tRNA methyltransferases have been clarifying the details of tRNA methyltransferases and have enabled these enzymes to be classified. In the final section, the evolution of modification enzymes is discussed.

  19. Identification of the RsmG methyltransferase target as 16S rRNA nucleotide G527 and characterization of Bacillus subtilis rsmG mutants

    DEFF Research Database (Denmark)

    Nishimura, Kenji; Johansen, Shanna K; Inaoka, Takashi

    2007-01-01

    The methyltransferase RsmG methylates the N7 position of nucleotide G535 in 16S rRNA of Bacillus subtilis (corresponding to G527 in Escherichia coli). Disruption of rsmG resulted in low-level resistance to streptomycin. A growth competition assay revealed that there are no differences in fitness...... between the rsmG mutant and parent strains under the various culture conditions examined. B. subtilis rsmG mutants emerged spontaneously at a relatively high frequency, 10(-6). Importantly, in the rsmG mutant background, high-level-streptomycin-resistant rpsL (encoding ribosomal protein S12) mutants...

  20. Conjugal transfer of aac(6')Ie-aph(2″)Ia gene from native species and mechanism of regulation and cross resistance in Enterococcus faecalis MCC3063 by real time-PCR.

    Science.gov (United States)

    Jaimee, G; Halami, P M

    2017-09-01

    High level aminoglycoside resistance (HLAR) in the lactic acid bacteria (LAB) derived from food animals is detrimental. The aim of this study was to investigate the localization and conjugal transfer of aminoglycoside resistance genes, aac(6')Ie-aph(2″)Ia and aph(3')IIIa in different Enterococcus species. The cross resistance patterns in Enterococcus faecalis MCC3063 to clinically important aminoglycosides by real time PCR were also studied. Southern hybridization experiments revealed the presence of aac(6')Ie-aph(2 ″ )Ia and aph(3')IIIa genes conferring HLAR in high molecular weight plasmids except in Lactobacillus plantarum. The plasmid encoded bifunctional aac(6')Ie-aph(2″)Ia gene was transferable from Enterococcus avium (n = 2), E. cecorum (n = 1), E. faecalis (n = 1) and Pediococcus lolii (n = 1) species into the recipient strain; E. faecalis JH2-2 by filter mating experiments thus indicating the possible risks of gene transfer into pathogenic strains. Molecular analysis of cross resistance patterns in native isolate of E. faecalis MCC3063 carrying aac(6')Ie-aph(2″)Ia and aph(3')IIIa gene was displayed by quantification of the mRNA levels in this study. For this, the culture was induced with increasing concentrations of gentamicin, kanamycin and streptomycin (2048, 4096, 8192, 16384 μg/mL) individually. The increasing concentrations of gentamicin and kanamycin induced the expression of the aac(6')Ie-aph(2″)Ia and aph(3')IIIa resistance genes, respectively. Interestingly, it was observed that induction with streptomycin triggered a significant fold increase in the expression of the aph(3')IIIa gene which otherwise was not known to modify the aminoglycoside. This is noteworthy as streptomycin was found to confer cross resistance to structurally unrelated kanamycin. Also, expression of the aph(3')IIIa gene when induced with streptomycin, revealed that bacteria harbouring this gene will be able to overcome streptomycin bactericidal action at

  1. Pan Drug-Resistant Environmental Isolate of Acinetobacter baumannii from Croatia.

    Science.gov (United States)

    Goic-Barisic, Ivana; Seruga Music, Martina; Kovacic, Ana; Tonkic, Marija; Hrenovic, Jasna

    2017-06-01

    Acinetobacter baumannii is an emerging nosocomial pathogen with also emerging resistance to different antibiotics. Multidrug and pan drug-resistant clinical isolates were reported worldwide. Here we report the first evidence of pan drug-resistant environmental isolate of A. baumannii. The isolate was recovered from the effluent of secondary treated municipal wastewater of the City of Zagreb, Croatia. The isolate was resistant to penicillins/β-lactamase inhibitors, carbapenems, fluoroquinolones, aminoglycosides, folate pathway inhibitors, and polymyxins, except intermediately susceptible to minocycline and tigecycline. Intrinsic chromosomally located bla OXA-51-like gene and acquired plasmid-located bla OXA-23-like gene were related to clinical isolates. Pan drug-resistant A. baumannii can occur in natural environments outside of the hospital. Secondary treated municipal wastewater represents a potential epidemiological reservoir of pan drug-resistant A. baumannii and carbapenem resistance gene.

  2. Association between TPMT*3C and decreased thiopurine S-methyltransferase activity in patients with neuromyelitis optica spectrum disorders in China.

    Science.gov (United States)

    Gong, Xiaoqing; Mei, Shenghui; Li, Xindi; Li, Xingang; Zhou, Heng; Liu, Yonghong; Zhou, Anna; Yang, Li; Zhao, Zhigang; Zhang, Xinghu

    2018-06-01

    Thiopurines are effective drugs in treating neuromyelitis optica spectrum disorders and other diseases. Thiopurines' toxicity is mainly imputed to thiopurine S-methyltransferase activity. In Chinese population, the most common and important variation of thiopurine S-methyltransferase is TPMT*3C (rs1142345). This study aims to reveal the association between thiopurine S-methyltransferase activity and genetic polymorphisms of thiopurine S-methyltransferase in patients with neuromyelitis optica spectrum disorders in China. A liquid chromatography tandem mass/mass method was used to evaluate the thiopurine S-methyltransferase activity by using 6-mercapthioprine as the substrate in human erythrocyte haemolysate via 1 h incubation at 37 °C to form its methylated product 6-methylmercaptopurine. The amount of 6-methylmercaptopurine was adjusted by haematocrit and normalized to 8 × 10 8 erythrocytes. The selected polymorphisms of thiopurine S-methyltransferase were identified using MassARRAY system (Sequenom) and multiple SNaPshot technique. In 69 patients with neuromyelitis optica spectrum disorders, thiopurine S-methyltransferase activity was 80.29-154.53 (127.51 ± 16.83) pmol/h/8 × 10 8 erythrocytes. TPMT*3C (rs1142345) was associated with lower thiopurine S-methyltransferase activity (BETA = -25.37, P = 0.011). Other selected variants were not associated with thiopurine S-methyltransferase activity. TPMT*3C affects TPMT activity in Chinese patients with neuromyelitis optica spectrum disorders. Further studies are warranted to confirm the results. TPRs = thiopurines; NMOSD = neuromyelitis optica spectrum disorders; TPMT = thiopurine S-methyltransferase; LC-MS/MS = liquid chromatography tandem mass/mass; 6-MMP = 6-methylmercaptopurine; IS = internal standard; SNP = single nucleotide polymorphism; MAF = minor allele frequency; HWE = Hardy-Weinberg equilibrium; BETA = regression coefficients; UTR-3 = untranslated region 3.

  3. Evolution of the Phosphatidylcholine Biosynthesis Pathways in Green Algae: Combinatorial Diversity of Methyltransferases.

    Science.gov (United States)

    Hirashima, Takashi; Toyoshima, Masakazu; Moriyama, Takashi; Sato, Naoki

    2018-01-01

    Phosphatidylcholine (PC) is one of the most common phospholipids in eukaryotes, although some green algae such as Chlamydomonas reinhardtii are known to lack PC. Recently, we detected PC in four species in the genus Chlamydomonas: C. applanata NIES-2202, C. asymmetrica NIES-2207, C. debaryana NIES-2212, and C. sphaeroides NIES-2242. To reveal the PC biosynthesis pathways in green algae and the evolutionary scenario involved in their diversity, we analyzed the PC biosynthesis genes in these four algae using draft genome sequences. Homology searches suggested that PC in these species is synthesized by phosphoethanolamine-N-methyltransferase (PEAMT) and/or phosphatidylethanolamine-N-methyltransferase (PEMT), both of which are absent in C. reinhardtii. Recombinant PEAMTs from these algae showed methyltransferase activity for phosphoethanolamine but not for monomethyl phosphoethanolamine in vitro, in contrast to land plant PEAMT, which catalyzes the three methylations from phosphoethanolamine to phosphocholine. This suggested an involvement of other methyltransferases in PC biosynthesis. Here, we characterized the putative phospholipid-N-methyltransferase (PLMT) genes of these species by genetic and phylogenetic analysis. Complementation assays using a PC biosynthesis-deficient yeast suggested that the PLMTs of these algae can synthesize PC from phosphatidylethanolamine. These results indicated that the PC biosynthesis pathways in green algae differ from those of land plants, although the enzymes involved are homologous. Phylogenetic analysis suggested that the PEAMTs and PLMTs in these algae were inherited from the common ancestor of green algae. The absence of PC biosynthesis in many Chlamydomonas species is likely a result of parallel losses of PEAMT and PLMT in this genus.

  4. Studies on N5-methyltetrahydrofolate-homocystein methyltransferase in normal and leukemia leukocytes.

    Science.gov (United States)

    Peytremann, R; Thorndike, J; Beck, W S

    1975-11-01

    A cobalamin-dependent N5-methyltetra-hydrofolate-homocysteine methyltransferase (methyl-transferase) was demonstrated in unfractioned extracts of human normal and leukemia leukocytes. Activity was substantially reduced in the absence of an added cobalamin derivative. Presumably, this residual activity reflects the endogeneous level of holoenzyme. Enzyme activity was notably higher in lymphoid cells than in myeloid cells. Thus, mean specific activities (+/-SD) were: chronic lymphocytic leukemia lymphocytes, 2.15+/-1.16; normal lymphocytes, 0.91+/-0.59; normal mature granulocytes, 0.15+/-0.10; chronic myelocytic leukemia granulocytes, barely detectable activity. Properties of leukocytes enzymes resembled those of methyltransferases previously studied in bacteria and other animal cells. Granulocytes and chronic myelocytic leukemia cells contain a factor or factors that inhibits Escherichia coli enzyme. The data suggest that the prominence of this cobalamin-dependent enzyme in lymphocytes and other mononuclear cell types may be related to their potential for cell division.

  5. Rapid increase in resistance to third generation cephalosporins, imipenem and co-resistance in Klebsiella pneumoniae from isolated from 7,140 blood-cultures (2010-2014) using EARS-Net data in Spain.

    Science.gov (United States)

    Aracil-García, Belén; Oteo-Iglesias, Jesús; Cuevas-Lobato, Óscar; Lara-Fuella, Noelia; Pérez-Grajera, Isabel; Fernández-Romero, Sara; Pérez-Vázquez, María; Campos, José

    2017-10-01

    An analysis was made about the evolution of resistance to 3rd generation cephalosporins, imipenem, and other antibiotics in invasive isolates of Klebsiella pneumoniae (K. pneumoniae) according to the Spanish EARS-Net database (2010-2014). Forty-two hospitals from 16 Autonomous Communities with an approximate population coverage of 33% participated. A total 7,140 pneumoniae corresponding to the same number of patients were studied. Overall resistance percentages (I+R) were: cefotaxime 15.8%, ceftazidime 13.7%, imipenem 1.7%, ciprofloxacin 20.1%, tobramycin 14.1%, gentamicin 10.4%, and amikacin 1.9%. Resistance to 3rd generation cephalosporins increased from 9.8% (2010) to 19% (2014); to ciprofloxacin from 15.4% (2010) to 19.6% (2014); to gentamicin from 6.2% (2010) to 10.3% (2014) and to tobramycin from 7.1% (2010) to 14.2% (2014) (presistance to 3rd generation cephalosporins, ciprofloxacin, and aminoglycosides increased from 3.3% (2010) to 9.7% (2014) (pResistance to imipenem also increased from 0.27% (2010) to 3.46% (2014) (presistant to imipenem, of which 104 (86%) produced carbapenemases: 74 OXA-48, 14 VIM, 9 KPC (6 KPC-2 and 3 KPC-3), 6 IMP, and 1 GES. Over the 5 year period (2010-2014), resistance to 3rd generation cephalosporins in invasive K. pneumoniae in Spain has doubled. The combined resistance to 3rd generation cephalosporins, ciprofloxacin, and aminoglycosides has tripled, and imipenem resistance has increased almost 13 times, mostly due to the spread of carbapenemase-producing isolates. Copyright © 2016 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  6. Antifungal amphiphilic aminoglycoside K20: bioactivities and mechanism of action

    Directory of Open Access Journals (Sweden)

    Sanjib K. Shrestha

    2014-12-01

    Full Text Available K20 is a novel amphiphilic antifungal aminoglycoside that is synthetically derived from the antibiotic kanamycin A. Reported here are investigations of K20’s antimicrobial activities, cytotoxicity, and fungicidal mechanism of action. In vitro growth inhibitory activities against a variety of human and plant pathogenic yeasts, filamentous fungi, and bacteria were determined using microbroth dilution assays and time-kill curve analyses, and hemolytic and animal cell cytotoxic activities were determined. Effects on Cryptococcus neoformans H-99 infectivity were determined with a preventive murine lung infection model. The antifungal mechanism of action was studied using intact fungal cells, yeast lipid mutants, and small unilamellar lipid vesicles. K20 exhibited broad-spectrum in vitro antifungal activities but not antibacterial activities. Pulmonary, single dose-administration of K20 reduced C. neoformans lung infection rates 4-fold compared to controls. Hemolysis and half-maximal cytotoxicities of mammalian cells occurred at concentrations that were 10 to 32-fold higher than fungicidal MICs. With fluorescein isothiocyanate, 20 to 25 mg/L K20 caused staining of >95% of C. neoformans and Fusarium graminearum cells and at 31.3 mg/L caused rapid leakage (30 to 80% in 15 min of calcein from preloaded small unilamellar lipid vesicles. K20 appears to be a broad-spectrum fungicide, capable of reducing the infectivity of C. neoformans, and exhibits low hemolytic activity and mammalian cell toxicity. It perturbs the plasma membrane by mechanisms that are lipid modulated. K20 is a novel amphiphilic aminoglycoside amenable to scalable production and a potential lead antifungal for therapeutic and crop protection applications.

  7. Azobenzene-aminoglycoside: Self-assembled smart amphiphilic nanostructures for drug delivery.

    Science.gov (United States)

    Deka, Smriti Rekha; Yadav, Santosh; Mahato, Manohar; Sharma, Ashwani Kumar

    2015-11-01

    Here, we have designed and synthesized a novel cationic amphiphilic stimuli-responsive azobenzene-aminoglycoside (a small molecule) conjugate, Azo-AG 5, and characterized it by UV and FTIR. Light responsive nature of Azo-AG 5 was assessed under UV-vis light. Self- assembly of Azo-AG 5 in aqueous solutions into nanostructures and their ability to act as drug carrier were also investigated. The nanostructures of Azo-AG 5 showed average hydrodynamic diameter of ∼ 255 nm with aminoglycoside moiety (neomycin) and 4-dimethylaminoazobenzene forming hydrophilic shell and hydrophobic core, respectively. In the hydrophobic core, eosin and aspirin were successfully encapsulated. Dynamic light scattering (DLS) measurements demonstrated that the nanoassemblies showed expansion and contraction on successive UV and visible light irradiations exhibiting reversible on-off switch for controlling the drug release behavior. Similar behavior was observed when these nanostructures were subjected to pH-change. In vitro drug release studies showed a difference in UV and visible light-mediated release pattern. It was observed that the release rate under UV irradiation was comparatively higher than that observed under visible light. Further, azoreductase-mediated cleavage of the azo moiety in Azo-AG 5 nanoassemblies resulted in the dismantling of the structures into aggregated microstructures. Azo-AG 5 nanostructures having positive surface charge (+9.74 mV) successfully interacted with pDNA and retarded its mobility on agarose gel. Stimuli responsiveness of nanostructures and their on-off switch like behavior ensure the great potential as controlled drug delivery systems and in other biomedical applications such as colon-specific delivery and gene delivery. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Genomic Analysis Reveals Distinct Concentration-Dependent Evolutionary Trajectories for Antibiotic Resistance in Escherichia coli

    Science.gov (United States)

    Mogre, Aalap; Sengupta, Titas; Veetil, Reshma T.; Ravi, Preethi; Seshasayee, Aswin Sai Narain

    2014-01-01

    Evolution of bacteria under sublethal concentrations of antibiotics represents a trade-off between growth and resistance to the antibiotic. To understand this trade-off, we performed in vitro evolution of laboratory Escherichia coli under sublethal concentrations of the aminoglycoside kanamycin over short time durations. We report that fixation of less costly kanamycin-resistant mutants occurred earlier in populations growing at lower sublethal concentration of the antibiotic, compared with those growing at higher sublethal concentrations; in the latter, resistant mutants with a significant growth defect persisted longer. Using deep sequencing, we identified kanamycin resistance-conferring mutations, which were costly or not in terms of growth in the absence of the antibiotic. Multiple mutations in the C-terminal end of domain IV of the translation elongation factor EF-G provided low-cost resistance to kanamycin. Despite targeting the same or adjacent residues of the protein, these mutants differed from each other in the levels of resistance they provided. Analysis of one of these mutations showed that it has little defect in growth or in synthesis of green fluorescent protein (GFP) from an inducible plasmid in the absence of the antibiotic. A second class of mutations, recovered only during evolution in higher sublethal concentrations of the antibiotic, deleted the C-terminal end of the ATP synthase shaft. This mutation confers basal-level resistance to kanamycin while showing a strong growth defect in the absence of the antibiotic. In conclusion, the early dynamics of the development of resistance to an aminoglycoside antibiotic is dependent on the levels of stress (concentration) imposed by the antibiotic, with the evolution of less costly variants only a matter of time. PMID:25281544

  9. A case of aminoglycosides induced retinal toxicity treated with megadoses of steroids and an intravitreal dexamethasone implant (Ozurdex(®)).

    Science.gov (United States)

    Hernández Pardines, F; Tapia-Quijada, H; Hueso-Abancens, J R

    2016-06-01

    The case is described of a patient who had a sudden loss of vision in her right eye after glaucoma surgery. A diagnosis of retinal toxicity due to tobramycin (an aminoglycoside) was reached, which was characterised by retinal whitening with a red cherry stain, macular oedema, and vasculitis that progressed to papillary and macular atrophy with arteriolar sclerosis. Given the severity of symptoms an early attempt was made with megadoses of steroids and an intravitreal dexamethasone implant (Ozurdex®, Allergan S.A.), without response. Aminoglycoside toxicity is a rare, idiosyncratic, very serious complication for which there is no effective treatment. Copyright © 2016 Sociedad Española de Oftalmología. Published by Elsevier España, S.L.U. All rights reserved.

  10. Complexation of anionic copolymers of acrylamide and N-(2-hydroxypropyl)methacrylamide with aminoglycoside antibiotics

    Science.gov (United States)

    Solovskii, M. V.; Tarabukina, E. B.; Amirova, A. I.; Zakharova, N. V.; Smirnova, M. Yu.; Gavrilova, I. I.

    2014-03-01

    The complexation of aminoglycoside antibiotics neomycin, gentamicin, kanamycin, and amikacin in the form of free bases with carboxyl- and sulfo-containing copolymers of acrylamide and N-(2-hydroxypropyl)methacrylamide (HPMA) in water and water-salt solutions is studied by means of viscometry, equilibrium dialysis, potentiometric titration, and molecular hydrodynamics. Factors influencing the stability of formed copolymer-antibiotic complexes and determinations of their toxicity are established.

  11. Mutations in Mll2, an H3K4 Methyltransferase, Result in Insulin Resistance and Impaired Glucose Tolerance in Mice

    Science.gov (United States)

    Schröter, David; Matthews, Helen C.; Bogani, Debora; Moir, Lee; Long, Anna; Church, Christopher; Hugill, Alison; Anstee, Quentin M.; Goldin, Rob; Thursz, Mark; Hollfelder, Florian; Cox, Roger D.

    2013-01-01

    We employed a random mutagenesis approach to identify novel monogenic determinants of type 2 diabetes. Here we show that haplo-insufficiency of the histone methyltransferase myeloid-lineage leukemia (Mll2/Wbp7) gene causes type 2 diabetes in the mouse. We have shown that mice heterozygous for two separate mutations in the SET domain of Mll2 or heterozygous Mll2 knockout mice were hyperglycaemic, hyperinsulinaemic and developed non-alcoholic fatty liver disease. Consistent with previous Mll2 knockout studies, mice homozygous for either ENU mutation (or compound heterozygotes) died during embryonic development at 9.5–14.5 days post coitum. Heterozygous deletion of Mll2 induced in the adult mouse results in a normal phenotype suggesting that changes in chromatin methylation during development result in the adult phenotype. Mll2 has been shown to regulate a small subset of genes, a number of which Neurod1, Enpp1, Slc27a2, and Plcxd1 are downregulated in adult mutant mice. Our results demonstrate that histone H3K4 methyltransferase Mll2 is a component of the genetic regulation necessary for glucose homeostasis, resulting in a specific disease pattern linking chromatin modification with causes and progression of type 2 diabetes, providing a basis for its further understanding at the molecular level. PMID:23826075

  12. Mutations in Mll2, an H3K4 methyltransferase, result in insulin resistance and impaired glucose tolerance in mice.

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    Michelle Goldsworthy

    Full Text Available We employed a random mutagenesis approach to identify novel monogenic determinants of type 2 diabetes. Here we show that haplo-insufficiency of the histone methyltransferase myeloid-lineage leukemia (Mll2/Wbp7 gene causes type 2 diabetes in the mouse. We have shown that mice heterozygous for two separate mutations in the SET domain of Mll2 or heterozygous Mll2 knockout mice were hyperglycaemic, hyperinsulinaemic and developed non-alcoholic fatty liver disease. Consistent with previous Mll2 knockout studies, mice homozygous for either ENU mutation (or compound heterozygotes died during embryonic development at 9.5-14.5 days post coitum. Heterozygous deletion of Mll2 induced in the adult mouse results in a normal phenotype suggesting that changes in chromatin methylation during development result in the adult phenotype. Mll2 has been shown to regulate a small subset of genes, a number of which Neurod1, Enpp1, Slc27a2, and Plcxd1 are downregulated in adult mutant mice. Our results demonstrate that histone H3K4 methyltransferase Mll2 is a component of the genetic regulation necessary for glucose homeostasis, resulting in a specific disease pattern linking chromatin modification with causes and progression of type 2 diabetes, providing a basis for its further understanding at the molecular level.

  13. Geographic distribution of methyltransferases of Helicobacter pylori: evidence of human host population isolation and migration

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    Vítor Jorge MB

    2009-09-01

    Full Text Available Abstract Background Helicobacter pylori colonizes the human stomach and is associated with gastritis, peptic ulcer, and gastric cancer. This ubiquitous association between H. pylori and humans is thought to be present since the origin of modern humans. The H. pylori genome encodes for an exceptional number of restriction and modifications (R-M systems. To evaluate if R-M systems are an adequate tool to determine the geographic distribution of H. pylori strains, we typed 221 strains from Africa, America, Asia, and Europe, and evaluated the expression of different 29 methyltransferases. Results Independence tests and logistic regression models revealed that ten R-M systems correlate with geographical localization. The distribution pattern of these methyltransferases may have been originated by co-divergence of regional H. pylori after its human host migrated out of Africa. The expression of specific methyltransferases in the H. pylori population may also reflect the genetic and cultural background of its human host. Methyltransferases common to all strains, M. HhaI and M. NaeI, are likely conserved in H. pylori, and may have been present in the bacteria genome since the human diaspora out of Africa. Conclusion This study indicates that some methyltransferases are useful geomarkers, which allow discrimination of bacterial populations, and that can be added to our tools to investigate human migrations.

  14. Integrated metabolo-proteomic approach to decipher the mechanisms by which wheat QTL (Fhb1 contributes to resistance against Fusarium graminearum.

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    Raghavendra Gunnaiah

    Full Text Available BACKGROUND: Resistance in plants to pathogen attack can be qualitative or quantitative. For the latter, hundreds of quantitative trait loci (QTLs have been identified, but the mechanisms of resistance are largely unknown. Integrated non-target metabolomics and proteomics, using high resolution hybrid mass spectrometry, were applied to identify the mechanisms of resistance governed by the fusarium head blight resistance locus, Fhb1, in the near isogenic lines derived from wheat genotype Nyubai. FINDINGS: The metabolomic and proteomic profiles were compared between the near isogenic lines (NIL with resistant and susceptible alleles of Fhb1 upon F. graminearum or mock-inoculation. The resistance-related metabolites and proteins identified were mapped to metabolic pathways. Metabolites of the shunt phenylpropanoid pathway such as hydroxycinnamic acid amides, phenolic glucosides and flavonoids were induced only in the resistant NIL, or induced at higher abundances in resistant than in susceptible NIL, following pathogen inoculation. The identities of these metabolites were confirmed, with fragmentation patterns, using the high resolution LC-LTQ-Orbitrap. Concurrently, the enzymes of phenylpropanoid biosynthesis such as cinnamyl alcohol dehydrogenase, caffeoyl-CoA O-methyltransferase, caffeic acid O-methyltransferase, flavonoid O-methyltransferase, agmatine coumaroyltransferase and peroxidase were also up-regulated. Increased cell wall thickening due to deposition of hydroxycinnamic acid amides and flavonoids was confirmed by histo-chemical localization of the metabolites using confocal microscopy. CONCLUSION: The present study demonstrates that the resistance in Fhb1 derived from the wheat genotype Nyubai is mainly associated with cell wall thickening due to deposition of hydroxycinnamic acid amides, phenolic glucosides and flavonoids, but not with the conversion of deoxynivalenol to less toxic deoxynivalenol 3-O-glucoside.

  15. Antimicrobial resistance mechanisms among Campylobacter.

    Science.gov (United States)

    Wieczorek, Kinga; Osek, Jacek

    2013-01-01

    Campylobacter jejuni and Campylobacter coli are recognized as the most common causative agents of bacterial gastroenteritis in the world. Humans most often become infected by ingesting contaminated food, especially undercooked chicken, but also other sources of bacteria have been described. Campylobacteriosis is normally a self-limiting disease. Antimicrobial treatment is needed only in patients with more severe disease and in those who are immunologically compromised. The most common antimicrobial agents used in the treatment of Campylobacter infections are macrolides, such as erythromycin, and fluoroquinolones, such as ciprofloxacin. Tetracyclines have been suggested as an alternative choice in the treatment of clinical campylobacteriosis but in practice are not often used. However, during the past few decades an increasing number of resistant Campylobacter isolates have developed resistance to fluoroquinolones and other antimicrobials such as macrolides, aminoglycosides, and beta-lactams. Trends in antimicrobial resistance have shown a clear correlation between use of antibiotics in the veterinary medicine and animal production and resistant isolates of Campylobacter in humans. In this review, the patterns of emerging resistance to the antimicrobial agents useful in treatment of the disease are presented and the mechanisms of resistance to these drugs in Campylobacter are discussed.

  16. Antibiotic resistance profile of Pseudomonas aeruginosa isolated from aquaculture and abattoir environments in urban communities

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    Isoken Henrietta Igbinosa

    2017-01-01

    Full Text Available Objective: To characterize multiple antibiotic resistance profile of Pseudomonas aeruginosa from aquaculture and abattoir environments. Methods: Wastewater samples were obtained from the abattoir and aquaculture environments between May 2016 and July 2016 and analysed using standard phenotypic, biochemical and PCR-based methods. Results: The mean pseudomonads count ranged from (4 × 102 ± 1.01 to (2 × 104 ± 0.10 colony-forming unit/mL in the aquaculture environment and (3 × 103 ± 0.00 to (1 × 105 ± 1.00 colony-forming unit/mL in the abattoir environment. A total of 96 isolates of Pseudomonas aeruginosa confirmed by PCR were thereafter selected from both aquaculture and abattoir environments and further characterized for their antimicrobial susceptibility profile by adopting the disc diffusion method. High level of resistance was observed against the aminoglycosides [gentamycin 64/96 (66.67% and kanamycin 52/96 (54.17%], monobactams [aztreonam 76/96 (79.17%], carbapenems [meropenem 52/96 (54.17%], tetracyclines [tetracycline 72/96 (75.00%] and cephems [ceftazidime 72/96 (75.00% and cefuroxime 48/96 (50.00%]. Multiple antibiotic resistant index of the respective isolates ranged from 0.4 to 0.8 while multidrug resistant profile of the isolates revealed that 28 of the respective isolates were resistant to ceftazidime, cefuroxime, gentamycin, kanamycin, aztreonam which belongs to cephems, aminoglycosides and monobactam class of antimicrobials. Conclusions: Findings from the present study therefore underscores the need for effective monitoring of the abattoir and aquaculture environments as they could be the significant source for spreading antibiotic resistant bacteria within the environment.

  17. The genome-wide identification and transcriptional levels of DNA methyltransferases and demethylases in globe artichoke.

    Science.gov (United States)

    Gianoglio, Silvia; Moglia, Andrea; Acquadro, Alberto; Comino, Cinzia; Portis, Ezio

    2017-01-01

    Changes to the cytosine methylation status of DNA, driven by the activity of C5 methyltransferases (C5-MTases) and demethylases, exert an important influence over development, transposon movement, gene expression and imprinting. Three groups of C5-MTase enzymes have been identified in plants, namely MET (methyltransferase 1), CMT (chromomethyltransferases) and DRM (domains rearranged methyltransferases). Here the repertoire of genes encoding C5-MTase and demethylase by the globe artichoke (Cynara cardunculus var. scolymus) is described, based on sequence homology, a phylogenetic analysis and a characterization of their functional domains. A total of ten genes encoding C5-MTase (one MET, five CMTs and four DRMs) and five demethylases was identified. An analysis of their predicted product's protein structure suggested an extensive level of conservation has been retained by the C5-MTases. Transcriptional profiling based on quantitative real time PCR revealed a number of differences between the genes encoding maintenance and de novo methyltransferases, sometimes in a tissue- or development-dependent manner, which implied a degree of functional specialization.

  18. The genome-wide identification and transcriptional levels of DNA methyltransferases and demethylases in globe artichoke.

    Directory of Open Access Journals (Sweden)

    Silvia Gianoglio

    Full Text Available Changes to the cytosine methylation status of DNA, driven by the activity of C5 methyltransferases (C5-MTases and demethylases, exert an important influence over development, transposon movement, gene expression and imprinting. Three groups of C5-MTase enzymes have been identified in plants, namely MET (methyltransferase 1, CMT (chromomethyltransferases and DRM (domains rearranged methyltransferases. Here the repertoire of genes encoding C5-MTase and demethylase by the globe artichoke (Cynara cardunculus var. scolymus is described, based on sequence homology, a phylogenetic analysis and a characterization of their functional domains. A total of ten genes encoding C5-MTase (one MET, five CMTs and four DRMs and five demethylases was identified. An analysis of their predicted product's protein structure suggested an extensive level of conservation has been retained by the C5-MTases. Transcriptional profiling based on quantitative real time PCR revealed a number of differences between the genes encoding maintenance and de novo methyltransferases, sometimes in a tissue- or development-dependent manner, which implied a degree of functional specialization.

  19. Nicotinamide -Methyltransferase in Health and Cancer

    Directory of Open Access Journals (Sweden)

    David B Ramsden

    2017-06-01

    Full Text Available Over the past decade, the roles of nicotinamide N -methyltransferase and its product 1-methyl nicotinamide have emerged from playing merely minor roles in phase 2 xenobiotic metabolism as actors in some of the most important scenes of human life. In this review, the structures of the gene, messenger RNA, and protein are discussed, together with the role of the enzyme in many of the common cancers that afflict people today.

  20. A Rapid and Efficient Assay for the Characterization of Substrates and Inhibitors of Nicotinamide N-Methyltransferase

    NARCIS (Netherlands)

    van Haren, Matthijs J; Sastre Torano, Javier; Sartini, Davide; Emanuelli, Monica; Parsons, Richard B; Martin, Nathaniel I

    2016-01-01

    Nicotinamide N-methyltransferase (NNMT) is one of the most abundant small molecule methyltransferases in the human body and is primarily responsible for the N-methylation of the nicotinamide (vitamin B3). Employing the cofactor S-adenosyl-l-methionine, NNMT transfers a methyl group to the pyridine

  1. Antibiotic resistance patterns of pediatric community-acquired urinary infections

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    Eliana Biondi Medeiros Guidoni

    Full Text Available Knowledge about antimicrobial resistance patterns of the etiological agents of urinary tract infections (UTIs is essential for appropriate therapy. Urinary isolates from symptomatic UTI cases attended at Santa Casa University Hospital of São Paulo from August 1986 to December 1989 and August 2004 to December 2005 were identified by conventional methods. Antimicrobial resistance testing was performed by Kirby Bauer's disc diffusion method. Among the 257 children, E. coli was found in 77%. A high prevalence of resistance was observed against ampicillin and TMP/SMX (55% and 51%. The antibiotic resistance rates for E. coli were: nitrofurantoin (6%, nalidixic acid (14%, 1st generation cephalosporin (13%, 3rd generation cephalosporins (5%, aminoglycosides (2%, norfloxacin (9% and ciprofloxacin (4%. We found that E. coli was the predominant bacterial pathogen of community-acquired UTIs. We also detected increasing resistance to TMP/SMX among UTI pathogens in this population.

  2. Molecular Basis for the Regulation of the H3K4 Methyltransferase Activity of PRDM9

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    Hong Wu

    2013-10-01

    Full Text Available PRDM9, a histone lysine methyltransferase, is a key determinant of the localization of meiotic recombination hot spots in humans and mice and the only vertebrate protein known to be involved in hybrid sterility. Here, we report the crystal structure of the PRDM9 methyltransferase domain in complex with a histone H3 peptide dimethylated on lysine 4 (H3K4me2 and S-adenosylhomocysteine (AdoHcy, which provides insights into the methyltransferase activity of PRDM proteins. We show that the genuine substrate of PRDM9 is histone H3 lysine 4 (H3K4 and that the enzyme possesses mono-, di-, and trimethylation activities. We also determined the crystal structure of PRDM9 in its autoinhibited state, which revealed a rearrangement of the substrate and cofactor binding sites by a concerted action of the pre-SET and post-SET domains, providing important insights into the regulatory mechanisms of histone lysine methyltransferase activity.

  3. An engineered split M.HhaI-zinc finger fusion lacks the intended methyltransferase specificity

    International Nuclear Information System (INIS)

    Meister, Glenna E.; Chandrasegaran, Srinivasan; Ostermeier, Marc

    2008-01-01

    The ability to site-specifically methylate DNA in vivo would have wide applicability to the study of basic biomedical problems as well as enable studies on the potential of site-specific DNA methylation as a therapeutic strategy for the treatment of diseases. Natural DNA methyltransferases lack the specificity required for these applications. Nomura and Barbas [W. Nomura, C.F. Barbas 3rd, In vivo site-specific DNA methylation with a designed sequence-enabled DNA methylase, J. Am. Chem. Soc. 129 (2007) 8676-8677] have reported that an engineered DNA methyltransferase comprised of fragments of M.HhaI methyltransferase and zinc finger proteins has very high specificity for the chosen target site. Our analysis of this engineered enzyme shows that the fusion protein methylates target and non-target sites with similar efficiency

  4. Increased susceptibility to chemotherapeutic alkylating agents of mice deficient in DNA repair methyltransferase.

    Science.gov (United States)

    Shiraishi, A; Sakumi, K; Sekiguchi, M

    2000-10-01

    O(6)-methylguanine-DNA methyltransferase plays vital roles in preventing induction of mutations and cancer as well as cell death related to alkylating agents. Mice defective in the MGMT: gene, encoding the methyltransferase, were used to evaluate cell death-inducing and tumorigenic activities of therapeutic agents which have alkylation potential. MGMT(-/-) mice were considerably more sensitive to dacarbazine, a monofunctional triazene, than were wild-type mice, in terms of survival. When dacarbazine was administered i.p. to 6-week-old mice and survival at 30 days was enumerated, LD(50) values of MGMT(-/-) and MGMT(+/+) mice were 20 and 450 mg/kg body wt, respectively. Increased sensitivity of MGMT(-/-) mice to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosou rea (ACNU), a bifunctional nitrosourea, was also noted. On the other hand, there was no difference in survival of MGMT(+/+) and MGMT(-/-) mice exposed to cyclophosphamide, a bifunctional nitrogen mustard. It appears that dacarbazine and ACNU produce O(6)-alkylguanine as a major toxic lesion, while cyclophosphamide yields other types of modifications in DNA which are not subjected to the action of the methyltransferase. MGMT(-/-) mice seem to be less refractory to the tumor-inducing effect of dacarbazine than are MGMT(+/+) mice. Thus, the level of O(6)-methylguanine-DNA methyltransferase activity is an important factor when determining susceptibility to drugs with the potential for alkylation.

  5. Two-dimensional solid-phase extraction strategy for the selective enrichment of aminoglycosides in milk.

    Science.gov (United States)

    Shen, Aijin; Wei, Jie; Yan, Jingyu; Jin, Gaowa; Ding, Junjie; Yang, Bingcheng; Guo, Zhimou; Zhang, Feifang; Liang, Xinmiao

    2017-03-01

    An orthogonal two-dimensional solid-phase extraction strategy was established for the selective enrichment of three aminoglycosides including spectinomycin, streptomycin, and dihydrostreptomycin in milk. A reversed-phase liquid chromatography material (C 18 ) and a weak cation-exchange material (TGA) were integrated in a single solid-phase extraction cartridge. The feasibility of two-dimensional clean-up procedure that experienced two-step adsorption, two-step rinsing, and two-step elution was systematically investigated. Based on the orthogonality of reversed-phase and weak cation-exchange procedures, the two-dimensional solid-phase extraction strategy could minimize the interference from the hydrophobic matrix existing in traditional reversed-phase solid-phase extraction. In addition, high ionic strength in the extracts could be effectively removed before the second dimension of weak cation-exchange solid-phase extraction. Combined with liquid chromatography and tandem mass spectrometry, the optimized procedure was validated according to the European Union Commission directive 2002/657/EC. A good performance was achieved in terms of linearity, recovery, precision, decision limit, and detection capability in milk. Finally, the optimized two-dimensional clean-up procedure incorporated with liquid chromatography and tandem mass spectrometry was successfully applied to the rapid monitoring of aminoglycoside residues in milk. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Complete genome sequence analysis of the fish pathogen Flavobacterium columnare provides insights into antibiotic resistance and pathogenicity related genes.

    Science.gov (United States)

    Zhang, Yulei; Zhao, Lijuan; Chen, Wenjie; Huang, Yunmao; Yang, Ling; Sarathbabu, V; Wu, Zaohe; Li, Jun; Nie, Pin; Lin, Li

    2017-10-01

    We analyzed here the complete genome sequences of a highly virulent Flavobacterium columnare Pf1 strain isolated in our laboratory. The complete genome consists of a 3,171,081 bp circular DNA with 2784 predicted protein-coding genes. Among these, 286 genes were predicted as antibiotic resistance genes, including 32 RND-type efflux pump related genes which were associated with the export of aminoglycosides, indicating inducible aminoglycosides resistances in F. columnare. On the other hand, 328 genes were predicted as pathogenicity related genes which could be classified as virulence factors, gliding motility proteins, adhesins, and many putative secreted proteases. These genes were probably involved in the colonization, invasion and destruction of fish tissues during the infection of F. columnare. Apparently, our obtained complete genome sequences provide the basis for the explanation of the interactions between the F. columnare and the infected fish. The predicted antibiotic resistance and pathogenicity related genes will shed a new light on the development of more efficient preventional strategies against the infection of F. columnare, which is a major worldwide fish pathogen. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Antibiotic sensitivity and resistance in children with urinary tract infection in Sanliurfa.

    Science.gov (United States)

    Abuhandan, Mahmut; Güzel, Bülent; Oymak, Yeşim; Çiftçi, Halil

    2013-06-01

    This study aimed to evaluate antibiotic resistance in the province of Şanliurfa and to observe any difference between antibiotic resistance rates. The study comprised 107 children who presented at the pediatric polyclinic with complaints of urinary tract infection with the diagnosis of urinary tract infection and whose urine cultures exhibited bacterial growth. The patients were analyzed with respect to the frequency of proliferating pathogens, sensitivity to the antibiotics used and the rates of developed resistance to the antibiotics. A total of 107 patients aged between 1 year and 15 years were included in the study, encompassing 14 (13.1%) males and 93 (86.9%) females. According to the urine culture results, proliferation of Escherichia coli (E. coli) was observed in 69 (64.5%), Klebsiella spp. in 13 (12.1%), Proteus mirabilis in 9 (8.4%), Staphylococcus aureus in 5 (4.7%), Pseudomonas aeruginosa in 5 (4.7%), Acinetobacter spp. in 3 (2.8%) and Enterococcus spp. in 3 (2.8%) patients. For proliferating E. coli, high resistance rates to ceftriaxone (39.5%), nitrofurantoin (19.7%), ampicillin-sulbactam (64.1%), co-trimoxazole (41.5%), amoxicillinclavulanate (51.7%) and cefuroxime (38.1%) were observed. All of isolated microorganisms were resistant to ampicillin-sulbactam, amoxicillin-clavulanate, co-trimoxazole, ceftriaxone, cefuroxime and cefoxitin in decreasing frequencies. The most effective antimicrobial agents were determined to be imipenem, sulpera-zone, quinolone and aminoglycosides. In our region, parenteral antibiotics that should be selected for the empirical treatment of UTIs in all age groups are the aminoglycosides and 3(rd) generation cephalosporines. In contrast to other studies, these results suggest that co-trimoxazole should be used for children aged 0-1, and 2(nd) generation cephalosporins should be used for the oral treatment of children aged 1-5 due to the low rate of resistance to nitrofurantoin in patients aged over 5 years.

  8. In Vitro Synergistic Effects of Double and Triple Combinations of β-Lactams, Vancomycin, and Netilmicin against Methicillin-Resistant Staphylococcus aureus Strains

    Science.gov (United States)

    Rochon-Edouard, Stéphanie; Pestel-Caron, Martine; Lemeland, Jean-François; Caron, François

    2000-01-01

    Several studies have previously reported synergistic effects between vancomycin and a given β-lactam or a given aminoglycoside against methicillin-resistant Staphylococcus aureus (MRSA) strains. The aim of our study was to exhaustively compare the effects of different combinations of a β-lactam, vancomycin, and/or an aminoglycoside against 32 clinical MRSA strains with different aminoglycoside susceptibility patterns. The effects of 26 different β-lactam–vancomycin and 8 different aminoglycoside-vancomycin combinations were first studied using a disk diffusion screening method. The best interactions with vancomycin were observed with either imipenem, cefazolin, or netilmicin. By checkerboard studies, imipenem-vancomycin and cefazolin-vancomycin each provided a synergistic bacteriostatic effect against 22 strains; the mean fractional inhibitory concentration (FIC) indexes were 0.35 and 0.46 for imipenem-vancomycin and cefazolin-vancomycin, respectively. The vancomycin-netilmicin combination provided an indifferent effect against all of the 32 strains tested; the mean of FIC index was 1.096. The mean concentrations of imipenem, cefazolin, netilmicin, and vancomycin at which FIC indexes were calculated were clinically achievable. Killing experiments were then performed using imipenem, cefazolin, netilmicin, and vancomycin at one-half of the MIC, alone and in different combinations, against 10 strains. The vancomycin-netilmicin regimen was rarely bactericidal, even against strains susceptible to netilmicin. The imipenem-vancomycin and cefazolin-vancomycin combinations were strongly bactericidal against six and five strains, respectively. The addition of netilmicin markedly enhanced the killing activity of the combination of cefazolin or imipenem plus vancomycin, but only for the MRSA strains against which the β-lactam–vancomycin combinations had no bactericidal effect. It is noteworthy that the latter strains were both susceptible to netilmicin and

  9. Identification of Aquifex aeolicus tRNA (m2(2G26) methyltransferase gene.

    Science.gov (United States)

    Takeda, Hiroshi; Hori, Hiroyuki; Endo, Yaeta

    2002-01-01

    The modifications of N2,N2-dimethylguanine (m2(2)G) are found in tRNAs and rRNAs from eukarya and archaea. In tRNAs, modification at position G26 is generated by tRNA (m2(2)G26) methyltransferase, which is encoded by the corresponding gene, trm1. This enzyme catalyzes the methyl-transfer from S-adenosyl-L-methionine to the semi-conserved residue, G26, via the intermediate modified base, m2G26. Recent genome sequencing project has been reported that the putative trm1 is encoded in the genome of Aquifex aeolicus, a hyper-thermophilic eubacterium as only one exception among eubacteria. In order to confirm whether this bacterial trm1 gene product is a real tRNA (m2(2)G26) methyltransferase or not, we expressed this protein by wheat germ in vitro cell-free translation system. Our biochemical analysis clearly showed that this gene product possessed tRNA (m2(2)G26) methyltransferase activity.

  10. Resistance to K. pneumoniae in young children with congenital heart defects

    Directory of Open Access Journals (Sweden)

    V. N. Ilina

    2015-10-01

    Full Text Available Klebsiella pneumoniae is one of the leading agents of nosocomial infections (NI. In Russia, Klebsiella pneumoniae is the third in frequency of gram-negative pathogen NI. For a long time one of the major clinically relevant mechanisms of acquired resistance to K. pneumoniae is multidrug resistance caused by extended spectrum -lactamase production (ESBL. Carbapenems show the greatest resistance to the action of ESB. However, now there exist registered strains of K.pneumoniae resistant to carbapenems. In connection with this in 2008 we conducted a prospective study on resistance to K. pneumoniae in young children being treated at ICU. It was found out that resistance to III-IV-generation cephalosporines, fluoroquinolones, aminoglycosides is determined by production of ESBL, while resistance to carbapenems occurs due to reduction of permeability of cell membranes, in combination with production of ESBL. Some features of patients colonized with multidrug-resistant strains of K. pneumoniae are described.

  11. Human Health Hazards from Antimicrobial-Resistant Escherichia coli of Animal Origin

    DEFF Research Database (Denmark)

    Hammerum, A. M.; Heuer, Ole Eske

    2009-01-01

    of antimicrobial agents in food animals may add to the burden of antimicrobial resistance in humans. Bacteria from the animal reservoir that carry resistance to antimicrobial agents that are regarded as highly or critically important in human therapy (e.g., aminoglycosides, fluoroquinolones, and third- and fourth......Because of the intensive use of antimicrobial agents in food animal production, meat is frequently contaminated with antimicrobial-resistant Escherichia coli. Humans can be colonized with E. coli of animal origin, and because of resistance to commonly used antimicrobial agents, these bacteria may...... cause infections for which limited therapeutic options are available. This may lead to treatment failure and can have serious consequences for the patient. Furthermore, E. coli of animal origin may act as a donor of antimicrobial resistance genes for other pathogenic E. coli. Thus, the intensive use...

  12. The activity of aminoglycoside antibiotics against Trypanosoma brucei.

    Science.gov (United States)

    Maina, N W; Kinyanjui, B; Onyango, J D; Auma, J E; Croj, S

    1998-01-01

    The trypanocidal activity of four aminoglycosides was determined against Trypanosoma brucei in vitro. The drug activity in descending order, was as follows; paromomycin kanamycin>gentamycin > neomycin. Paromomycin bad the highest activity and the concentration that inhibited 50% of trypanosome growth (IC50) was 11.4microM. The effect of paromomycin on the causative agents of the East African form of sleeping sickness - T.b. rhodesiense KETRI 265, 2285, 2545, 2562 and EATRO 110,112, 1152 was subsequently assessed. Variations sensitivities between the trypanosome populations were observed and IC50 values ranging from 13.01 to 43.06 microM recorded. However, when paromomycin was administered intraperitoneally (i.p) at 500 mg/kg, it was not effective in curing mice infected with T. b. rhodesienseKETRI 2545 the most drug-sensitive isolate in vitro. Lack of in vivo activity may be because the trypanosome is an extracellular parasite. The pharmacokinetics of paromomycin in the mouse model need to be determined.

  13. The Order Bacillales Hosts Functional Homologs of the Worrisome cfr Antibiotic Resistance Gene

    DEFF Research Database (Denmark)

    Hansen, Lykke H.; Planellas, Mercè H.; Long, Katherine S.

    2012-01-01

    The cfr gene encodes the Cfr methyltransferase that methylates a single adenine in the peptidyl transferase region of bacterial ribosomes. The methylation provides resistance to several classes of antibiotics that include drugs of clinical and veterinary importance. This paper describes a first...

  14. Biosynthesis of estragole and methyl-eugenol in sweet basil (Ocimum basilicum L). Developmental and chemotypic association of allylphenol O-methyltransferase activities.

    Science.gov (United States)

    Lewinsohn, E; Ziv-Raz, I; Dudai, N; Tadmor, Y; Lastochkin, E; Larkov, O; Chaimovitsh, D; Ravid, U; Putievsky, E; Pichersky, E; Shoham, Y

    2000-12-07

    Sweet basil (Ocimum basilicum L., Lamiaceae) is a common herb, used for culinary and medicinal purposes. The essential oils of different sweet basil chemotypes contain various proportions of the allyl phenol derivatives estragole (methyl chavicol), eugenol, and methyl eugenol, as well as the monoterpene alcohol linalool. To monitor the developmental regulation of estragole biosynthesis in sweet basil, an enzymatic assay for S-adenosyl-L-methionine (SAM):chavicol O-methyltransferase activity was developed. Young leaves display high levels of chavicol O-methyltransferase activity, but the activity was negligible in older leaves, indicating that the O-methylation of chavicol primarily occurs early during leaf development. The O-methyltransferase activities detected in different sweet basil genotypes differed in their substrate specificities towards the methyl acceptor substrate. In the high-estragole-containing chemotype R3, the O-methyltransferase activity was highly specific for chavicol, while eugenol was virtually not O-methylated. In contrast, chemotype 147/97, that contains equal levels of estragole and methyl eugenol, displayed O-methyltransferase activities that accepted both chavicol and eugenol as substrates, generating estragole and methyl eugenol, respectively. Chemotype SW that contains high levels of eugenol, but lacks both estragole and methyl eugenol, had apparently no allylphenol dependent O-methyltransferase activities. These results indicate the presence of at least two types of allylphenol-specific O-methyltransferase activities in sweet basil chemotypes, one highly specific for chavicol; and a different one that can accept eugenol as a substrate. The relative availability and substrate specificities of these O-methyltransferase activities biochemically rationalizes the variation in the composition of the essential oils of these chemotypes.

  15. Crystal structure of MboIIA methyltransferase

    OpenAIRE

    Osipiuk, Jerzy; Walsh, Martin A.; Joachimiak, Andrzej

    2003-01-01

    DNA methyltransferases (MTases) are sequence-specific enzymes which transfer a methyl group from S-adenosyl-l-methionine (AdoMet) to the amino group of either cytosine or adenine within a recognized DNA sequence. Methylation of a base in a specific DNA sequence protects DNA from nucleolytic cleavage by restriction enzymes recognizing the same DNA sequence. We have determined at 1.74 Å resolution the crystal structure of a β-class DNA MTase MboIIA (M·MboIIA) from the bacterium Moraxella bovis,...

  16. Preliminary X-ray analysis of twinned crystals of sarcosine dimethylglycine methyltransferase from Halorhodospira halochoris

    International Nuclear Information System (INIS)

    Kallio, Juha Pekka; Jänis, Janne; Nyyssölä, Antti; Hakulinen, Nina; Rouvinen, Juha

    2009-01-01

    The crystallization and preliminary X-ray diffraction analysis of sarcosine dimethylglycine methyltransferase from H. halochoris is reported. Sarcosine dimethylglycine methyltransferase (EC 2.1.1.157) is an enzyme from the extremely halophilic anaerobic bacterium Halorhodospira halochoris. This enzyme catalyzes the twofold methylation of sarcosine to betaine, with S-adenosylmethionine (AdoMet) as the methyl-group donor. This study presents the crystallization and preliminary X-ray analysis of recombinant sarcosine dimethylglycine methyltransferase produced in Escherichia coli. Mass spectroscopy was used to determine the purity and homogeneity of the enzyme material. Two different crystal forms, which initially appeared to be hexagonal and tetragonal, were obtained. However, on analyzing the diffraction data it was discovered that both crystal forms were pseudo-merohedrally twinned. The true crystal systems were monoclinic and orthorhombic. The monoclinic crystal diffracted to a maximum of 2.15 Å resolution and the orthorhombic crystal diffracted to 1.8 Å resolution

  17. A glutamate/aspartate switch controls product specificity in a protein arginine methyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Debler, Erik W.; Jain, Kanishk; Warmack, Rebeccah A.; Feng, You; Clarke, Steven G.; Blobel, Günter; Stavropoulos, Pete

    2016-02-08

    Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-L-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs.

  18. Targeting MLL1 H3K4 methyltransferase activity in mixed-lineage leukemia.

    Science.gov (United States)

    Cao, Fang; Townsend, Elizabeth C; Karatas, Hacer; Xu, Jing; Li, Li; Lee, Shirley; Liu, Liu; Chen, Yong; Ouillette, Peter; Zhu, Jidong; Hess, Jay L; Atadja, Peter; Lei, Ming; Qin, Zhaohui S; Malek, Sami; Wang, Shaomeng; Dou, Yali

    2014-01-23

    Here we report a comprehensive characterization of our recently developed inhibitor MM-401 that targets the MLL1 H3K4 methyltransferase activity. MM-401 is able to specifically inhibit MLL1 activity by blocking MLL1-WDR5 interaction and thus the complex assembly. This targeting strategy does not affect other mixed-lineage leukemia (MLL) family histone methyltransferases (HMTs), revealing a unique regulatory feature for the MLL1 complex. Using MM-401 and its enantiomer control MM-NC-401, we show that inhibiting MLL1 methyltransferase activity specifically blocks proliferation of MLL cells by inducing cell-cycle arrest, apoptosis, and myeloid differentiation without general toxicity to normal bone marrow cells or non-MLL cells. More importantly, transcriptome analyses show that MM-401 induces changes in gene expression similar to those of MLL1 deletion, supporting a predominant role of MLL1 activity in regulating MLL1-dependent leukemia transcription program. We envision broad applications for MM-401 in basic and translational research. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. The diversity of antimicrobial resistance genes among staphylococci of animal origin.

    Science.gov (United States)

    Wendlandt, Sarah; Feßler, Andrea T; Monecke, Stefan; Ehricht, Ralf; Schwarz, Stefan; Kadlec, Kristina

    2013-08-01

    Staphylococci of animal origin harbor a wide variety of resistance genes. So far, more than 40 different resistance genes have been identified in staphylococci from animals. This includes genes that confer resistance to virtually all classes of antimicrobial agents approved for use in animals, such as penicillins, cephalosporins, tetracyclines, macrolides, lincosamides, phenicols, aminoglycosides, aminocyclitols, pleuromutilins, and diaminopyrimidines. The gene products of some of these resistance genes confer resistance to only specific members of a class of antimicrobial agents, whereas others confer resistance to the entire class or even to members of different classes of antimicrobial agents. The resistance mechanisms specified by the resistance genes fall into three major categories: (i) enzymatic inactivation, (ii) active efflux, or (iii) protection/modification/replacement of the cellular target sites of the antimicrobial agents. Mobile genetic elements, in particular plasmids and transposons, play a major role as carriers of antimicrobial resistance genes in animal staphylococci. They facilitate the exchange of resistance genes with staphylococci of human origin but also with other Gram-positive bacteria. Copyright © 2013 Elsevier GmbH. All rights reserved.

  20. Arabidopsis DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity

    International Nuclear Information System (INIS)

    Song, Yuan; Wu, Keqiang; Dhaubhadel, Sangeeta; An, Lizhe; Tian, Lining

    2010-01-01

    DNA methyltransferase2 (DNMT2) is always deemed to be enigmatic, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. Here we show that Arabidopsis DNA methyltransferase2 (AtDNMT2) is localized in nucleus and associates with histone deacetylation. Bimolecular fluorescence complementation and pull-down assays show AtDNMT2 interacts with type-2 histone deacetylases (AtHD2s), a unique type of histone deacetylase family in plants. Through analyzing the expression of AtDNMT2: ss-glucuronidase (GUS) fusion protein, we demonstrate that AtDNMT2 has the ability to repress gene expression at transcription level. Meanwhile, the expression of AtDNMT2 gene is altered in athd2c mutant plants. We propose that AtDNMT2 possibly involves in the activity of histone deacetylation and plant epigenetic regulatory network.

  1. Arabidopsis DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity

    Energy Technology Data Exchange (ETDEWEB)

    Song, Yuan [Key Laboratory of Arid and Grassland Agroecology, Ministry of Education, School of Life Science, Lanzhou University, Lanzhou 730000 (China); Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada); Wu, Keqiang [Institute of Plant Biology, National Taiwan University, Taipei 106, Taiwan (China); Dhaubhadel, Sangeeta [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada); An, Lizhe, E-mail: lizhean@lzu.edu.cn [Key Laboratory of Arid and Grassland Agroecology, Ministry of Education, School of Life Science, Lanzhou University, Lanzhou 730000 (China); Tian, Lining, E-mail: tianl@agr.gc.ca [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada)

    2010-05-28

    DNA methyltransferase2 (DNMT2) is always deemed to be enigmatic, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. Here we show that Arabidopsis DNA methyltransferase2 (AtDNMT2) is localized in nucleus and associates with histone deacetylation. Bimolecular fluorescence complementation and pull-down assays show AtDNMT2 interacts with type-2 histone deacetylases (AtHD2s), a unique type of histone deacetylase family in plants. Through analyzing the expression of AtDNMT2: ss-glucuronidase (GUS) fusion protein, we demonstrate that AtDNMT2 has the ability to repress gene expression at transcription level. Meanwhile, the expression of AtDNMT2 gene is altered in athd2c mutant plants. We propose that AtDNMT2 possibly involves in the activity of histone deacetylation and plant epigenetic regulatory network.

  2. Antibiotic resistance pattern in uropathogens

    Directory of Open Access Journals (Sweden)

    Gupta V

    2002-01-01

    Full Text Available Uropathogenic strains from inpatient and outpatient departments were studied from April 1997 to March 1999 for their susceptibility profiles. The various isolates were Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Acinetobacter baumanii and Enterococcus faecalis. Antibiotic susceptibility pattern of these isolates revealed that for outpatients, first generation cephalosporins, nitrofurantoin, norfloxacin/ciprofloxacin were effective for treatment of urinary tract infection but for inpatients, parenteral therapy with newer aminoglycosides and third generation cephalosporins need to be advocated as the organisms for nosocomial UTI exhibit a high degree of drug resistance. Trimethoprim and sulphamethoxazole combination was not found to be effective for the treatment of urinary tract infections as all the uropathogens from inpatients and outpatients showed high degree of resistance to co-trimoxazole. Culture and sensitivity of the isolates from urine samples should be done as a routine before advocating the therapy.

  3. Mechanisms of antibiotic resistance in Staphylococcus aureus.

    Science.gov (United States)

    Pantosti, Annalisa; Sanchini, Andrea; Monaco, Monica

    2007-06-01

    Staphylococcus aureus can exemplify better than any other human pathogen the adaptive evolution of bacteria in the antibiotic era, as it has demonstrated a unique ability to quickly respond to each new antibiotic with the development of a resistance mechanism, starting with penicillin and methicillin, until the most recent, linezolid and daptomycin. Resistance mechanisms include enzymatic inactivation of the antibiotic (penicillinase and aminoglycoside-modification enzymes), alteration of the target with decreased affinity for the antibiotic (notable examples being penicillin-binding protein 2a of methicillin-resistant S. aureus and D-Ala-D-Lac of peptidoglycan precursors of vancomycin-resistant strains), trapping of the antibiotic (for vancomycin and possibly daptomycin) and efflux pumps (fluoroquinolones and tetracycline). Complex genetic arrays (staphylococcal chromosomal cassette mec elements or the vanA operon) have been acquired by S. aureus through horizontal gene transfer, while resistance to other antibiotics, including some of the most recent ones (e.g., fluoroquinolones, linezolid and daptomycin) have developed through spontaneous mutations and positive selection. Detection of the resistance mechanisms and their genetic basis is an important support to antibiotic susceptibility surveillance in S. aureus.

  4. Association between antimicrobial resistance in Escherichia coli isolates from food animals and blood stream isolates from humans in Europe: an ecological study.

    Science.gov (United States)

    Vieira, Antonio R; Collignon, Peter; Aarestrup, Frank M; McEwen, Scott A; Hendriksen, Rene S; Hald, Tine; Wegener, Henrik C

    2011-12-01

    In addition to medical antimicrobial usage, the use of antimicrobials in food animals contributes to the occurrence of resistance among some bacterial species isolated from infections in humans. Recently, several studies have indicated that a large proportion of Escherichia coli causing infections in humans, especially those resistant to antimicrobials, have an animal origin. We analyzed the correlation between the prevalence of antimicrobial resistance in E. coli isolates from blood stream infections in humans and in E. coli isolates from poultry, pigs, and cattle between 2005 and 2008 for 11 countries, using available surveillance data. We also assessed the correlation between human antimicrobial usage and the occurrence of resistance in E. coli isolates from blood stream infections. Strong and significant correlations between prevalences of resistance to ampicillin (r=0.94), aminoglycosides (r=0.72), third-generation cephalosporins (r=0.76), and fluoroquinolones (r=0.68) were observed for human and poultry E. coli isolates. Similar significant correlations were observed for ampicillin (r=0.91), aminoglycosides (r=0.73), and fluoroquinolone resistance (r=0.74) in pig and human isolates. In cattle isolates, only ampicillin resistance (r=0.72) was significantly correlated to human isolates. When usage of antimicrobials in humans was analyzed with antimicrobial resistance among human isolates, only correlations between fluoroquinolones (r=0.90) and third-generation cephalosporins (r=0.75) were significant. Resistance in E. coli isolates from food animals (especially poultry and pigs) was highly correlated with resistance in isolates from humans. This supports the hypothesis that a large proportion of resistant E. coli isolates causing blood stream infections in people may be derived from food sources.

  5. Streptococcus suis, an emerging drug-resistant animal and human pathogen

    Directory of Open Access Journals (Sweden)

    Claudio ePalmieri

    2011-11-01

    Full Text Available Streptococcus suis, a major porcine pathogen, has been receiving growing attention not only for its role in severe and increasingly reported infections in humans, but also for its involvement in drug resistance. Recent studies and the analysis of sequenced genomes have been providing important insights into the S. suis resistome, and have resulted in the identification of resistance determinants for tetracyclines, macrolides, aminoglycosides, chloramphenicol, antifolate drugs, streptothricin, and cadmium salts. Resistance gene-carrying genetic elements described so far include integrative and conjugative elements, transposons, genomic islands, phages, and chimeric elements. Some of these elements are similar to those reported in major streptococcal pathogens such as Streptococcus pyogenes, Streptococcus pneumoniae, and Streptococcus agalactiae and share the same chromosomal insertion sites. The available information strongly suggests that S. suis is an important antibiotic resistance reservoir that can contribute to the spread of resistance genes to the above-mentioned streptococci. S. suis is thus a paradigmatic example of possible intersections between animal and human resistomes.

  6. Antibiotic resistance in Escherichia coli strains isolated from Antarctic bird feces, water from inside a wastewater treatment plant, and seawater samples collected in the Antarctic Treaty area

    Science.gov (United States)

    Rabbia, Virginia; Bello-Toledo, Helia; Jiménez, Sebastián; Quezada, Mario; Domínguez, Mariana; Vergara, Luis; Gómez-Fuentes, Claudio; Calisto-Ulloa, Nancy; González-Acuña, Daniel; López, Juana; González-Rocha, Gerardo

    2016-06-01

    Antibiotic resistance is a problem of global concern and is frequently associated with human activity. Studying antibiotic resistance in bacteria isolated from pristine environments, such as Antarctica, extends our understanding of these fragile ecosystems. Escherichia coli strains, important fecal indicator bacteria, were isolated on the Fildes Peninsula (which has the strongest human influence in Antarctica), from seawater, bird droppings, and water samples from inside a local wastewater treatment plant. The strains were subjected to molecular typing with pulsed-field gel electrophoresis to determine their genetic relationships, and tested for antibiotic susceptibility with disk diffusion tests for several antibiotic families: β-lactams, quinolones, aminoglycosides, tetracyclines, phenicols, and trimethoprim-sulfonamide. The highest E. coli count in seawater samples was 2400 cfu/100 mL. Only strains isolated from seawater and the wastewater treatment plant showed any genetic relatedness between groups. Strains of both these groups were resistant to β-lactams, aminoglycosides, tetracycline, and trimethoprim-sulfonamide.In contrast, strains from bird feces were susceptible to all the antibiotics tested. We conclude that naturally occurring antibiotic resistance in E. coli strains isolated from Antarctic bird feces is rare and the bacterial antibiotic resistance found in seawater is probably associated with discharged treated wastewater originating from Fildes Peninsula treatment plants.

  7. Arsenic (+3 oxidation state) methyltransferase and the inorganic arsenic methylation phenotype

    International Nuclear Information System (INIS)

    Li Jiaxin; Waters, Stephen B.; Drobna, Zuzana; Devesa, Vicenta; Styblo, Miroslav; Thomas, David J.

    2005-01-01

    Inorganic arsenic is enzymatically methylated; hence, its ingestion results in exposure to the parent compound and various methylated arsenicals. Both experimental and epidemiological evidences suggest that some of the adverse health effects associated with chronic exposure to inorganic arsenic may be mediated by these methylated metabolites. If i As methylation is an activation process, then the phenotype for inorganic arsenic methylation may determine risk associated with exposure to this metalloid. We examined inorganic arsenic methylation phenotypes and arsenic (+3 oxidation state) methyltransferase genotypes in four species: three that methylate inorganic arsenic (human (Homo sapiens), rat (Rattus norwegicus), and mouse (Mus musculus)) and one that does not methylate inorganic arsenic (chimpanzee, Pan troglodytes). The predicted protein products from arsenic (+3 oxidation state) methyltransferase are similar in size for rat (369 amino acid residues), mouse (376 residues), and human (375 residues). By comparison, a 275-nucleotide deletion beginning at nucleotide 612 in the chimpanzee gene sequence causes a frameshift that leads to a nonsense mutation for a premature stop codon after amino acid 205. The null phenotype for inorganic arsenic methylation in the chimpanzee is likely due to the deletion in the gene for arsenic (+3 oxidation state) methyltransferase that yields an inactive truncated protein. This lineage-specific loss of function caused by the deletion event must have occurred in the Pan lineage after Homo-Pan divergence about 5 million years ago

  8. Structural insights into mechanisms of the small RNA methyltransferase HEN1

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Ying; Ji, Lijuan; Huang, Qichen; Vassylyev, Dmitry G.; Chen, Xuemei; Ma, Jin-Biao; (UAB); (UCR)

    2010-02-22

    RNA silencing is a conserved regulatory mechanism in fungi, plants and animals that regulates gene expression and defence against viruses and transgenes. Small silencing RNAs of {approx}20-30 nucleotides and their associated effector proteins, the Argonaute family proteins, are the central components in RNA silencing. A subset of small RNAs, such as microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs in animals and siRNAs in Drosophila, requires an additional crucial step for their maturation; that is, 2'-O-methylation on the 3' terminal nucleotide. A conserved S-adenosyl-L-methionine-dependent RNA methyltransferase, HUA ENHANCER 1 (HEN1), and its homologues are responsible for this specific modification. Here we report the 3.1 {angstrom} crystal structure of full-length HEN1 from Arabidopsis in complex with a 22-nucleotide small RNA duplex and cofactor product S-adenosyl-L-homocysteine. Highly cooperative recognition of the small RNA substrate by multiple RNA binding domains and the methyltransferase domain in HEN1 measures the length of the RNA duplex and determines the substrate specificity. Metal ion coordination by both 2' and 3' hydroxyls on the 3'-terminal nucleotide and four invariant residues in the active site of the methyltransferase domain suggests a novel Mg{sup 2+}-dependent 2'-O-methylation mechanism.

  9. Rauvolfia serpentina N-methyltransferases involved in ajmaline and Nβ -methylajmaline biosynthesis belong to a gene family derived from γ-tocopherol C-methyltransferase.

    Science.gov (United States)

    Cázares-Flores, Paulo; Levac, Dylan; De Luca, Vincenzo

    2016-08-01

    Ajmaline biosynthesis in Rauvolfia serpentina has been one of the most studied monoterpenoid indole alkaloid (MIA) pathways within the plant family Apocynaceae. Detailed molecular and biochemical information on most of the steps involved in the pathway has been generated over the last 30 years. Here we report the identification, molecular cloning and functional expression in Escherichia coli of two R. serpentinacDNAs that are part of a recently discovered γ-tocopherol-like N-methyltransferase (γ-TLMT) family and are involved in indole and side-chain N-methylation of ajmaline. Recombinant proteins showed remarkable substrate specificity for molecules with an ajmalan-type backbone and strict regiospecific N-methylation. Furthermore, N-methyltransferase gene transcripts and enzyme activity were enriched in R. serpentina roots which correlated with accumulation of ajmaline alkaloid. This study elucidates the final step in the ajmaline biosynthetic pathway and describes the enzyme responsible for the formation of Nβ -methylajmaline, an unusual charged MIA found in R. serpentina. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  10. The arginine methyltransferase Rmt2 is enriched in the nucleus and co-purifies with the nuclear porins Nup49, Nup57 and Nup100

    International Nuclear Information System (INIS)

    Olsson, Ida; Berrez, Jean-Marc; Leipus, Arunas; Ostlund, Cecilia; Mutvei, Ann

    2007-01-01

    Arginine methylation is a post-translational modification of proteins implicated in RNA processing, protein compartmentalization, signal transduction, transcriptional regulation and DNA repair. In a screen for proteins associated with the nuclear envelope in the yeast Saccharomyces cerevisiae, we have identified the arginine methyltransferase Rmt2, previously shown to methylate the ribosomal protein L12. By indirect immunofluorescence and subcellular fractionations we demonstrate here that Rmt2 has nuclear and cytoplasmic localizations. Biochemical analysis of a fraction enriched in nuclei reveals that nuclear Rmt2 is resistant to extractions with salt and detergent, indicating an association with structural components. This was supported by affinity purification experiments with TAP-tagged Rmt2. Rmt2 was found to co-purify with the nucleoporins Nup49, Nup57 and Nup100, revealing a novel link between arginine methyltransferases and the nuclear pore complex. In addition, a genome-wide transcription study of the rmt2Δ mutant shows significant downregulation of the transcription of MYO1, encoding the Type II myosin heavy chain required for cytokinesis and cell separation

  11. Histone H3 lysine 36 methyltransferase mobilizes NER factors to regulate tolerance against alkylation damage in fission yeast.

    Science.gov (United States)

    Lim, Kim Kiat; Nguyen, Thi Thuy Trang; Li, Adelicia Yongling; Yeo, Yee Phan; Chen, Ee Sin

    2018-04-09

    The Set2 methyltransferase and its target, histone H3 lysine 36 (H3K36), affect chromatin architecture during the transcription and repair of DNA double-stranded breaks. Set2 also confers resistance against the alkylating agent, methyl methanesulfonate (MMS), through an unknown mechanism. Here, we show that Schizosaccharomyces pombe (S. pombe) exhibit MMS hypersensitivity when expressing a set2 mutant lacking the catalytic histone methyltransferase domain or a H3K36R mutant (reminiscent of a set2-null mutant). Set2 acts synergistically with base excision repair factors but epistatically with nucleotide excision repair (NER) factors, and determines the timely nuclear accumulation of the NER initiator, Rhp23, in response to MMS. Set2 facilitates Rhp23 recruitment to chromatin at the brc1+ locus, presumably to repair alkylating damage and regulate the expression of brc1+ in response to MMS. Set2 also show epistasis with DNA damage checkpoint proteins; regulates the activation of Chk1, a DNA damage response effector kinase; and acts in a similar functional group as proteins involved in homologous recombination. Consistently, Set2 and H3K36 ensure the dynamicity of Rhp54 in DNA repair foci formation after MMS treatment. Overall, our results indicate a novel role for Set2/H3K36me in coordinating the recruitment of DNA repair machineries to timely manage alkylating damage.

  12. Metagenomic profiles of antibiotic resistance genes (ARGs) between human impacted estuary and deep ocean sediments.

    Science.gov (United States)

    Chen, Baowei; Yang, Ying; Liang, Ximei; Yu, Ke; Zhang, Tong; Li, Xiangdong

    2013-11-19

    Knowledge of the origins and dissemination of antibiotic resistance genes (ARGs) is essential for understanding modern resistomes in the environment. The mechanisms of the dissemination of ARGs can be revealed through comparative studies on the metagenomic profiling of ARGs between relatively pristine and human-impacted environments. The deep ocean bed of the South China Sea (SCS) is considered to be largely devoid of anthropogenic impacts, while the Pearl River Estuary (PRE) in south China has been highly impacted by intensive human activities. Commonly used antibiotics (sulfamethazine, norfloxacin, ofloxacin, tetracycline, and erythromycin) have been detected through chemical analysis in the PRE sediments, but not in the SCS sediments. In the relatively pristine SCS sediments, the most prevalent and abundant ARGs are those related to resistance to macrolides and polypeptides, with efflux pumps as the predominant mechanism. In the contaminated PRE sediments, the typical ARG profiles suggest a prevailing resistance to antibiotics commonly used in human health and animal farming (including sulfonamides, fluoroquinolones, and aminoglycosides), and higher diversity in both genotype and resistance mechanism than those in the SCS. In particular, antibiotic inactivation significantly contributed to the resistance to aminoglycosides, β-lactams, and macrolides observed in the PRE sediments. There was a significant correlation in the levels of abundance of ARGs and those of mobile genetic elements (including integrons and plasmids), which serve as carriers in the dissemination of ARGs in the aquatic environment. The metagenomic results from the current study support the view that ARGs naturally originate in pristine environments, while human activities accelerate the dissemination of ARGs so that microbes would be able to tolerate selective environmental stress in response to anthropogenic impacts.

  13. Determination of stability constants of aminoglycoside antibiotics with their metal complexes

    Science.gov (United States)

    Tiwow, Vanny M. A.

    2014-03-01

    One group of aminoglycoside antibiotics contains aminosugars. The aminosugar neomycin B with its derivate product neamine (2-Deoxy-4-0-(2,6-diamino-2,6-dideoxy-α-D-glucopyranosyl)-D-Streptamine) was identified as a free ligands and metal complexes. In particular, the stability constants of metal complexes by potentiometric titration techniques were investigated. Our previous study had determined the acid dissociation constants of these aminosugars with few metal complexes in fair depth. In this work, the complexation of two pyridine-containing amino alcohols and an amino sugar (neamine) have been measured potentiometrically. For instance, the stability constant of copper(II) complexation were determine and the model system generated an excellent fit. Stability constants with several metals have been determined and will be reported.

  14. Clinical and molecular analysis of a four-generation Chinese family with aminoglycoside-induced and nonsyndromic hearing loss associated with the mitochondrial 12S rRNA C1494T mutation

    International Nuclear Information System (INIS)

    Wang Qiuju; Li Qingzhong; Han Dongyi; Zhao Yali; Zhao Lidong; Qian Yaping; Yuan Hu; Li Ronghua; Zhai Suoqiang; Young Wieyen; Guan Minxin

    2006-01-01

    We report here the clinical, genetic, and molecular characterization of a four-generation Chinese family with aminoglycoside-induced and nonsyndromic hearing loss. Five of nine matrilineal relatives had aminoglycoside-induced hearing loss. These matrilineal relatives exhibited variable severity and audiometric configuration of hearing impairment, despite sharing some common features: being bilateral and having sensorineural hearing impairment. Sequence analysis of mitochondrial DNA (mtDNA) in the pedigree identified 16 variants and the homoplasmic 12S rRNA C1494T mutation, which was associated with hearing loss in the other large Chinese family. In fact, the occurrence of the C1494T mutation in these genetically unrelated pedigrees affected by hearing impairment strongly indicated that this mutation is involved in the pathogenesis of aminoglycoside-induced and nonsyndromic hearing loss. However, incomplete penetrance of hearing loss indicated that the C1494T mutation itself is not sufficient to produce a clinical phenotype but requires the involvement of modifier factors for the phenotypic expression. Those mtDNA variants, showing no evolutional conservation, may not have a potential modifying role in the pathogenesis of the C1494T mutation. However, nuclear background seems to contribute to the phenotypic variability of matrilineal relatives in this family. Furthermore, aminoglycosides modulate the expressivity and penetrance of deafness associated with the C1494T mutation in this family

  15. Transcriptome Profiling of Antimicrobial Resistance in Pseudomonas aeruginosa.

    Science.gov (United States)

    Khaledi, Ariane; Schniederjans, Monika; Pohl, Sarah; Rainer, Roman; Bodenhofer, Ulrich; Xia, Boyang; Klawonn, Frank; Bruchmann, Sebastian; Preusse, Matthias; Eckweiler, Denitsa; Dötsch, Andreas; Häussler, Susanne

    2016-08-01

    Emerging resistance to antimicrobials and the lack of new antibiotic drug candidates underscore the need for optimization of current diagnostics and therapies to diminish the evolution and spread of multidrug resistance. As the antibiotic resistance status of a bacterial pathogen is defined by its genome, resistance profiling by applying next-generation sequencing (NGS) technologies may in the future accomplish pathogen identification, prompt initiation of targeted individualized treatment, and the implementation of optimized infection control measures. In this study, qualitative RNA sequencing was used to identify key genetic determinants of antibiotic resistance in 135 clinical Pseudomonas aeruginosa isolates from diverse geographic and infection site origins. By applying transcriptome-wide association studies, adaptive variations associated with resistance to the antibiotic classes fluoroquinolones, aminoglycosides, and β-lactams were identified. Besides potential novel biomarkers with a direct correlation to resistance, global patterns of phenotype-associated gene expression and sequence variations were identified by predictive machine learning approaches. Our research serves to establish genotype-based molecular diagnostic tools for the identification of the current resistance profiles of bacterial pathogens and paves the way for faster diagnostics for more efficient, targeted treatment strategies to also mitigate the future potential for resistance evolution. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  16. Recruitment of DNA methyltransferase I to DNA repair sites

    Science.gov (United States)

    Mortusewicz, Oliver; Schermelleh, Lothar; Walter, Joachim; Cardoso, M. Cristina; Leonhardt, Heinrich

    2005-01-01

    In mammalian cells, the replication of genetic and epigenetic information is directly coupled; however, little is known about the maintenance of epigenetic information in DNA repair. Using a laser microirradiation system to introduce DNA lesions at defined subnuclear sites, we tested whether the major DNA methyltransferase (Dnmt1) or one of the two de novo methyltransferases (Dnmt3a, Dnmt3b) are recruited to sites of DNA repair in vivo. Time lapse microscopy of microirradiated mammalian cells expressing GFP-tagged Dnmt1, Dnmt3a, or Dnmt3b1 together with red fluorescent protein-tagged proliferating cell nuclear antigen (PCNA) revealed that Dnmt1 and PCNA accumulate at DNA damage sites as early as 1 min after irradiation in S and non-S phase cells, whereas recruitment of Dnmt3a and Dnmt3b was not observed. Deletion analysis showed that Dnmt1 recruitment was mediated by the PCNA-binding domain. These data point to a direct role of Dnmt1 in the restoration of epigenetic information during DNA repair. PMID:15956212

  17. Genomic survey, gene expression analysis and structural modeling suggest diverse roles of DNA methyltransferases in legumes.

    Directory of Open Access Journals (Sweden)

    Rohini Garg

    Full Text Available DNA methylation plays a crucial role in development through inheritable gene silencing. Plants possess three types of DNA methyltransferases (MTases, namely Methyltransferase (MET, Chromomethylase (CMT and Domains Rearranged Methyltransferase (DRM, which maintain methylation at CG, CHG and CHH sites. DNA MTases have not been studied in legumes so far. Here, we report the identification and analysis of putative DNA MTases in five legumes, including chickpea, soybean, pigeonpea, Medicago and Lotus. MTases in legumes could be classified in known MET, CMT, DRM and DNA nucleotide methyltransferases (DNMT2 subfamilies based on their domain organization. First three MTases represent DNA MTases, whereas DNMT2 represents a transfer RNA (tRNA MTase. Structural comparison of all the MTases in plants with known MTases in mammalian and plant systems have been reported to assign structural features in context of biological functions of these proteins. The structure analysis clearly specified regions crucial for protein-protein interactions and regions important for nucleosome binding in various domains of CMT and MET proteins. In addition, structural model of DRM suggested that circular permutation of motifs does not have any effect on overall structure of DNA methyltransferase domain. These results provide valuable insights into role of various domains in molecular recognition and should facilitate mechanistic understanding of their function in mediating specific methylation patterns. Further, the comprehensive gene expression analyses of MTases in legumes provided evidence of their role in various developmental processes throughout the plant life cycle and response to various abiotic stresses. Overall, our study will be very helpful in establishing the specific functions of DNA MTases in legumes.

  18. Molecular Basis of Resistance to Selected Antimicrobial Agents in the Emerging Zoonotic Pathogen Streptococcus suis.

    Science.gov (United States)

    Gurung, Mamata; Tamang, Migma Dorji; Moon, Dong Chan; Kim, Su-Ran; Jeong, Jin-Ha; Jang, Geum-Chan; Jung, Suk-Chan; Park, Yong-Ho; Lim, Suk-Kyung

    2015-07-01

    Characterization of 227 Streptococcus suis strains isolated from pigs during 2010 to 2013 showed high levels of resistance to clindamycin (95.6%), tilmicosin (94.7%), tylosin (93.8%), oxytetracycline (89.4%), chlortetracycline (86.8%), tiamulin (72.7%), neomycin (70.0%), enrofloxacin (56.4%), penicillin (56.4%), ceftiofur (55.9%), and gentamicin (55.1%). Resistance to tetracyclines, macrolides, aminoglycosides, and fluoroquinolone was attributed to the tet gene, erm(B), erm(C), mph(C), and mef(A) and/or mef(E) genes, aph(3')-IIIa and aac(6')-Ie-aph(2″)-Ia genes, and single point mutations in the quinolone resistance-determining region of ParC and GyrA, respectively. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  19. Infective endocarditis due to Enterobacter cloacae resistant to third- and fourth-generation cephalosporins.

    Science.gov (United States)

    Yoshino, Yusuke; Okugawa, Shu; Kimura, Satoshi; Makita, Eiko; Seo, Kazunori; Koga, Ichiro; Matsunaga, Naohisa; Kitazawa, Takatoshi; Ota, Yasuo

    2015-04-01

    We report the case of using a long-term combination of meropenem and amikacin to treat infective endocarditis caused by Enterobacter cloacae resistant to third- and fourth-generation cephalosporins. Multi-drug resistant Gram-negative bacilli, such as the E. cloacae in our study, may become possible pathogens of infective endocarditis. Our experience with this case indicates that long-term use of a combination of β-lactam and aminoglycosides might represent a suitable management option for future infective endocarditis cases due to non-Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, Kingella spp. (HACEK group) Gram-negative bacilli such as ours. Copyright © 2012. Published by Elsevier B.V.

  20. A fluorescence resonance energy transfer-based method for histone methyltransferases

    DEFF Research Database (Denmark)

    Devkota, Kanchan; Lohse, Brian; Nyby Jakobsen, Camilla

    2015-01-01

    A simple dye–quencher fluorescence resonance energy transfer (FRET)-based assay for methyltransferases was developed and used to determine kinetic parameters and inhibitory activity at EHMT1 and EHMT2. Peptides mimicking the truncated histone H3 tail were functionalized in each end with a dye...

  1. Metabolism of S-adenosylmethionine in rat hepatocytes: transfer of methyl group from S-adenosylmethionine by methyltransferase reactions

    International Nuclear Information System (INIS)

    Tsukada, K.; Abe, T.; Kuwahata, T.; Mitsui, K.

    1985-01-01

    Treatment of rats with a methionine diet leads not only to a marked increase of S-adenosylmethionine synthetase in liver, but also to the increase of glycine, guanidoacetate and betaine-homocysteine methyltransferases. The activity of tRNA methyltransferase decreased with the increased amounts of methionine in the diets. However, the activities of phospholipids and S-adenosylmethionine-homocysteine methyltransferases did not show any significant change. When hepatocarcinogenesis induced by 2-fluorenylacetamide progresses, the activities of glycine and guanidoacetate methyltransferases in rat liver decreased, and could not be detected in tumorous areas 8 months after treatment. The levels of S-adenosylmethionine in the liver also decreased to levels of one-fifth of control animals at 8 months. The uptake and metabolism of [methyl- 3 H]-methionine and -S-adenosylmethionine have been investigated by in vivo and isolated hepatocytes. The uptake of methionine and transfer of methyl group to phospholipid in the cells by methionine were remarkably higher than those by S-adenosylmethionine. These results indicate that phospholipids in hepatocytes accept methyl group from S-adenosylmethionine immediately, when it is synthesized from methionine, before mixing its pool in the cells. 39 references, 1 figure, 2 tables

  2. Neuronal DNA Methyltransferases: Epigenetic Mediators between Synaptic Activity and Gene Expression?

    Science.gov (United States)

    Bayraktar, Gonca; Kreutz, Michael R

    2018-04-01

    DNMT3A and 3B are the main de novo DNA methyltransferases (DNMTs) in the brain that introduce new methylation marks to non-methylated DNA in postmitotic neurons. DNA methylation is a key epigenetic mark that is known to regulate important cellular processes in neuronal development and brain plasticity. Accumulating evidence disclosed rapid and dynamic changes in DNA methylation of plasticity-relevant genes that are important for learning and memory formation. To understand how DNMTs contribute to brain function and how they are regulated by neuronal activity is a prerequisite for a deeper appreciation of activity-dependent gene expression in health and disease. This review discusses the functional role of de novo methyltransferases and in particular DNMT3A1 in the adult brain with special emphasis on synaptic plasticity, memory formation, and brain disorders.

  3. Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry

    DEFF Research Database (Denmark)

    Vranken, Charlotte; Deen, Jochem; Dirix, Lieve

    2014-01-01

    We demonstrate an approach to optical DNA mapping, which enables near single-molecule characterization of whole bacteriophage genomes. Our approach uses a DNA methyltransferase enzyme to target labelling to specific sites and copper-catalysed azide-alkyne cycloaddition to couple a fluorophore...... to the DNA. We achieve a labelling efficiency of ∼70% with an average labelling density approaching one site every 500 bp. Such labelling density bridges the gap between the output of a typical DNA sequencing experiment and the long-range information derived from traditional optical DNA mapping. We lay...... the foundations for a wider-scale adoption of DNA mapping by screening 11 methyltransferases for their ability to direct sequence-specific DNA transalkylation; the first step of the DNA labelling process and by optimizing reaction conditions for fluorophore coupling via a click reaction. Three of 11 enzymes...

  4. Widespread acquisition of antimicrobial resistance among Campylobacter isolates from UK retail poultry and evidence for clonal expansion of resistant lineages.

    Science.gov (United States)

    Wimalarathna, Helen M L; Richardson, Judith F; Lawson, Andy J; Elson, Richard; Meldrum, Richard; Little, Christine L; Maiden, Martin C J; McCarthy, Noel D; Sheppard, Samuel K

    2013-07-15

    Antimicrobial resistance is increasing among clinical Campylobacter cases and is common among isolates from other sources, specifically retail poultry - a major source of human infection. In this study the antimicrobial susceptibility of isolates from a UK-wide survey of Campylobacter in retail poultry in 2001 and 2004-5 was investigated. The occurrence of phenotypes resistant to tetracycline, quinolones (ciprofloxacin and naladixic acid), erythromycin, chloramphenicol and aminoglycosides was quantified. This was compared with a phylogeny for these isolates based upon Multi Locus Sequence Typing (MLST) to investigate the pattern of antimicrobial resistance acquisition. Antimicrobial resistance was present in all lineage clusters, but statistical testing showed a non-random distribution. Erythromycin resistance was associated with Campylobacter coli. For all antimicrobials tested, resistant isolates were distributed among relatively distant lineages indicative of widespread acquisition. There was also evidence of clustering of resistance phenotypes within lineages; indicative of local expansion of resistant strains. These results are consistent with the widespread acquisition of antimicrobial resistance among chicken associated Campylobacter isolates, either through mutation or horizontal gene transfer, and the expansion of these lineages as a proportion of the population. As Campylobacter are not known to multiply outside of the host and long-term carriage in humans is extremely infrequent in industrialized countries, the most likely location for the proliferation of resistant lineages is in farmed chickens.

  5. A Simple Model for Inducing Optimal Increase of SDF-1 with Aminoglycoside Ototoxicity

    Directory of Open Access Journals (Sweden)

    Hyun Mi Ju

    2017-01-01

    Full Text Available Objectives. As a homing factor of stem cell, stromal derived factor-1 (SDF-1 is important for the regenerative research in ototoxicity. Mice models with aminoglycoside ototoxicity have been widely used to study the regeneration capacity of MSCs in repair of cochlear injury. We developed a mouse model with maximal increase in SDF-1 levels in the inner ear, according to the “one-shot” doses of kanamycin and furosemide. Methods. C57BL/6 mice had kanamycin (420, 550, and 600 mg/kg dissolved in PBS, followed by an intraperitoneal injection of furosemide (130 mg/kg. The injuries of inner ear were measured with hearing thresholds, histology, and outer hair cell counts at 0, 3, 5, 7, 10, and 14 days before the sacrifice. The levels of SDF-1 in the inner ear were tested by real-time RT-PCR and immunohistochemistry. Results. There were a significant reduction in hearing thresholds and a maximal increase of SDF-1 levels in the furosemide 130 mg/kg + kanamycin 550 mg/kg group, but severe hearing deterioration over time was observed in the furosemide 130 mg/kg + kanamycin 600 mg/kg group and four mice were dead. SDF-1 was detected mostly in the stria vascularis and organ of Corti showing the highest increase in expression. Conclusion. We observed optimal induction of the stem cell homing factor in the newly generated aminoglycoside-induced ototoxicity mouse model using a “one-shot” protocol. This study regarding high SDF-1 levels in our mouse model of ototoxicity would play a major role in the development of therapeutic agents using MSC homing.

  6. Investigation into complexing of phthalexone S with praseodymium ions and some aminoglycoside antibiotics

    International Nuclear Information System (INIS)

    Alykov, N.M.

    1981-01-01

    Complex formation of phthalexone S (Phth) with praseodymium ion and some aminoglycoside antibiotics (Ab) in aqueous ethanol solutions (1:1) has been examined photometrically at 619 mm. It has been shown that compounds with the ratios of Ab:Pr:Phth=1:2:8, 1:1:4, 1:1:3 are formed depending on the number of amino groups and structure of the antibiotics. The molar absorptivities and solubility products for the complexes have been calculated. The complex formation scheme is given. A procedure has been developed of determining 0.01-10 μg of antibiotics in 1 ml of a biological material with a relative error of less than 10% [ru

  7. A mouse speciation gene encodes a meiotic histone H3 methyltransferase

    Czech Academy of Sciences Publication Activity Database

    Mihola, Ondřej; Trachtulec, Zdeněk; Vlček, Čestmír; Schimenti, J.C.; Forejt, Jiří

    2009-01-01

    Roč. 323, č. 5912 (2009), s. 373-375 ISSN 0036-8075 Institutional research plan: CEZ:AV0Z50520514 Keywords : hybrid sterility * histone H3K4 methyltransferase * Prdm9 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 29.747, year: 2009

  8. Biology of Acinetobacter baumannii: Pathogenesis, Antibiotic Resistance Mechanisms, and Prospective Treatment Options

    Science.gov (United States)

    Lee, Chang-Ro; Lee, Jung Hun; Park, Moonhee; Park, Kwang Seung; Bae, Il Kwon; Kim, Young Bae; Cha, Chang-Jun; Jeong, Byeong Chul; Lee, Sang Hee

    2017-01-01

    Acinetobacter baumannii is undoubtedly one of the most successful pathogens responsible for hospital-acquired nosocomial infections in the modern healthcare system. Due to the prevalence of infections and outbreaks caused by multi-drug resistant A. baumannii, few antibiotics are effective for treating infections caused by this pathogen. To overcome this problem, knowledge of the pathogenesis and antibiotic resistance mechanisms of A. baumannii is important. In this review, we summarize current studies on the virulence factors that contribute to A. baumannii pathogenesis, including porins, capsular polysaccharides, lipopolysaccharides, phospholipases, outer membrane vesicles, metal acquisition systems, and protein secretion systems. Mechanisms of antibiotic resistance of this organism, including acquirement of β-lactamases, up-regulation of multidrug efflux pumps, modification of aminoglycosides, permeability defects, and alteration of target sites, are also discussed. Lastly, novel prospective treatment options for infections caused by multi-drug resistant A. baumannii are summarized. PMID:28348979

  9. Human METTL12 is a mitochondrial methyltransferase that modifies citrate synthase.

    Science.gov (United States)

    Rhein, Virginie F; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2017-06-01

    The protein methylome in mammalian mitochondria has been little studied until recently. Here, we describe that lysine-368 of human citrate synthase is methylated and that the modifying enzyme, localized in the mitochondrial matrix, is methyltransferase-like protein 12 (METTL12), a member of the family of 7β-strand methyltransferases. Lysine-368 is near the active site of citrate synthase, but removal of methylation has no effect on its activity. In mitochondria, it is possible that some or all of the enzymes of the citric acid cycle, including citrate synthase, are organized in metabolons to facilitate the channelling of substrates between participating enzymes. Thus, possible roles for the methylation of Lys-368 are in controlling substrate channelling itself, or in influencing protein-protein interactions in the metabolon. © 2017 The Authors FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  10. Enhancer of zeste homologue 2 plays an important role in neuroblastoma cell survival independent of its histone methyltransferase activity.

    Science.gov (United States)

    Bate-Eya, Laurel T; Gierman, Hinco J; Ebus, Marli E; Koster, Jan; Caron, Huib N; Versteeg, Rogier; Dolman, M Emmy M; Molenaar, Jan J

    2017-04-01

    Neuroblastoma is predominantly characterised by chromosomal rearrangements. Next to V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma Derived Homolog (MYCN) amplification, chromosome 7 and 17q gains are frequently observed. We identified a neuroblastoma patient with a regional 7q36 gain, encompassing the enhancer of zeste homologue 2 (EZH2) gene. EZH2 is the histone methyltransferase of lysine 27 of histone H3 (H3K27me3) that forms the catalytic subunit of the polycomb repressive complex 2. H3K27me3 is commonly associated with the silencing of genes involved in cellular processes such as cell cycle regulation, cellular differentiation and cancer. High EZH2 expression correlated with poor prognosis and overall survival independent of MYCN amplification status. Unexpectedly, treatment of 3 EZH2-high expressing neuroblastoma cell lines (IMR32, CHP134 and NMB), with EZH2-specific inhibitors (GSK126 and EPZ6438) resulted in only a slight G1 arrest, despite maximum histone methyltransferase activity inhibition. Furthermore, colony formation in cell lines treated with the inhibitors was reduced only at concentrations much higher than necessary for complete inhibition of EZH2 histone methyltransferase activity. Knockdown of the complete protein with three independent shRNAs resulted in a strong apoptotic response and decreased cyclin D1 levels. This apoptotic response could be rescued by overexpressing EZH2ΔSET, a truncated form of wild-type EZH2 lacking the SET transactivation domain necessary for histone methyltransferase activity. Our findings suggest that high EZH2 expression, at least in neuroblastoma, has a survival function independent of its methyltransferase activity. This important finding highlights the need for studies on EZH2 beyond its methyltransferase function and the requirement for compounds that will target EZH2 as a complete protein. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Preliminary characterization of (nucleoside-2′-O-)-methyltransferase crystals from Meaban and Yokose flaviviruses

    International Nuclear Information System (INIS)

    Mastrangelo, Eloise; Bollati, Michela; Milani, Mario; Lamballeire, Xavier de; Brisbare, Nadege; Dalle, Karen; Lantez, Violaine; Egloff, Marie-Pierre; Coutard, Bruno; Canard, Bruno; Gould, Ernest; Forrester, Naomi; Bolognesi, Martino

    2006-01-01

    Two methyltransferases from flaviviruses (Meaban and Yokose viruses) have been overexpressed and crystallized. Diffraction data and characterization of the two crystal forms are presented, together with a preliminary molecular-replacement solution for both enzymes. Viral methyltranferases (MTase) are involved in the third step of the mRNA-capping process, transferring a methyl group from S-adenosyl-l-methionine (SAM) to the capped mRNA. MTases are classified into two groups: (guanine-N7)-methyltransferases (N7MTases), which add a methyl group onto the N7 atom of guanine, and (nucleoside-2′-O-)-methyltransferases (2′OMTases), which add a methyl group to a ribose hydroxyl. The MTases of two flaviviruses, Meaban and Yokose viruses, have been overexpressed, purified and crystallized in complex with SAM. Characterization of the crystals together with details of preliminary X-ray diffraction data collection (at 2.8 and 2.7 Å resolution, respectively) are reported here. The sequence homology relative to Dengue virus 2′OMTase and the structural conservation of specific residues in the putative active sites suggest that both enzymes belong to the 2′OMTase subgroup

  12. Preliminary characterization of (nucleoside-2′-O-)-methyltransferase crystals from Meaban and Yokose flaviviruses

    Energy Technology Data Exchange (ETDEWEB)

    Mastrangelo, Eloise; Bollati, Michela; Milani, Mario [Department of Biomolecular Sciences and Biotechnology, CNR-INFM, University of Milano, Via Celoria 26, 20133 Milano (Italy); Lamballeire, Xavier de; Brisbare, Nadege [Unité des Virus Emergents, Faculté de Médecine, 27 Boulevard Jean Moulin, 13005 Marseille (France); Dalle, Karen; Lantez, Violaine; Egloff, Marie-Pierre; Coutard, Bruno; Canard, Bruno [Laboratoire Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS ESIL, Case 932, 163 Avenue de Luminy, 13288 Marseille CEDEX 9 (France); Gould, Ernest; Forrester, Naomi [CEH Oxford, Mansfield Road, Oxford OX1 3SR (United Kingdom); Bolognesi, Martino, E-mail: martino.bolognesi@unimi.it [Department of Biomolecular Sciences and Biotechnology, CNR-INFM, University of Milano, Via Celoria 26, 20133 Milano (Italy)

    2006-08-01

    Two methyltransferases from flaviviruses (Meaban and Yokose viruses) have been overexpressed and crystallized. Diffraction data and characterization of the two crystal forms are presented, together with a preliminary molecular-replacement solution for both enzymes. Viral methyltranferases (MTase) are involved in the third step of the mRNA-capping process, transferring a methyl group from S-adenosyl-l-methionine (SAM) to the capped mRNA. MTases are classified into two groups: (guanine-N7)-methyltransferases (N7MTases), which add a methyl group onto the N7 atom of guanine, and (nucleoside-2′-O-)-methyltransferases (2′OMTases), which add a methyl group to a ribose hydroxyl. The MTases of two flaviviruses, Meaban and Yokose viruses, have been overexpressed, purified and crystallized in complex with SAM. Characterization of the crystals together with details of preliminary X-ray diffraction data collection (at 2.8 and 2.7 Å resolution, respectively) are reported here. The sequence homology relative to Dengue virus 2′OMTase and the structural conservation of specific residues in the putative active sites suggest that both enzymes belong to the 2′OMTase subgroup.

  13. MALDI-TOF MS as a Tool To Detect a Nosocomial Outbreak of Extended-Spectrum-β-Lactamase- and ArmA Methyltransferase-Producing Enterobacter cloacae Clinical Isolates in Algeria.

    Science.gov (United States)

    Khennouchi, Nour Chems el Houda; Loucif, Lotfi; Boutefnouchet, Nafissa; Allag, Hamoudi; Rolain, Jean-Marc

    2015-10-01

    Enterobacter cloacae is among the most important pathogens responsible for nosocomial infections and outbreaks. In this study, 77 Enterobacter isolates were collected: 27 isolates from Algerian hospitals (in Constantine, Annaba, and Skikda) and 50 isolates from Marseille, France. All strains were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Antibiotic susceptibility testing was performed by the disk diffusion method. PCR was used to detect extended-spectrum-beta-lactamase (ESBL)-encoding, fluoroquinolone resistance-encoding, and aminoglycoside-modifying enzyme (AME) genes. Epidemiological typing was performed using MALDI-TOF MS with data mining approaches, along with multilocus sequence typing (MLST). Sixty-eight isolates (27 from Algeria, 41 from Marseille) were identified by MALDI-TOF MS as E. cloacae. Resistance to antibiotics in the Algerian isolates was significantly higher than that in the strains from Marseille, especially for beta-lactams and aminoglycosides. Eighteen of the 27 Algerian isolates and 11 of the 41 Marseille isolates possessed at least one ESBL-encoding gene: blaCTX-M and/or blaTEM. AME genes were detected in 20 of the 27 Algerian isolates and 8 of the 41 Marseille isolates [ant(2″)-Ia, aac(6')-Ib-cr, aadA1, aadA2, and armA]. Conjugation experiments showed that armA was carried on a transferable plasmid. MALDI-TOF typing showed three separate clusters according to the geographical distribution and species level. An MLST-based phylogenetic tree showed a clade of 14 E. cloacae isolates from a urology unit clustering together in the MALDI-TOF dendrogram, suggesting the occurrence of an outbreak in this unit. In conclusion, the ability of MALDI-TOF to biotype strains was confirmed, and surveillance measures should be implemented, especially for Algerian patients hospitalized in France. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  14. Crystallization and preliminary crystallographic analysis of tRNA (m7G46) methyltransferase from Escherichia coli

    International Nuclear Information System (INIS)

    Liu, Qi; Gao, Yang; Yang, Weili; Zhou, Huihao; Gao, Yongxiang; Zhang, Xiao; Teng, Maikun; Niu, Liwen

    2008-01-01

    tRNA (m 7 G46) methyltransferase from E. coli was overexpressed, purified and crystallized. Diffraction data were collected to 2.04 Å resolution. Transfer RNA (tRNA) (m 7 G46) methyltransferase (TrmB) belongs to the Rossmann-fold methyltransferase (RFM) family and uses S-adenosyl-l-methionine (SAM) as the methyl-group donor to catalyze the formation of N 7 -methylguanosine (m 7 G) at position 46 in the variable loop of tRNAs. After attempts to crystallize full-length Escherichia coli TrmB (EcTrmB) failed, a truncated protein lacking the first 32 residues of the N-terminus but with an additional His 6 tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 3350 (PEG 3350) as precipitant at 283 K. An X-ray diffraction data set was collected using a single flash-cooled crystal that belonged to space group P2 1

  15. Methylation of 23S rRNA nucleotide G748 by RlmAII methyltransferase renders Streptococcus pneumoniae telithromycin susceptible.

    Science.gov (United States)

    Takaya, Akiko; Sato, Yoshiharu; Shoji, Tatsuma; Yamamoto, Tomoko

    2013-08-01

    Several posttranscriptional modifications of bacterial rRNAs are important in determining antibiotic resistance or sensitivity. In all Gram-positive bacteria, dimethylation of nucleotide A2058, located in domain V of 23S rRNA, by the dimethyltransferase Erm(B) results in low susceptibility and resistance to telithromycin (TEL). However, this is insufficient to produce high-level resistance to TEL in Streptococcus pneumoniae. Inactivation of the methyltransferase RlmA(II), which methylates the N-1 position of nucleotide G748, located in hairpin 35 of domain II of 23S rRNA, results in increased resistance to TEL in erm(B)-carrying S. pneumoniae. Sixteen TEL-resistant mutants (MICs, 16 to 32 μg/ml) were obtained from a clinically isolated S. pneumoniae strain showing low TEL susceptibility (MIC, 2 μg/ml), with mutation resulting in constitutive dimethylation of A2058 because of nucleotide differences in the regulatory region of erm(B) mRNA. Primer extension analysis showed that the degree of methylation at G748 in all TEL-resistant mutants was significantly reduced by a mutation in the gene encoding RlmA(II) to create a stop codon or change an amino acid residue. Furthermore, RNA footprinting with dimethyl sulfate and a molecular modeling study suggested that methylation of G748 may contribute to the stable interaction of TEL with domain II of 23S rRNA, even after dimethylation of A2058 by Erm(B). This novel finding shows that methylation of G748 by RlmA(II) renders S. pneumoniae TEL susceptible.

  16. Detection of Specific Solvent Rearrangement Regions of an Enzyme: NMR and ITC Studies with Aminoglycoside Phosphotransferase(3 )-IIIa

    International Nuclear Information System (INIS)

    Ozen, C.; Norris, Adrianne; Land, Miriam Louise; Tjioe, Elina; Serpersu, Engin H

    2008-01-01

    This work describes differential effects of solvent in complexes of the aminoglycoside phosphotransferase(3 and cent;)-IIIa (APH) with different aminoglycosides and the detection of change in solvent structure at specific sites away from substrates. Binding of kanamycins to APH occurs with a larger negative and cent;H in H2O relative to D2O ( and cent; and cent;H(H2O-D2O) < 0), while the reverse is true for neomycins. Unusually large negative and cent;Cp values were observed for binding of aminoglycosides to APH. and cent;Cp for the APHneomycin complex was -1.6 kcal and acirc;mol-1 and acirc;deg-1. A break at 30 C was observed in the APH-kanamycin complex yielding and cent;Cp values of -0.7 kcal and acirc;mol-1 and acirc;deg-1 and -3.8 kcal and acirc;mol-1 and acirc;deg-1 below and above 30 C, respectively. Neither the change in accessible surface area ( and cent;ASA) nor contributions from heats of ionization were sufficient to explain the large negative and cent;Cp values. Most significantly, 15N-1H HSQC experiments showed that temperature-dependent shifts of the backbone amide protons of Leu 88, Ser 91, Cys 98, and Leu143 revealed a break at 30 C only in the APH-kanamycin complex in spectra collected between 21 C and 38 C. These amino acids represent solVent reorganization sites that experience a change in solvent structure in their immediate environment as structurally different ligands bind to the enzyme. These residues were away from the substrate binding site and distributed in three hydrophobic patches in APH. Overall, our results show that a large number of factors affect and cent;Cp and binding of structurally different ligand groups cause different solvent structure in the active site as well as differentially affecting specific sites away from the ligand binding site

  17. Crystal structures of the methyltransferase and helicase from the ZIKA 1947 MR766 Uganda strain

    Energy Technology Data Exchange (ETDEWEB)

    Bukrejewska, Malgorzata; Derewenda, Urszula; Radwanska, Malwina; Engel, Daniel A.; Derewenda, Zygmunt S.

    2017-08-15

    Two nonstructural proteins encoded byZika virusstrain MR766 RNA, a methyltransferase and a helicase, were crystallized and their structures were solved and refined at 2.10 and 2.01 Å resolution, respectively. The NS5 methyltransferase contains a boundS-adenosyl-L-methionine (SAM) co-substrate. The NS3 helicase is in the apo form. Comparison with published crystal structures of the helicase in the apo, nucleotide-bound and single-stranded RNA (ssRNA)-bound states suggests that binding of ssRNA to the helicase may occur through conformational selection rather than induced fit.

  18. Use of antimicrobials in veterinary medicine and mechanisms of resistance.

    Science.gov (United States)

    Schwarz, S; Chaslus-Dancla, E

    2001-01-01

    This review deals with the application of antimicrobial agents in veterinary medicine and food animal production and the possible consequences arising from the widespread and multipurpose use of antimicrobials. The various mechanisms that bacteria have developed to escape the inhibitory effects of the antimicrobials most frequently used in the veterinary field are reported in detail. Resistance of bacteria to tetracyclines, macrolide-lincosamide-streptogramin antibiotics, beta-lactam antibiotics, aminoglycosides, sulfonamides, trimethoprim, fluoroquinolones and chloramphenicol/florfenicol is described with regard to enzymatic inactivation, decreased intracellular drug accumulation and modification/protection/replacement of the target sites. In addition, basic information is given about mobile genetic elements which carry the respective resistance genes, such as plasmids, transposons, and gene cassettes/integrons, and their ways of spreading via conjugation, mobilisation, transduction, and transformation.

  19. Noncompetitive inhibition of indolethylamine-N-methyltransferase by N,N-dimethyltryptamine and N,N-dimethylaminopropyltryptamine.

    Science.gov (United States)

    Chu, Uyen B; Vorperian, Sevahn K; Satyshur, Kenneth; Eickstaedt, Kelsey; Cozzi, Nicholas V; Mavlyutov, Timur; Hajipour, Abdol R; Ruoho, Arnold E

    2014-05-13

    Indolethylamine-N-methyltransferase (INMT) is a Class 1 transmethylation enzyme known for its production of N,N-dimethyltryptamine (DMT), a hallucinogen with affinity for various serotonergic, adrenergic, histaminergic, dopaminergic, and sigma-1 receptors. DMT is produced via the action of INMT on the endogenous substrates tryptamine and S-adenosyl-l-methionine (SAM). The biological, biochemical, and selective small molecule regulation of INMT enzyme activity remain largely unknown. Kinetic mechanisms for inhibition of rabbit lung INMT (rabINMT) by the product, DMT, and by a new novel tryptamine derivative were determined. After Michaelis-Menten and Lineweaver-Burk analyses had been applied to study inhibition, DMT was found to be a mixed competitive and noncompetitive inhibitor when measured against tryptamine. The novel tryptamine derivative, N-[2-(1H-indol-3-yl)ethyl]-N',N'-dimethylpropane-1,3-diamine (propyl dimethyl amino tryptamine or PDAT), was shown to inhibit rabINMT by a pure noncompetitive mechanism when measured against tryptamine with a Ki of 84 μM. No inhibition by PDAT was observed at 2 mM when it was tested against structurally similar Class 1 methyltransferases, such as human phenylethanolamine-N-methyltransferase (hPNMT) and human nicotinamide-N-methyltransferase (hNNMT), indicating selectivity for INMT. The demonstration of noncompetitive mechanisms for INMT inhibition implies the presence of an inhibitory allosteric site. In silico analyses using the computer modeling software Autodock and the rabINMT sequence threaded onto the human INMT (hINMT) structure (Protein Data Bank entry 2A14 ) identified an N-terminal helix-loop-helix non-active site binding region of the enzyme. The energies for binding of DMT and PDAT to this region of rabINMT, as determined by Autodock, were -6.34 and -7.58 kcal/mol, respectively. Assessment of the allosteric control of INMT may illuminate new biochemical pathway(s) underlying the biology of INMT.

  20. The relationship between the use of flucloxacillin, vancomycin, aminoglycosides and ciprofloxacin and the susceptibility patterns of coagulase-negative staphylococci recovered from blood cultures.

    NARCIS (Netherlands)

    Mulder, JG; Kosterink, JGW; Degener, JE

    1997-01-01

    Antibiotic use is a cause of selection of multiresistant bacterial strains. Over three years (1990-1992) we studied the relation between the use of flucloxacillin, vancomycin, aminoglycosides and ciprofloxacin and the susceptibility of coagulase-negative staphylococci (CNS) recovered from blood

  1. Surveillance of multidrug resistant suppurative infection causing bacteria in hospitalized patients in an Indian tertiary care hospital

    OpenAIRE

    Nabakishore Nayak; Rajesh K. Lenka; Rabindra N. Padhy

    2014-01-01

    Objective: To examine antibiograms of a cohort of suppurative bacteria isolated from wound-swabs from hospitalized patients of all economic groups of a typical Indian teaching hospital. Methods: In surveillance, antibiotic resistance patterns of 10 species of suppurative bacteria isolated from wound-swabs over a period of 24 months were recorded. Those were subjected to antibiotic sensitivity test, using 16 prescribed antibiotics of 5 different groups (3 aminoglycosides, 4 beta-lactams, 3 ...

  2. Abundance and distribution of antibiotic resistance genes in a full-scale anaerobic-aerobic system alternately treating ribostamycin, spiramycin and paromomycin production wastewater.

    Science.gov (United States)

    Tang, Mei; Dou, Xiaomin; Wang, Chunyan; Tian, Zhe; Yang, Min; Zhang, Yu

    2017-12-01

    The occurrence of antibiotic-resistant bacteria and antibiotic resistance genes (ARGs) has been intensively investigated for wastewater treatment systems treating single class of antibiotic in recent years. However, the impacts of alternately occurring antibiotics in antibiotic production wastewater on the behavior of ARGs in biological treatment systems were not well understood yet. Herein, techniques including high-capacity quantitative PCR and quantitative PCR (qPCR) were used to investigate the behavior of ARGs in an anaerobic-aerobic full-scale system. The system alternately treated three kinds of antibiotic production wastewater including ribostamycin, spiramycin and paromomycin, which referred to stages 1, 2 and 3. The aminoglycoside ARGs (52.1-79.3%) determined using high-capacity quantitative PCR were the most abundant species in all sludge samples of the three stages. The total relative abundances of macrolide-lincosamide-streptogramin (MLS) resistance genes and aminoglycoside resistance genes measured using qPCR were significantly higher (P  0.05) in both aerobic and anaerobic sludge samples. In aerobic sludge, one acetyltransferase gene (aacA4) and the other three nucleotidyltransferase genes (aadB, aadA and aadE) exhibited positive correlations with intI1 (r 2  = 0.83-0.94; P < 0.05), implying the significance of horizontal transfer in their proliferation. These results and facts will be helpful to understand the abundance and distribution of ARGs from antibiotic production wastewater treatment systems.

  3. The histone methyltransferase SET8 is required for S-phase progression

    DEFF Research Database (Denmark)

    Jørgensen, Stine; Elvers, Ingegerd; Trelle, Morten Beck

    2008-01-01

    Chromatin structure and function is influenced by histone posttranslational modifications. SET8 (also known as PR-Set7 and SETD8) is a histone methyltransferase that monomethylates histonfe H4-K20. However, a function for SET8 in mammalian cell proliferation has not been determined. We show...

  4. Clinical Pharmacogenetics Implementation Consortium guidelines for thiopurine methyltransferase genotype and thiopurine dosing

    DEFF Research Database (Denmark)

    Relling, M V; Gardner, E E; Sandborn, W J

    2011-01-01

    Thiopurine methyltransferase (TPMT) activity exhibits monogenic co-dominant inheritance, with ethnic differences in the frequency of occurrence of variant alleles. With conventional thiopurine doses, homozygous TPMT-deficient patients (~1 in 178 to 1 in 3,736 individuals with two nonfunctional TP...

  5. In vitro susceptibility and resistance phenotypes in contemporary Enterobacter isolates in a university hospital in Crete, Greece.

    Science.gov (United States)

    Maraki, Sofia; Vardakas, Konstantinos Z; Samonis, George; Perdikis, Dimitrios; Mavromanolaki, Viktoria Eirini; Kofteridis, Diamantis P; Falagas, Matthew E

    2017-06-01

    To study the evolution in the susceptibility of Enterobacter spp. in Crete, Greece from 2010 to 2015. Non-duplicate isolates were studied using automated systems. Phenotypic confirmatory tests were applied. A total of 939 Enterobacter isolates were included. Colistin was the most active antibiotic (97.9%) followed by imipenem (96.1%), gentamicin (95.7%), tigecycline (91.8%), cefepime (89.4%), chloramphenicol (85.8%), fosfomycin (85.5%), trimethoprim/sulfamethoxazole (83.3%) and piperacillin/tazobactam (73.3%). Antibiotic resistance did not increase during the study period for most antibiotics. Lower susceptibility was observed among multidrug-resistant strains and carbapenem-nonsusceptible isolates. AmpC was the most common resistant mechanism (21%); carbapenemases (3.7%) and aminoglycoside-modifying enzymes (6.5%) were also detected. A significant proportion of Enterobacter spp. was resistant to several antibiotics, most notably β-lactams.

  6. O6-methylguanine-DNA methyltransferase in wild-type and ada mutants of Escherichia coli

    International Nuclear Information System (INIS)

    Mitra, S.; Pal, B.C.; Foote, R.S.

    1982-01-01

    O 6 -Methylguanine-DNA methyltransferase is induced in Escherichia coli during growth in low levels of N-methyl-N'-nitro-N-nitrosoguanidine. We have developed a sensitive assay for quantitating low levels of this activity with a synthetic DNA substrate containing 3 H-labeled O 6 -methylguanine as the only modified base. Although both wild-type and adaptation-deficient (ada) mutants of E. coli contained low but comparable numbers (from 13 to 60) of the enzyme molecules per cell, adaptation treatment caused a significant increase of the enzyme in the wild type but not in the ada mutants, suggesting that the ada mutation is in a regulatory locus and not in the structural gene for the methyltransferase

  7. A WWW-based information system on resistance of bacteria to antibiotics.

    Science.gov (United States)

    Schindler, J; Schindler, Z; Schindler, J

    1998-01-01

    The information system on resistance of bacteria to antibiotics (WARN--World Antibiotic Resistance Network) is implemented as a WWW server at Charles University in Prague (http:/(/)www.warn.cas.cz). Its main goal is to give information about problems of antibiotic resistance of bacteria and to process data on isolated strains. The WARN web-site contains six main topics. Four of them form the core of the system: Topics of Interest bring information on selected timely topics in antibiotic resistance--pneumococci, staphylococci, beta-lactamases, glycopeptide--and aminoglycoside resistance. Global Monitor brings references and reports on resistance in the world as well as recommended method of surveillance. The topic Data contains raw data on strains in particular countries and hospitals. Data can be viewed in their original form as a list of records (strains) or processed to provide statistics about the resistance rates in the selected country or hospital respectively. The topic Search allows one to search for one or several terms in the whole document. Counts of accessed pages show, that there is a standing demand for information about the serious problems of antibiotic therapy of infectious diseases.

  8. Effects of nicotinamide N-methyltransferase on PANC-1 cells proliferation, metastatic potential and survival under metabolic stress.

    Science.gov (United States)

    Yu, Tao; Wang, Yong-Tao; Chen, Pan; Li, Yu-Hua; Chen, Yi-Xin; Zeng, Hang; Yu, Ai-Ming; Huang, Min; Bi, Hui-Chang

    2015-01-01

    Aberrant expression of Nicotinamide N-methyltransferase (NNMT) has been reported in pancreatic cancer. However, the role of NNMT in pancreatic cancer development remains elusive. Therefore, the present study was to investigate the impact of NNMT on pancreatic cancer cell proliferation, metastatic potential and survival under metabolic stress. Pancreatic cancer cell line PANC-1 was transfected with NNMT expression plasmid or small interfering RNA of NNMT to overexpress or knockdown intracellular NNMT expression, respectively. Rate of cell proliferation was monitored. Transwell migration and matrigel invasion assays were conducted to assess cell migration and invasion capacity. Resistance to glucose deprivation, sensitivity to glycolytic inhibition, mitochondrial inhibtion and resistance to rapamycin were examined to evaluate cell survival under metabolic stress. NNMT silencing markedly reduced cell proliferation, whereas NNMT overexpression promoted cell growth moderately. Knocking down NNMT also significantly suppressed the migration and invasion capacities of PANC-1 cells. Conversely, NNMT upregulation enhanced cell migration and invasion capacities. In addition, NNMT knockdown cells were much less resistant to glucose deprivation and rapamycin as well as glycolytic inhibitor 2-deoxyglucose whereas NNMT-expressing cells showed opposite effects although the effects were not so striking. These data sugguest that NNMT plays an important role in PANC-1 cell proliferation, metastatic potential and survival under metabolic stress. © 2015 S. Karger AG, Basel.

  9. Effects of Nicotinamide N-Methyltransferase on PANC-1 Cells Proliferation, Metastatic Potential and Survival Under Metabolic Stress

    Directory of Open Access Journals (Sweden)

    Tao Yu

    2015-01-01

    Full Text Available Background: Aberrant expression of Nicotinamide N-methyltransferase (NNMT has been reported in pancreatic cancer. However, the role of NNMT in pancreatic cancer development remains elusive. Therefore, the present study was to investigate the impact of NNMT on pancreatic cancer cell proliferation, metastatic potential and survival under metabolic stress. Methods: Pancreatic cancer cell line PANC-1 was transfected with NNMT expression plasmid or small interfering RNA of NNMT to overexpress or knockdown intracellular NNMT expression, respectively. Rate of cell proliferation was monitored. Transwell migration and matrigel invasion assays were conducted to assess cell migration and invasion capacity. Resistance to glucose deprivation, sensitivity to glycolytic inhibition, mitochondrial inhibtion and resistance to rapamycin were examined to evaluate cell survival under metabolic stress. Results: NNMT silencing markedly reduced cell proliferation, whereas NNMT overexpression promoted cell growth moderately. Knocking down NNMT also significantly suppressed the migration and invasion capacities of PANC-1 cells. Conversely, NNMT upregulation enhanced cell migration and invasion capacities. In addition, NNMT knockdown cells were much less resistant to glucose deprivation and rapamycin as well as glycolytic inhibitor 2-deoxyglucose whereas NNMT-expressing cells showed opposite effects although the effects were not so striking. Conclusions: These data sugguest that NNMT plays an important role in PANC-1 cell proliferation, metastatic potential and survival under metabolic stress.

  10. Crystallization and preliminary crystallographic analysis of tRNA (m{sup 7}G46) methyltransferase from Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Qi; Gao, Yang; Yang, Weili; Zhou, Huihao; Gao, Yongxiang; Zhang, Xiao; Teng, Maikun, E-mail: mkteng@ustc.edu.cn; Niu, Liwen, E-mail: mkteng@ustc.edu.cn [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230027 (China); Key Laboratory of Structural Biology, Chinese Academy of Sciences, 96 Jinzhai Road, Hefei, Anhui 230027 (China)

    2008-08-01

    tRNA (m{sup 7}G46) methyltransferase from E. coli was overexpressed, purified and crystallized. Diffraction data were collected to 2.04 Å resolution. Transfer RNA (tRNA) (m{sup 7}G46) methyltransferase (TrmB) belongs to the Rossmann-fold methyltransferase (RFM) family and uses S-adenosyl-l-methionine (SAM) as the methyl-group donor to catalyze the formation of N{sup 7}-methylguanosine (m{sup 7}G) at position 46 in the variable loop of tRNAs. After attempts to crystallize full-length Escherichia coli TrmB (EcTrmB) failed, a truncated protein lacking the first 32 residues of the N-terminus but with an additional His{sub 6} tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 3350 (PEG 3350) as precipitant at 283 K. An X-ray diffraction data set was collected using a single flash-cooled crystal that belonged to space group P2{sub 1}.

  11. A Reverse Genetics Approach for the Design of Methyltransferase-Defective Live Attenuated Avian Metapneumovirus Vaccines.

    Science.gov (United States)

    Zhang, Yu; Sun, Jing; Wei, Yongwei; Li, Jianrong

    2016-01-01

    Avian metapneumovirus (aMPV), also known as avian pneumovirus or turkey rhinotracheitis virus, is the causative agent of turkey rhinotracheitis and is associated with swollen head syndrome in chickens. aMPV belongs to the family Paramyxoviridae which includes many important human pathogens such as human respiratory syncytial virus (RSV), human metapneumovirus (hMPV), and human parainfluenza virus type 3 (PIV3). The family also includes highly lethal emerging pathogens such as Nipah virus and Hendra virus, as well as agriculturally important viruses such as Newcastle disease virus (NDV). For many of these viruses, there is no effective vaccine. Here, we describe a reverse genetics approach to develop live attenuated aMPV vaccines by inhibiting the viral mRNA cap methyltransferase. The viral mRNA cap methyltransferase is an excellent target for the attenuation of paramyxoviruses because it plays essential roles in mRNA stability, efficient viral protein translation and innate immunity. We have described in detail the materials and methods used to generate recombinant aMPVs that lack viral mRNA cap methyltransferase activity. We have also provided methods to evaluate the genetic stability, pathogenesis, and immunogenicity of live aMPV vaccine candidates in turkeys.

  12. The novel kasugamycin 2'-N-acetyltransferase gene aac(2')-IIa, carried by the IncP island, confers kasugamycin resistance to rice-pathogenic bacteria.

    Science.gov (United States)

    Yoshii, Atsushi; Moriyama, Hiromitsu; Fukuhara, Toshiyuki

    2012-08-01

    Kasugamycin (KSM), a unique aminoglycoside antibiotic, has been used in agriculture for many years to control not only rice blast caused by the fungus Magnaporthe grisea but also rice bacterial grain and seedling rot or rice bacterial brown stripe caused by Burkholderia glumae or Acidovorax avenae subsp. avenae, respectively. Since both bacterial pathogens are seed-borne and cause serious injury to rice seedlings, the emergence of KSM-resistant B. glumae and A. avenae isolates highlights the urgent need to understand the mechanism of resistance to KSM. Here, we identified a novel gene, aac(2')-IIa, encoding a KSM 2'-N-acetyltransferase from both KSM-resistant pathogens but not from KSM-sensitive bacteria. AAC(2')-IIa inactivates KSM, although it reveals no cross-resistance to other aminoglycosides. The aac(2')-IIa gene from B. glumae strain 5091 was identified within the IncP genomic island inserted into the bacterial chromosome, indicating the acquisition of this gene by horizontal gene transfer. Although excision activity of the IncP island and conjugational gene transfer was not detected under the conditions tested, circular intermediates containing the aac(2')-IIa gene were detected. These results indicate that the aac(2')-IIa gene had been integrated into the IncP island of a donor bacterial species. Molecular detection of the aac(2')-IIa gene could distinguish whether isolates are resistant or susceptible to KSM. This may contribute to the production of uninfected rice seeds and lead to the effective control of these pathogens by KSM.

  13. Incidence of high-level evernimicin resistance in Enterococcus faecium among food animals and humans

    DEFF Research Database (Denmark)

    Aarestrup, Frank Møller; McNicholas, P. M.

    2002-01-01

    Six high-level evernimicin-resistant Enterococcus faecium isolates were identified among 304 avilarnycin-resistant E. faecium isolates from animals and 404 stool samples from humans with diarrhea. All four animal isolates, and one of the human isolates, were able to transfer resistance...... to a susceptible E. faecium strain. The resulting transconjugants all tested positive for the presence of emtA, a gene encoding a methyltransferase previously linked with high-level evernimicin resistance. The four transconjugants derived from animal isolates all carried the same plasmid, while a differently sized...... plasmid was found in the isolate from humans. This study demonstrated a low incidence of high-level evernimicin resistance mediated by the emtA gene in different E. faecium isolates of animal and human origin....

  14. Microbiology and antimicrobial susceptibility of otitis externa: a changing pattern of antimicrobial resistance.

    Science.gov (United States)

    Heward, E; Cullen, M; Hobson, J

    2018-04-01

    Otitis externa is a common presentation to secondary care otolaryngology clinics. Despite this, few studies have investigated the microbiology and antimicrobial resistance of otitis externa. This study aimed to examine these issues. Analysis identified 302 swabs taken from 217 patients (100 male, 117 female), between 1 January 2015 and 30 March 2016, at our rapid access otolaryngology clinic. In total, 315 organisms were isolated; the most frequent was Pseudomonas aeruginosa (31.1 per cent), followed by candida species (22.9 per cent) and Staphylococcus aureus (11.7 per cent). P aeruginosa was sensitive to ciprofloxacin in 97.7 per cent of cases and to gentamicin in 78.4 per cent. Compared with studies worldwide, the relative proportions of different organisms causing otitis externa and the patterns of antimicrobial resistance differ. Increasing resistance of P aeruginosa to aminoglycosides demonstrates a changing pattern of antimicrobial resistance that has not been previously reported. Reassuringly, quinolone antibiotics remain highly effective when treating P aeruginosa.

  15. Molecular cloning and functional expression of a stress-induced multifunctional O-methyltransferase with pinosylvin methyltransferase activity from Scots pine (Pinus sylvestris L.).

    Science.gov (United States)

    Chiron, H; Drouet, A; Claudot, A C; Eckerskorn, C; Trost, M; Heller, W; Ernst, D; Sandermann, H

    2000-12-01

    Formation of pinosylvin (PS) and pinosylvin 3-O-monomethyl ether (PSM), as well as the activities of stilbene synthase (STS) and S-adenosyl-1-methionine (SAM):pinosylvin O-methyltransferase (PMT), were induced strongly in needles of Scots pine seedlings upon ozone treatment, as well as in cell suspension cultures of Scots pine upon fungal elicitation. A SAM-dependent PMT protein was purified and partially characterised. A cDNA encoding PMT was isolated from an ozone-induced Scots pine cDNA library. Southern blot analysis of the genomic DNA suggested the presence of a gene family. The deduced protein sequence showed the typical highly conserved regions of O-methyltransferases (OMTs), and average identities of 20-56% to known OMTs. PMT expressed in Escherichia coli corresponded to that of purified PMT (40 kDa) from pine cell cultures. The recombinant enzyme catalysed the methylation of PS, caffeic acid, caffeoyl-CoA and quercetin. Several other substances, such as astringenin, resveratrol, 5-OH-ferulic acid, catechol and luteolin, were also methylated. Recombinant PMT thus had a relatively broad substrate specificity. Treatment of 7-year old Scots pine trees with ozone markedly increased the PMT mRNA level. Our results show that PMT represents a new SAM-dependent OMT for the methylation of stress-induced pinosylvin in Scots pine needles.

  16. Whole genome analysis of linezolid resistance in Streptococcus pneumoniae reveals resistance and compensatory mutations

    Directory of Open Access Journals (Sweden)

    Légaré Danielle

    2011-10-01

    Full Text Available Abstract Background Several mutations were present in the genome of Streptococcus pneumoniae linezolid-resistant strains but the role of several of these mutations had not been experimentally tested. To analyze the role of these mutations, we reconstituted resistance by serial whole genome transformation of a novel resistant isolate into two strains with sensitive background. We sequenced the parent mutant and two independent transformants exhibiting similar minimum inhibitory concentration to linezolid. Results Comparative genomic analyses revealed that transformants acquired G2576T transversions in every gene copy of 23S rRNA and that the number of altered copies correlated with the level of linezolid resistance and cross-resistance to florfenicol and chloramphenicol. One of the transformants also acquired a mutation present in the parent mutant leading to the overexpression of an ABC transporter (spr1021. The acquisition of these mutations conferred a fitness cost however, which was further enhanced by the acquisition of a mutation in a RNA methyltransferase implicated in resistance. Interestingly, the fitness of the transformants could be restored in part by the acquisition of altered copies of the L3 and L16 ribosomal proteins and by mutations leading to the overexpression of the spr1887 ABC transporter that were present in the original linezolid-resistant mutant. Conclusions Our results demonstrate the usefulness of whole genome approaches at detecting major determinants of resistance as well as compensatory mutations that alleviate the fitness cost associated with resistance.

  17. Expression of DNA methyltransferases is influenced by growth hormone in the long-living Ames dwarf mouse in vivo and in vitro.

    Science.gov (United States)

    Armstrong, Vanessa L; Rakoczy, Sharlene; Rojanathammanee, Lalida; Brown-Borg, Holly M

    2014-08-01

    Methyltransferase expression and DNA methylation are linked to aging and age-related disease. We utilized 3-, 12-, and 24-month-old Ames dwarf and their wild-type siblings to examine the genotype and age-related differences in the expression of methyltransferase enzymes related to DNA methylation in the liver, glycine-N-methyltransferase and DNA methyltransferase (DNMT). We found that DNMT proteins and transcripts are differentially expressed in dwarf mice compared with wild-type siblings that can be attributed to age and/or genotype. However, DNMT1 protein expression is drastically reduced compared with wild-type controls at every age. DNMT3a protein levels coincide with differences observed in DNMT activity. Growth hormone appears to modulate expression of DNMT1 and 3a in dwarf liver tissue and primary hepatocytes. Therefore, growth hormone may contribute to age-related processes, DNA methylation, and, ultimately, longevity. © The Author 2013. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. Time-dependent inactivation of human phenylethanolamine N-methyltransferase by 7-isothiocyanatotetrahydroisoquinoline

    Science.gov (United States)

    Wu, Qian; Caine, Joanne M.; Thomson, Stuart A.; Slavica, Meri; Grunewald, Gary L.

    2009-01-01

    Inhibitors of phenylethanolamine N-methyltransferase [PNMT, the enzyme that catalyzes the final step in the biosynthesis of epinephrine (Epi)] may be of use in determining the role of Epi in the central nervous system. Here we describe the synthesis and characterization of 7-SCN tetrahydroisoquinoline as an affinity label for human PNMT. PMID:19171483

  19. Multi-clonal evolution of multi-drug-resistant/extensively drug-resistant Mycobacterium tuberculosis in a high-prevalence setting of Papua New Guinea for over three decades

    Science.gov (United States)

    Bainomugisa, Arnold; Lavu, Evelyn; Hiashiri, Stenard; Majumdar, Suman; Honjepari, Alice; Moke, Rendi; Dakulala, Paison; Hill-Cawthorne, Grant A.; Pandey, Sushil; Marais, Ben J.; Coulter, Chris; Coin, Lachlan

    2018-01-01

    An outbreak of multi-drug resistant (MDR) tuberculosis (TB) has been reported on Daru Island, Papua New Guinea. Mycobacterium tuberculosis strains driving this outbreak and the temporal accrual of drug resistance mutations have not been described. Whole genome sequencing of 100 of 165 clinical isolates referred from Daru General Hospital to the Supranational reference laboratory, Brisbane, during 2012–2015 revealed that 95 belonged to a single modern Beijing sub-lineage strain. Molecular dating suggested acquisition of streptomycin and isoniazid resistance in the 1960s, with potentially enhanced virulence mediated by an mycP1 mutation. The Beijing sub-lineage strain demonstrated a high degree of co-resistance between isoniazid and ethionamide (80/95; 84.2 %) attributed to an inhA promoter mutation combined with inhA and ndh coding mutations. Multi-drug resistance, observed in 78/95 samples, emerged with the acquisition of a typical rpoB mutation together with a compensatory rpoC mutation in the 1980s. There was independent acquisition of fluoroquinolone and aminoglycoside resistance, and evidence of local transmission of extensively drug resistant (XDR) strains from 2009. These findings underline the importance of whole genome sequencing in informing an effective public health response to MDR/XDR TB. PMID:29310751

  20. The Novel Kasugamycin 2′-N-Acetyltransferase Gene aac(2′)-IIa, Carried by the IncP Island, Confers Kasugamycin Resistance to Rice-Pathogenic Bacteria

    Science.gov (United States)

    Moriyama, Hiromitsu; Fukuhara, Toshiyuki

    2012-01-01

    Kasugamycin (KSM), a unique aminoglycoside antibiotic, has been used in agriculture for many years to control not only rice blast caused by the fungus Magnaporthe grisea but also rice bacterial grain and seedling rot or rice bacterial brown stripe caused by Burkholderia glumae or Acidovorax avenae subsp. avenae, respectively. Since both bacterial pathogens are seed-borne and cause serious injury to rice seedlings, the emergence of KSM-resistant B. glumae and A. avenae isolates highlights the urgent need to understand the mechanism of resistance to KSM. Here, we identified a novel gene, aac(2′)-IIa, encoding a KSM 2′-N-acetyltransferase from both KSM-resistant pathogens but not from KSM-sensitive bacteria. AAC(2′)-IIa inactivates KSM, although it reveals no cross-resistance to other aminoglycosides. The aac(2′)-IIa gene from B. glumae strain 5091 was identified within the IncP genomic island inserted into the bacterial chromosome, indicating the acquisition of this gene by horizontal gene transfer. Although excision activity of the IncP island and conjugational gene transfer was not detected under the conditions tested, circular intermediates containing the aac(2′)-IIa gene were detected. These results indicate that the aac(2′)-IIa gene had been integrated into the IncP island of a donor bacterial species. Molecular detection of the aac(2′)-IIa gene could distinguish whether isolates are resistant or susceptible to KSM. This may contribute to the production of uninfected rice seeds and lead to the effective control of these pathogens by KSM. PMID:22660700

  1. Molecular phylogenetics and comparative modeling of HEN1, a methyltransferase involved in plant microRNA biogenesis

    Directory of Open Access Journals (Sweden)

    Obarska Agnieszka

    2006-01-01

    Full Text Available Abstract Background Recently, HEN1 protein from Arabidopsis thaliana was discovered as an essential enzyme in plant microRNA (miRNA biogenesis. HEN1 transfers a methyl group from S-adenosylmethionine to the 2'-OH or 3'-OH group of the last nucleotide of miRNA/miRNA* duplexes produced by the nuclease Dicer. Previously it was found that HEN1 possesses a Rossmann-fold methyltransferase (RFM domain and a long N-terminal extension including a putative double-stranded RNA-binding motif (DSRM. However, little is known about the details of the structure and the mechanism of action of this enzyme, and about its phylogenetic origin. Results Extensive database searches were carried out to identify orthologs and close paralogs of HEN1. Based on the multiple sequence alignment a phylogenetic tree of the HEN1 family was constructed. The fold-recognition approach was used to identify related methyltransferases with experimentally solved structures and to guide the homology modeling of the HEN1 catalytic domain. Additionally, we identified a La-like predicted RNA binding domain located C-terminally to the DSRM domain and a domain with a peptide prolyl cis/trans isomerase (PPIase fold, but without the conserved PPIase active site, located N-terminally to the catalytic domain. Conclusion The bioinformatics analysis revealed that the catalytic domain of HEN1 is not closely related to any known RNA:2'-OH methyltransferases (e.g. to the RrmJ/fibrillarin superfamily, but rather to small-molecule methyltransferases. The structural model was used as a platform to identify the putative active site and substrate-binding residues of HEN and to propose its mechanism of action.

  2. Crystal structure of arginine methyltransferase 6 from Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Chongyuan Wang

    Full Text Available Arginine methylation plays vital roles in the cellular functions of the protozoan Trypanosoma brucei. The T. brucei arginine methyltransferase 6 (TbPRMT6 is a type I arginine methyltransferase homologous to human PRMT6. In this study, we report the crystal structures of apo-TbPRMT6 and its complex with the reaction product S-adenosyl-homocysteine (SAH. The structure of apo-TbPRMT6 displays several features that are different from those of type I PRMTs that were structurally characterized previously, including four stretches of insertion, the absence of strand β15, and a distinct dimerization arm. The comparison of the apo-TbPRMT6 and SAH-TbPRMT6 structures revealed the fine rearrangements in the active site upon SAH binding. The isothermal titration calorimetry results demonstrated that SAH binding greatly increases the affinity of TbPRMT6 to a substrate peptide derived from bovine histone H4. The western blotting and mass spectrometry results revealed that TbPRMT6 methylates bovine histone H4 tail at arginine 3 but cannot methylate several T. brucei histone tails. In summary, our results highlight the structural differences between TbPRMT6 and other type I PRMTs and reveal that the active site rearrangement upon SAH binding is important for the substrate binding of TbPRMT6.

  3. YebU is a m5C methyltransferase specific for 16 S rRNA nucleotide 1407

    DEFF Research Database (Denmark)

    Andersen, Niels Møller; Douthwaite, Stephen

    2006-01-01

    generally require specific enzymes, and only one m5C rRNA methyltransferase, RsmB (formerly Fmu) that methylates nucleotide C967, has previously been identified. BLAST searches of the E.coli genome revealed a single gene, yebU, with sufficient similarity to rsmB to encode a putative m5C RNA...... methyltransferase. This suggested that the yebU gene product modifies C1407 and/or C1962. Here, we analysed the E.coli rRNAs by matrix assisted laser desorption/ionization mass spectrometry and show that inactivation of the yebU gene leads to loss of methylation at C1407 in 16 S rRNA, but does not interfere...

  4. Antimicrobial resistance in Salmonella spp. recovered from patients admitted to six different hospitals in Tehran, Iran from 2007 to 2008

    DEFF Research Database (Denmark)

    Tajbakhsh, Mercedeh; Hendriksen, Rene S.; Nochi, Zahra

    2012-01-01

    were screened for the presence of Salmonella, serotyped, tested for antimicrobial susceptibility using disk diffusion and examined for the presence of relevant resistance genes and integrons by PCR. A total of 1,120 patients were screened for the presence of Salmonella. Out of 71 Salmonella isolates...... recovered, the following serovars were identified: 17 Typhi, 14 Paratyphi C, 13 Enteritidis, 11 Paratyphi B, 10 Paratyphi A and six Infantis. Most resistance was observed towards sulfamethoxazole (30%), tetracyclines (25%), nalidixic acid (22%), spectinomycin (17%), trimethoprim (15%), ampicillin (14......%) and kanamycin (14%). The tetracycline resistance genes tet(A), tet(B), and tet(G) were found in 28%, 14% and 6% of the tetracycline resistant isolates, respectively. The genes aadA, aadB, strA, strB and aphA1-Iab were present in 83%, 55%, 34%, 1% and 17% of the aminoglycoside resistant isolates, respectively...

  5. Aminoglycoside-induced and non-syndromic hearing loss is associated with the G7444A mutation in the mitochondrial COI/tRNASer(UCN) genes in two Chinese families

    International Nuclear Information System (INIS)

    Zhu Yi; Qian Yaping; Tang Xiaowen; Wang Jindan; Yang Li; Liao Zhisu; Li Ronghua; Ji Jinzhang; Li Zhiyuan; Chen Jianfu; Choo, Daniel I.; Lu Jianxin; Guan Minxin

    2006-01-01

    We report here the clinical, genetic, and molecular characterization of two Chinese families with aminoglycoside induced and non-syndromic hearing impairment. Clinical and genetic evaluations revealed the variable severity and age-of-onset in hearing impairment in these families. Strikingly, there were extremely low penetrances of hearing impairment in these Chinese families. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the distinct sets of mtDNA polymorphism, in addition to the identical G7444A mutation associated with hearing loss. Indeed, the G7444A mutation in the CO1 gene and the precursor of tRNA Ser(UCN) gene is present in homoplasmy only in the maternal lineage of those pedigrees but not other members of these families and 164 Chinese controls. Their mitochondrial genomes belong to the Eastern Asian haplogroups C5a and D4a, respectively. In fact, the occurrence of the G7444A mutation in these several genetically unrelated subjects affected by hearing impairment strongly indicates that this mutation is involved in the pathogenesis of hearing impairment. However, there was the absence of other functionally significant mtDNA mutations in two Chinese pedigrees carrying the G7444A mutation. Therefore, nuclear modifier gene(s) or aminoglycoside(s) may play a role in the phenotypic expression of the deafness-associated G7444A mutation in these Chinese pedigrees

  6. MicroRNA-29a Alleviates Bile Duct Ligation Exacerbation of Hepatic Fibrosis in Mice through Epigenetic Control of Methyltransferases

    Directory of Open Access Journals (Sweden)

    Ya-Ling Yang

    2017-01-01

    Full Text Available MicroRNA-29 (miR-29 is found to modulate hepatic stellate cells’ (HSCs activation and, thereby, reduces liver fibrosis pathogenesis. Histone methyltransferase regulation of epigenetic reactions reportedly participates in hepatic fibrosis. This study is undertaken to investigate the miR-29a regulation of the methyltransferase signaling and epigenetic program in hepatic fibrosis progression. miR-29a transgenic mice (miR-29aTg mice and wild-type littermates were subjected to bile duct-ligation (BDL to develop cholestatic liver fibrosis. Primary HSCs were transfected with a miR-29a mimic and antisense inhibitor. Profibrogenic gene expression, histone methyltransferases and global genetic methylation were probed with real-time quantitative RT-PCR, immunohistochemical stain, Western blot and ELISA. Hepatic tissue in miR-29aTg mice displayed weak fibrotic matrix as evidenced by Sirius Red staining concomitant with low fibrotic matrix collagen 1α1 expression within affected tissues compared to the wild-type mice. miR-29a overexpression reduced the BDL exaggeration of methyltransferases, DNMT1, DNMT3b and SET domain containing 1A (SET1A expression. It also elevated phosphatase and tensin homolog deleted on chromosome 10 (PTEN signaling within liver tissue. In vitro, miR-29a mimic transfection lowered collagen 1α1, DNMT1, DNMT3b and SET1A expression in HSCs. Gain of miR-29a signaling resulted in DNA hypomethylation and high PTEN expression. This study shines a new light on miR-29a inhibition of methyltransferase, a protective effect to maintain the DNA hypomethylation state that decreases fibrogenic activities in HSC. These robust analyses also highlight the miR-29a regulation of epigenetic actions to ameliorate excessive fibrosis during cholestatic liver fibrosis development.

  7. Identification of the yeast gene encoding the tRNA m1G methyltransferase responsible for modification at position 9.

    Science.gov (United States)

    Jackman, Jane E; Montange, Rebecca K; Malik, Harmit S; Phizicky, Eric M

    2003-05-01

    Methylation of tRNA at the N-1 position of guanosine to form m(1)G occurs widely in nature. It occurs at position 37 in tRNAs from all three kingdoms, and the methyltransferase that catalyzes this reaction is known from previous work of others to be critically important for cell growth in Escherichia coli and the yeast Saccharomyces cerevisiae. m(1)G is also widely found at position 9 in eukaryotic tRNAs, but the corresponding methyltransferase was unknown. We have used a biochemical genomics approach with a collection of purified yeast GST-ORF fusion proteins to show that m(1)G(9) formation of yeast tRNA(Gly) is associated with ORF YOL093w, named TRM10. Extracts lacking Trm10p have undetectable levels of m(1)G(9) methyltransferase activity but retain normal m(1)G(37) methyltransferase activity. Yeast Trm10p purified from E. coli quantitatively modifies the G(9) position of tRNA(Gly) in an S-adenosylmethionine-dependent fashion. Trm10p is responsible in vivo for most if not all m(1)G(9) modification of tRNAs, based on two results: tRNA(Gly) purified from a trm10-Delta/trm10-Delta strain is lacking detectable m(1)G; and a primer extension block occurring at m(1)G(9) is removed in trm10-Delta/trm10-Delta-derived tRNAs for all 9 m(1)G(9)-containing species that were testable by this method. There is no obvious growth defect of trm10-Delta/trm10-Delta strains. Trm10p bears no detectable resemblance to the yeast m(1)G(37) methyltransferase, Trm5p, or its orthologs. Trm10p homologs are found widely in eukaryotes and many archaea, with multiple homologs in several metazoans, including at least three in humans.

  8. Friend of Prmt1, a novel chromatin target of protein arginine methyltransferases

    NARCIS (Netherlands)

    T.B. van Dijk (Thamar); N. Gillemans (Nynke); C. Stein (Claudia); P. Fanis (Pavlos); J.A.A. Demmers (Jeroen); M.P.C. van de Corput (Mariëtte); J. Essers (Jeroen); F.G. Grosveld (Frank); U.M. Bauer (Uta-Maria); J.N.J. Philipsen (Sjaak)

    2010-01-01

    textabstractWe describe the isolation and characterization of Friend of Prmt1 (Fop), a novel chromatin target of protein arginine methyltransferases. Human Fop is encoded by C1orf77, a gene of previously unknown function. We show that Fop is tightly associated with chromatin, and that it is modified

  9. Antimicrobial resistance of Pseudomonas spp. isolated from wastewater and wastewater-impacted marine coastal zone.

    Science.gov (United States)

    Luczkiewicz, Aneta; Kotlarska, Ewa; Artichowicz, Wojciech; Tarasewicz, Katarzyna; Fudala-Ksiazek, Sylwia

    2015-12-01

    In this study, species distribution and antimicrobial susceptibility of cultivated Pseudomonas spp. were studied in influent (INF), effluent (EFF), and marine outfall (MOut) of wastewater treatment plant (WWTP). The susceptibility was tested against 8 antimicrobial classes, active against Pseudomonas spp.: aminoglycosides, carbapenems, broad-spectrum cephalosporins from the 3rd and 4th generation, extended-spectrum penicillins, as well as their combination with the β-lactamase inhibitors, monobactams, fluoroquinolones, and polymyxins. Among identified species, resistance to all antimicrobials but colistin was shown by Pseudomonas putida, the predominant species in all sampling points. In other species, resistance was observed mainly against ceftazidime, ticarcillin, ticarcillin-clavulanate, and aztreonam, although some isolates of Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas pseudoalcaligenes, and Pseudomonas protegens showed multidrug-resistance (MDR) phenotype. Among P. putida, resistance to β-lactams and to fluoroquinolones as well as multidrug resistance become more prevalent after wastewater treatment, but the resistance rate decreased in marine water samples. Obtained data, however, suggests that Pseudomonas spp. are equipped or are able to acquire a wide range of antibiotic resistance mechanisms, and thus should be monitored as possible source of resistance genes.

  10. Dnmt3a is an epigenetic mediator of adipose insulin resistance

    DEFF Research Database (Denmark)

    You, Dongjoo; Nilsson, Emma; Tenen, Danielle E.

    2017-01-01

    Insulin resistance results from an intricate interaction between genetic make-up and environment, and thus may be orchestrated by epigenetic mechanisms like DNA methylation. Here, we demonstrate that DNA methyltransferase 3a (Dnmt3a) is both necessary and sufficient to mediate insulin resistance...... in cultured mouse and human adipocytes. Furthermore, adipose-specific Dnmt3a knock-out mice are protected from diet-induced insulin resistance and glucose intolerance without accompanying changes in adiposity. Unbiased gene profiling studies revealed Fgf21 as a key negatively regulated Dnmt3a target gene...... in adipocytes with concordant changes in DNA methylation at the Fgf21 promoter region. Consistent with this, Fgf21 can rescue Dnmt3a-mediated insulin resistance, and DNA methylation at the FGF21 locus was elevated in human subjects with diabetes and correlated negatively with expression of FGF21 in human...

  11. Mitochondrial targeting of human O6-methylguanine DNA methyltransferase protects against cell killing by chemotherapeutic alkylating agents.

    Science.gov (United States)

    Cai, Shanbao; Xu, Yi; Cooper, Ryan J; Ferkowicz, Michael J; Hartwell, Jennifer R; Pollok, Karen E; Kelley, Mark R

    2005-04-15

    DNA repair capacity of eukaryotic cells has been studied extensively in recent years. Mammalian cells have been engineered to overexpress recombinant nuclear DNA repair proteins from ectopic genes to assess the impact of increased DNA repair capacity on genome stability. This approach has been used in this study to specifically target O(6)-methylguanine DNA methyltransferase (MGMT) to the mitochondria and examine its impact on cell survival after exposure to DNA alkylating agents. Survival of human hematopoietic cell lines and primary hematopoietic CD34(+) committed progenitor cells was monitored because the baseline repair capacity for alkylation-induced DNA damage is typically low due to insufficient expression of MGMT. Increased DNA repair capacity was observed when K562 cells were transfected with nuclear-targeted MGMT (nucl-MGMT) or mitochondrial-targeted MGMT (mito-MGMT). Furthermore, overexpression of mito-MGMT provided greater resistance to cell killing by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) than overexpression of nucl-MGMT. Simultaneous overexpression of mito-MGMT and nucl-MGMT did not enhance the resistance provided by mito-MGMT alone. Overexpression of either mito-MGMT or nucl-MGMT also conferred a similar level of resistance to methyl methanesulfonate (MMS) and temozolomide (TMZ) but simultaneous overexpression in both cellular compartments was neither additive nor synergistic. When human CD34(+) cells were infected with oncoretroviral vectors that targeted O(6)-benzylguanine (6BG)-resistant MGMT (MGMT(P140K)) to the nucleus or the mitochondria, committed progenitors derived from infected cells were resistant to 6BG/BCNU or 6BG/TMZ. These studies indicate that mitochondrial or nuclear targeting of MGMT protects hematopoietic cells against cell killing by BCNU, TMZ, and MMS, which is consistent with the possibility that mitochondrial DNA damage and nuclear DNA damage contribute equally to alkylating agent-induced cell killing during chemotherapy.

  12. Interactions within the mammalian DNA methyltransferase family

    Directory of Open Access Journals (Sweden)

    Ehrenhofer-Murray Ann E

    2003-05-01

    Full Text Available Abstract Background In mammals, epigenetic information is established and maintained via the postreplicative methylation of cytosine residues by the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Dnmt1 is required for maintenance methylation whereas Dnmt3a and Dnmt3b are responsible for de novo methylation. Contrary to Dnmt3a or Dnmt3b, the isolated C-terminal region of Dnmt1 is catalytically inactive, despite the presence of the sequence motifs typical of active DNA methyltransferases. Deletion analysis has revealed that a large part of the N-terminal domain is required for enzymatic activity. Results The role played by the N-terminal domain in this regulation has been investigated using the yeast two-hybrid system. We show here the presence of an intra-molecular interaction in Dnmt1 but not in Dnmt3a or Dnmt3b. This interaction was confirmed by immunoprecipitation and was localized by deletion mapping. Furthermore, a systematic analysis of interactions among the Dnmt family members has revealed that DNMT3L interacts with the C-terminal domain of Dnmt3a and Dnmt3b. Conclusions The lack of methylating ability of the isolated C-terminal domain of Dnmt1 could be explained in part by a physical interaction between N- and C-terminal domains that apparently is required for activation of the catalytic domain. Our deletion analysis suggests that the tertiary structure of Dnmt1 is important in this process rather than a particular sequence motif. Furthermore, the interaction between DNMT3L and the C-terminal domains of Dnmt3a and Dnmt3b suggests a mechanism whereby the enzymatically inactive DNMT3L brings about the methylation of its substrate by recruiting an active methylase.

  13. Interactions within the mammalian DNA methyltransferase family

    Science.gov (United States)

    Margot, Jean B; Ehrenhofer-Murray, Ann E; Leonhardt, Heinrich

    2003-01-01

    Background In mammals, epigenetic information is established and maintained via the postreplicative methylation of cytosine residues by the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Dnmt1 is required for maintenance methylation whereas Dnmt3a and Dnmt3b are responsible for de novo methylation. Contrary to Dnmt3a or Dnmt3b, the isolated C-terminal region of Dnmt1 is catalytically inactive, despite the presence of the sequence motifs typical of active DNA methyltransferases. Deletion analysis has revealed that a large part of the N-terminal domain is required for enzymatic activity. Results The role played by the N-terminal domain in this regulation has been investigated using the yeast two-hybrid system. We show here the presence of an intra-molecular interaction in Dnmt1 but not in Dnmt3a or Dnmt3b. This interaction was confirmed by immunoprecipitation and was localized by deletion mapping. Furthermore, a systematic analysis of interactions among the Dnmt family members has revealed that DNMT3L interacts with the C-terminal domain of Dnmt3a and Dnmt3b. Conclusions The lack of methylating ability of the isolated C-terminal domain of Dnmt1 could be explained in part by a physical interaction between N- and C-terminal domains that apparently is required for activation of the catalytic domain. Our deletion analysis suggests that the tertiary structure of Dnmt1 is important in this process rather than a particular sequence motif. Furthermore, the interaction between DNMT3L and the C-terminal domains of Dnmt3a and Dnmt3b suggests a mechanism whereby the enzymatically inactive DNMT3L brings about the methylation of its substrate by recruiting an active methylase. PMID:12777184

  14. The management of multidrug-resistant Enterobacteriaceae.

    Science.gov (United States)

    Bassetti, Matteo; Peghin, Maddalena; Pecori, Davide

    2016-12-01

    Multidrug-resistant (MDR) Enterobacteriaceae are often related to the production of extended-spectrum b-lactamases (ESBLs) and carbapenemase-producing Enterobacteriaceae (CRE), and represent an increasing global threat. Recommendations for the therapeutic management of MDR-related infections, however, are mainly derived from retrospective and nonrandomized prospective studies. The aim of this review is to discuss the challenges in the treatment of patients with infections because of MDR Enterobacteriaceae and provide an expert opinion while awaiting for more definitive data. To avoid the selection of carbapenemase-producing Enterobacteriaceae, carbapenem-sparing strategies should be considered. B-lactams/b-lactamase inhibitors, mainly piperacillin-tazobactam, minimum inhibitory concentration (MIC) 16/4mg/ml or less represents the best alternative to carbapenems for the treatment of ESBL-producing strains. Overall, combination therapy may be preferred over monotherapy for CRE. The combination of a carbapenem-containing regimen with colistin or high-dose tigecycline or aminoglycoside can be administered at high-dose prolonged infusion with therapeutic drug monitoring for the treatment of CRE with MIC for meropenem 8-16 mg/l or less. For MIC higher than 8-16 mg/l, the use of meropenem should be avoided and various combination therapies based on the in-vitro susceptibility of antimicrobials (e.g., colistin, high-dose tigecycline, fosfomycin, and aminoglycosides) should be selected. Carbapenem-sparing strategies should be used, when feasible, for ESBL infections. The majority of available nonrandomized studies highlight that combination for CRE seem to offer some therapeutic advantage over monotherapy. Strict infection control measures toward MDR Gram-negative pathogens remain necessary while awaiting for new treatment options.

  15. Antibiotic resistance of staphylococci from humans, food and different animal species according to data of the Hungarian resistance monitoring system in 2001.

    Science.gov (United States)

    Kaszanyitzky, Eva J; Jánosi, Sz; Egyed, Zsuzsanna; Agost, Gizella; Semjén, G

    2003-01-01

    Based on data of the Hungarian resistance monitoring system the antibiotic resistance of Staphylococcus strains of human and animal origin was studied. No methicillin-resistant staphylococci harbouring mecA gene were isolated from animals in 2001. Penicillin resistance, mediated by penicillinase production, was the most frequent among Staphylococcus aureus strains isolated from humans (96%), from bovine mastitis (55%), from foods (45%) and from dogs. In staphylococci isolated from animals low resistance percentages to aminoglycosides (0-2%), fluoroquinolones (0.5-3%) and sulphonamides (0.5-4%) were found but in strains isolated humans these figures were higher (1-14%, 5-18% and 3-31%, respectively). The most frequent antibiotic resistance profiles of strains isolated from animals and food were penicillin/tetracycline, penicillin/lincomycin and penicillin/lincomycin/tetracycline. Penicillin/tetracycline resistance was exhibited by strains from mastitis (3), samples from the meat industry (31), poultry flocks (1), poultry industry (1), noodle (1) and horses (2). Penicillin/lincomycin resistance was found in 10 Staphylococcus strains from mastitis, 1 from the dairy industry, 1 from the meat industry and 6 from dogs. Isolates from mastitis (2), from the dairy industry (2), from pigs (1), from the meat industry (1) and from poultry (1) harboured penicillin/lincomycin/tetracycline resistance pattern. Multiresistant strains were usually isolated only from one and sometimes from two animal species; therefore, the spread of defined resistant strains (clones) among different animal species could not be demonstrated. These results also suggest that the transfer of antibiotic resistance of S. aureus from animals to humans probably occurs less frequently than is generally assumed.

  16. Resistance pattern of clinical isolates of staphylococcus aureus against five groups of antibiotics

    International Nuclear Information System (INIS)

    Farzana, K.; Hameed, A.

    2006-01-01

    Among the samples received in pathology laboratory, Pakistan institute of Medical Science, Islamabad, 5069 samples had bacterial growth, among these 2580 (51%) samples were Gram-positive cocci and 1688 were Staphylococcus aureus during a period of two years. Out of these Gram-positive cocci 56% were resistant to penicillin group, 27% were resistant to cephalosporin group, 22% were resistant to aminoglycoside group 15% were resistant to quinolone group and 31% were resistant to other antibiotics (cotrimaxazole, erythromycin, aztreonam, vancomycin, nitrofurantion and meropenam). Antibio-grams of Gram-positive cocci were determined against various antibiotics by disc diffusion method. The rate of resistance to most of the antibiotics such as ampicillin, piperacillin, carbenicillin, penicillin, cephradine, cefotaxime, erythromycin, ceclor, ofloxacin, pefloxacin, ciprofloxacin, cotrimexazole (septran), gentamicin, meropenem, ceftazidime, erythromycin, tobramycin, enoxacin was higher when tested against the isolates collected from pus as compared to those from blood and urine. Antibiotic resistant strains were more prevalent in pus samples than other clinical isolates (blood and urine). The randomly selected 155 strains of Staphylococcus aureus when tested against five groups of antibiotics showed resistance rate against ampicillin (92%), cephradine (92%), cephradine (60%), and gentamicin (58%). However intermediate resistance was found in case of vancomicin (38%), in hospitalized and non-hospitalized patients. (author)

  17. Novel pathways for ameliorating the fitness cost of gentamicin resistant small colony variants

    DEFF Research Database (Denmark)

    Vestergaard, Martin; Paulander, Wilhelm Erik Axel; Leng, Bingfeng

    2016-01-01

    Small colony variants (SCVs) of the human pathogen Staphylococcus aureus are associated with persistent infections. Phenotypically, SCVs are characterized by slow growth and they can arise upon interruption of the electron transport chain that consequently reduce membrane potential and thereby...... limit uptake of aminoglycosides (e.g., gentamicin). In this study, we have examined the pathways by which the fitness cost of SCVs can be ameliorated. Five gentamicin resistant SCVs derived from S. aureus JE2 were independently selected on agar plates supplemented with gentamicin. The SCVs carried...... mutations in the menaquinone and hemin biosynthesis pathways, which caused a significant reduction in exponential growth rates relative to wild type (WT; 0.59-0.72) and reduced membrane potentials. Fifty independent lineages of the low-fitness, resistant mutants were serially passaged for up to 500...

  18. Occurrence of transferable antibiotic resistances in commercialized ready-to-eat mealworms (Tenebrio molitor L.).

    Science.gov (United States)

    Osimani, Andrea; Cardinali, Federica; Aquilanti, Lucia; Garofalo, Cristiana; Roncolini, Andrea; Milanović, Vesna; Pasquini, Marina; Tavoletti, Stefano; Clementi, Francesca

    2017-12-18

    The present study aimed to assess the occurrence of transferable determinants conferring resistance to tetracyclines, macrolide-lincosamide-streptogramin B, vancomycin, beta-lactams, and aminoglycosides in 40 samples of commercialized edible mealworms (Tenebrio molitor L.) purchased from European Union (EU) and non-EU producers. A high prevalence of tet(K) was observed in all of the samples assayed, with percentages of PCR-based positivity that ranged from 80% (samples from Thailand) to 100% (samples from the Netherlands, Belgium and France). For macrolides, erm(B) prevailed, being detected in 57.5% of the samples assayed, whereas erm(A) and erm(C) were detected with lower frequencies. Genes for resistance to vancomycin were only detected in samples produced in France and Belgium, with 90% and 10% of the samples being positive for vanA, respectively. Beta-lactamase genes were found with low occurrence, whereas the gene aac-aph, conferring high resistance to aminoglycosides, was found in 40% of the samples produced in the Netherlands and Belgium and 20% of the samples produced in Thailand. The results of Principal Coordinate Analysis and Principal Component Analysis depicted a clean separation of the samples collected from the four producers based on the distribution of the 12 AR determinants considered. Given the growing interest on the use of mealworms as a novel protein source, AR detection frequencies found in the present study suggest further investigation into the use of antibiotics during rearing of this insect species and more extensive studies focused on the factors that can affect the diffusion of transferable ARs in the production chain. Until such studies are completed, prudent use of antibiotics during rearing of edible insects is recommended. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Will new antimicrobials overcome resistance among Gram-negatives?

    Science.gov (United States)

    Bassetti, Matteo; Ginocchio, Francesca; Mikulska, Małgorzata; Taramasso, Lucia; Giacobbe, Daniele Roberto

    2011-10-01

    The spread of resistance among Gram-positive and Gram-negative bacteria represents a growing challenge for the development of new antimicrobials. The pace of antibiotic drug development has slowed during the last decade and, especially for Gram-negatives, clinicians are facing a dramatic shortage in the availability of therapeutic options to face the emergency of the resistance problem throughout the world. In this alarming scenario, although there is a shortage of compounds reaching the market in the near future, antibiotic discovery remains one of the keys to successfully stem and maybe overcome the tide of resistance. Analogs of already known compounds and new agents belonging to completely new classes of antimicrobials are in early stages of development. Novel and promising anti-Gram-negative antimicrobials belong both to old (cephalosporins, carbapenems, β-lactamase inhibitors, monobactams, aminoglycosides, polymyxin analogues and tetracycline) and completely new antibacterial classes (boron-containing antibacterial protein synthesis inhibitors, bis-indoles, outer membrane synthesis inhibitors, antibiotics targeting novel sites of the 50S ribosomal subunit and antimicrobial peptides). However, all of these compounds are still far from being introduced into clinical practice. Therefore, infection control policies and optimization in the use of already existing molecules are still the most effective approaches to reduce the spread of resistance and preserve the activity of antimicrobials.

  20. A NOVEL S-ADENOSYL-L-METHIONINE: ARSENIC (III) METHYLTRANSFERASE FROM RAT LIVER CYTOSOL

    Science.gov (United States)

    A Novel S-Adenosyl-L-methionine: Arsenic(III) Methyltransferase from Rat Liver CytosolShan Lin, Qing Shi, F. Brent Nix, Miroslav Styblo, Melinda A. Beck, Karen M. Herbin-Davis, Larry L. Hall, Josef B. Simeonsson, and David J. Thomas S-adenosyl-L-methionine (AdoMet): ar...

  1. Catechol-O-methyltransferase gene methylation and substance use in adolescents : the TRAILS study

    NARCIS (Netherlands)

    van der Knaap, L. J.; Schaefer, J. M.; Franken, I. H. A.; Verhulst, F. C.; van Oort, F. V. A.; Riese, H.

    Substance use often starts in adolescence and poses a major problem for society and individual health. The dopamine system plays a role in substance use, and catechol-O-methyltransferase (COMT) is an important enzyme that degrades dopamine. The Val(108/158)Met polymorphism modulates COMT activity

  2. Catechol-O-methyltransferase gene methylation and substance use in adolescents: The TRAILS study

    NARCIS (Netherlands)

    L.J. van der Knaap (Lisette); J.M. Schäfer (Johanna); I.H.A. Franken (Ingmar); F.C. Verhulst (Frank); F.V.A. van Oort (Floor); H. Riese (Harriëtte)

    2014-01-01

    textabstractSubstance use often starts in adolescence and poses a major problem for society and individual health. The dopamine system plays a role in substance use, and catechol-O-methyltransferase (COMT) is an important enzyme that degrades dopamine. The Val108/158Met polymorphism

  3. Multiple drug resistance of Aeromonas hydrophila isolates from Chicken samples collected from Mhow and Indore city of Madhyapradesh

    Directory of Open Access Journals (Sweden)

    Kaskhedikar

    2009-02-01

    Full Text Available Fourteen antibacterial agents belonging to 9 different groups of antibiotics viz. aminoglycosides, cephalosporins, nitrofurantoin, fluroquinolones, chloramphenicol, sulphonamides, tetracyclines, penicillin and polymixin were used for in vitro sensitivity testing of Aeromonas hydrophila isolated from fifteen samples of chicken collected from retail shops in Mhow city. The sensitivity (100% was attributed to ciprofloxacin, cefuroxime, ceftriaxone, cephotaxime, chloramphenicol, gentamycin, kanamycin, nitrofurantoin, nalidixic acid and ofloxacin followed by oxytetracycline (50%. All the isolates were resistant to ampicillin and colistin antibiotics. That means, none of the isolates were found to be sensitive for penicillin and polymixin group of antibiotics. Multiple drug resistance was also observed in all A. hydrophila isolates. Out of total isolates, 100% were resistant to two antimicrobial drugs and 50% to three drugs. [Vet. World 2009; 2(1.000: 31-32

  4. Defining the Relationship Between Phenotypic and Genotypic Resistance Profiles of Multidrug-Resistant Enterobacterial Clinical Isolates.

    Science.gov (United States)

    Galal, Lamis; Abdel Aziz, Neveen A; Hassan, Walaa M

    2018-05-11

    Fluoroquinolones and aminoglycosides offer effective therapy for extended-spectrum beta-lactamase (ESBL)-producing enterobacterial infections, but their usefulness is threatened by increasing resistant strains. This study was conducted to demonstrate the phenotypic outcomes of the coexistence of genetic determinants mediating resistance to extended-spectrum cephalosporins and quinolones in enterobacterial isolates collected from patients with health-care-associated infections in Egypt. ESBL phenotype was determined using double-disk synergy test (DDST). The PCR technique was used to detect the presence of the genes mediating quinolone resistance (qnr and aac(6')-Ib-cr) and coexistence with ESBL genes. We also examined the association between the genetic makeup of the isolates and their resistance profiles including effect on MIC results. Phenotypically ESBLs were detected in 60-82% of the enterobacterial isolates. ESBL, qnr and aac(6')-Ib-cr genes were detected with the following percentages in Citrobacter isolates (69%, 69%, and 43%, respectively), E.coli isolates (65%, 70%, and 45%, respectively), Enterobacter isolates (56%, 67%, and 33%, respectively), and finally Klebsiella isolates (42%, 66%, and 25%, respectively). The coexistence of these multiresistant genetic elements significantly increased the MIC values of the tested antibiotics from different classes. We suggest using blaTEM, blaCTX-M-15, qnr, and aac(6')-Ib-cr genes for better and faster prediction of suitable antibiotic therapy with effective doses against ESBL-producing isolates harboring plasmid-mediated quinolone resistance (PMQR) determinants. Amikacin, meropenem, gentamicin, and imipenem seem to be better choices of treatment for such life-threatening infections, because of their remaining highest activity.

  5. The phenotypic and molecular assessment of the non-conserved Arabidopsis MICRORNA163/S-ADENOSYL-METHYLTRANSFERASE regulatory module during biotic stress.

    Science.gov (United States)

    Litholdo, Celso Gaspar; Eamens, Andrew Leigh; Waterhouse, Peter Michael

    2018-04-01

    In plants, microRNAs (miRNAs) have evolved in parallel to the protein-coding genes that they target for expression regulation, and miRNA-directed gene expression regulation is central to almost every cellular process. MicroRNA, miR163, is unique to the Arabidopsis genus and is processed into a 24-nucleotide (nt) mature small regulatory RNA (sRNA) from a single precursor transcript transcribed from a single locus, the MIR163 gene. The MIR163 locus is a result of a recent inverted duplication event of one of the five closely related S-ADENOSYL-METHYLTRANSFERASE genes that the mature miR163 sRNA targets for expression regulation. Currently, however, little is known about the role of the miR163/S-ADENOSYL-METHYLTRANSFERASE regulatory module in response to biotic stress. Here, we document the expression domains of MIR163 and the S-ADENOSYL-METHYLTRANSFERASE target genes following fusion of their putative promoter sequences to the β-glucuronidase (GUS) reporter gene and subsequent in planta expression. Further, we report on our phenotypic and molecular assessment of Arabidopsis thaliana plants with altered miR163 accumulation, namely the mir163-1 and mir163-2 insertion knockout mutants and the miR163 overexpression line, the MIR163-OE plant. Finally, we reveal miR163 accumulation and S-ADENOSYL-METHYLTRANSFERASE target gene expression post treatment with the defence elicitors, salicylic acid and jasmonic acid, and following Fusarium oxysporum infection, wounding, and herbivory attack. Together, the work presented here provides a comprehensive new biological insight into the role played by the Arabidopsis genus-specific miR163/S-ADENOSYL-METHYLTRANSFERASE regulatory module in normal A. thaliana development and during the exposure of A. thaliana plants to biotic stress.

  6. Lack of phosphatidylethanolamine N-methyltransferase in mice does not promote fatty acid oxidation in skeletal muscle.

    Science.gov (United States)

    Tasseva, Guergana; van der Veen, Jelske N; Lingrell, Susanne; Jacobs, René L; Vance, Dennis E; Vance, Jean E

    2016-02-01

    Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine (PE) to phosphatidylcholine (PC) in the liver. Mice lacking PEMT are protected from high-fat diet-induced obesity and insulin resistance, and exhibit increased whole-body energy expenditure and oxygen consumption. Since skeletal muscle is a major site of fatty acid oxidation and energy utilization, we determined if rates of fatty acid oxidation/oxygen consumption in muscle are higher in Pemt(-/-) mice than in Pemt(+/+) mice. Although PEMT is abundant in the liver, PEMT protein and activity were undetectable in four types of skeletal muscle. Moreover, amounts of PC and PE in the skeletal muscle were not altered by PEMT deficiency. Thus, we concluded that any influence of PEMT deficiency on skeletal muscle would be an indirect consequence of lack of PEMT in liver. Neither the in vivo rate of fatty acid uptake by muscle nor the rate of fatty acid oxidation in muscle explants and cultured myocytes depended upon Pemt genotype. Nor did PEMT deficiency increase oxygen consumption or respiratory function in skeletal muscle mitochondria. Thus, the increased whole body oxygen consumption in Pemt(-/-) mice, and resistance of these mice to diet-induced weight gain, are not primarily due to increased capacity of skeletal muscle for utilization of fatty acids as an energy source. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

  7. Structure and possible mechanism of the CcbJ methyltransferase from Streptomyces caelestis

    Czech Academy of Sciences Publication Activity Database

    Bauer, J.; Ondrovičová, G.; Najmanová, Lucie; Pevala, V.; Kameník, Zdeněk; Koštan, J.; Janata, Jiří; Kutejová, Eva

    2014-01-01

    Roč. 70, APR 2014 (2014), s. 943-957 ISSN 0907-4449 R&D Projects: GA MŠk(CZ) EE2.3.30.0003; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : CATECHOL-O-METHYLTRANSFERASE * SN2-LIKE TRANSITION-STATE * CRYSTAL-STRUCTURES Subject RIV: CE - Biochemistry Impact factor: 7.232, year: 2013

  8. Catecholamine-o-methyltransferase polymorphisms are associated with postoperative pain intensity.

    LENUS (Irish Health Repository)

    Lee, Peter J

    2011-02-01

    single nucleotide polymorphisms (SNPs) in the genes for catecholamine-O-methyltransferase (COMT), μ-opioid receptor and GTP cyclohydrolase (GCH1) have been linked to acute and chronic pain states. COMT polymorphisms are associated with experimental pain sensitivity and a chronic pain state. No such association has been identified perioperatively. We carried out a prospective observational clinical trial to examine associations between these parameters and the development of postoperative pain in patients undergoing third molar (M3) extraction.

  9. Antibiotic resistance potential of the healthy preterm infant gut microbiome

    Directory of Open Access Journals (Sweden)

    Graham Rose

    2017-01-01

    Full Text Available Background Few studies have investigated the gut microbiome of infants, fewer still preterm infants. In this study we sought to quantify and interrogate the resistome within a cohort of premature infants using shotgun metagenomic sequencing. We describe the gut microbiomes from preterm but healthy infants, characterising the taxonomic diversity identified and frequency of antibiotic resistance genes detected. Results Dominant clinically important species identified within the microbiomes included C. perfringens, K. pneumoniae and members of the Staphylococci and Enterobacter genera. Screening at the gene level we identified an average of 13 antimicrobial resistance genes per preterm infant, ranging across eight different antibiotic classes, including aminoglycosides and fluoroquinolones. Some antibiotic resistance genes were associated with clinically relevant bacteria, including the identification of mecA and high levels of Staphylococci within some infants. We were able to demonstrate that in a third of the infants the S. aureus identified was unrelated using MLST or metagenome assembly, but low abundance prevented such analysis within the remaining samples. Conclusions We found that the healthy preterm infant gut microbiomes in this study harboured a significant diversity of antibiotic resistance genes. This broad picture of resistances and the wider taxonomic diversity identified raises further caution to the use of antibiotics without consideration of the resident microbial communities.

  10. Rising prevalence of antimicrobial resistance in urinary tract infections during pregnancy: Necessity for exploring newer treatment options

    Directory of Open Access Journals (Sweden)

    Meher Rizvi

    2011-01-01

    Full Text Available Background: Urinary tract infections (UTI are one of the most common medical complications of pregnancy. The emergence of drug resistance and particularly the Extended-spectrum beta-lactamase production by Escherichia coli and methicillin resistance in Staphylococci, limits the choice of antimicrobials. Materials and Methods: Patients in different stages of pregnancy with or without symptoms of urinary tract infection attending the antenatal clinic of obstetrics and gynaecology were screened for significant bacteriuria, by standard loop method on 5% sheep blood agar and teepol lactose agar. Isolates were identified by using standard biochemical tests and antimicrobial susceptibility testing was done using Kirby Bauer disc diffusion method. Results: A total of 4290 (51.2% urine samples from pregnant females showed growth on culture. Prevalence of asymptomatic bacteriuria 3210 (74.8% was higher than symptomatic UTI 1080 (25.2%. Escherichia coli was the most common pathogen accounting for 1800 (41.9% of the urinary isolates. Among the gram-positive cocci, coagulase negative species of Staphylococci 270 (6.4% were the most common pathogen. Significantly high resistance was shown by the gram negative bacilli as well as gram positive cocci to the β-lactam group of antimicrobials, flouroquinolones and aminoglycosides. Most alarming was the presence of ESBL in 846 (47% isolates of Escherichia coli and 344 (36.9% isolates of Klebsiella pneumoniae, along with the presence of methicillin resistance in 41% of Staphylococcus species and high-level aminoglycoside resistance in 45(30% isolates of Enterococcus species. Glycopeptides and carbepenems were the only group of drugs to which all the strains of gram positive cocci and gram negative bacilli were uniformly sensitive, respectively. Conclusions: Regular screening should be done for the presence of symptomatic or asymptomatic bacteriuria in pregnancy and specific guidelines should be issued for testing

  11. Systematic Review on Global Epidemiology of Methicillin-Resistant Staphylococcus pseudintermedius: Inference of Population Structure from Multilocus Sequence Typing Data

    DEFF Research Database (Denmark)

    dos Santos, Teresa Pires; Damborg, Peter; Moodley, Arshnee

    2016-01-01

    Background and rationale: Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is a major cause of infections in dogs, also posing a zoonotic risk to humans. This systematic review aimed to determine the global epidemiology of MRSP and provide new insights into the population structure...... the MLST database for this species. Analysis of MLST data was performed with eBURST and ClonalFrame, and the proportion of MRSP isolates resistant to selected antimicrobial drugs was determined for the most predominant clonal complexes. Results: Fifty-eight studies published over the last 10 years were....... In Europe, CC258, which is more frequently susceptible to enrofloxacin and aminoglycosides, and more frequently resistant to sulphonamides/trimethoprim than CC71, is increasingly reported in various countries. CC68, previously described as the epidemic North American clone, is frequently reported...

  12. Subcellular mechanisms involved in apoptosis induced by aminoglycoside antibiotics: Insights on p53, proteasome and endoplasmic reticulum

    Energy Technology Data Exchange (ETDEWEB)

    Denamur, Sophie; Boland, Lidvine [Université catholique de Louvain, Louvain Drug Research Institute, Cellular and Molecular Pharmacology, UCL B1.73.05, avenue E. Mounier, 73 – B1200 Brussels (Belgium); Beyaert, Maxime [Université catholique de Louvain, de Duve Institute, Laboratory of Physiological Chemistry, UCL B1.75.08, avenue Hippocrate, 75 B -1200 Brussels (Belgium); Verstraeten, Sandrine L. [Université catholique de Louvain, Louvain Drug Research Institute, Cellular and Molecular Pharmacology, UCL B1.73.05, avenue E. Mounier, 73 – B1200 Brussels (Belgium); Fillet, Marianne [University of Liege, CIRM, Department of Pharmacy, Laboratory for the Analysis of Medicines, Quartier Hopital, Avenue Hippocrate, 15, B36, Tower 4, 4000 Liège 1 (Belgium); Tulkens, Paul M. [Université catholique de Louvain, Louvain Drug Research Institute, Cellular and Molecular Pharmacology, UCL B1.73.05, avenue E. Mounier, 73 – B1200 Brussels (Belgium); Bontemps, Françoise [Université catholique de Louvain, de Duve Institute, Laboratory of Physiological Chemistry, UCL B1.75.08, avenue Hippocrate, 75 B -1200 Brussels (Belgium); Mingeot-Leclercq, Marie-Paule [Université catholique de Louvain, Louvain Drug Research Institute, Cellular and Molecular Pharmacology, UCL B1.73.05, avenue E. Mounier, 73 – B1200 Brussels (Belgium)

    2016-10-15

    Gentamicin, an aminoglycoside used to treat severe bacterial infections, may cause acute renal failure. In the renal cell line LLC-PK1, gentamicin accumulates in lysosomes, induces alterations of their permeability, and triggers the mitochondrial pathway of apoptosis via activation of caspase-9 and -3 and changes in Bcl-2 family proteins. Early ROS production in lysosomes has been associated with gentamicin induced lysosomal membrane permeabilization. In order to better understand the multiple interconnected pathways of gentamicin-induced apoptosis and ensuing renal cell toxicity, we investigated the effect of gentamicin on p53 and p21 levels. We also studied the potential effect of gentamicin on proteasome by measuring the chymotrypsin-, trypsin- and caspase-like activities, and on endoplasmic reticulum by determining phopho-eIF2α, caspase-12 activation and GRP78 and 94. We observed an increase in p53 levels, which was dependent on ROS production. Accumulation of p53 resulted in accumulation of p21 and of phospho-eIF2α. These effects could be related to an impairment of proteasome as we demonstrated an inhibition of trypsin-and caspase-like activities. Moderate endoplasmic reticulum stress could also participate to cellular toxicity induced by gentamicin, with activation of caspase-12 without change in GRP74 and GRP98. All together, these data provide new mechanistic insights into the apoptosis induced by aminoglycoside antibiotics on renal cell lines. - Highlights: • Gentamicin induces apoptosis through p53 pathway. • Gentamicin inhibits proteosomal activity. • Gentamicin activates caspase-12.

  13. Subcellular mechanisms involved in apoptosis induced by aminoglycoside antibiotics: Insights on p53, proteasome and endoplasmic reticulum

    International Nuclear Information System (INIS)

    Denamur, Sophie; Boland, Lidvine; Beyaert, Maxime; Verstraeten, Sandrine L.; Fillet, Marianne; Tulkens, Paul M.; Bontemps, Françoise; Mingeot-Leclercq, Marie-Paule

    2016-01-01

    Gentamicin, an aminoglycoside used to treat severe bacterial infections, may cause acute renal failure. In the renal cell line LLC-PK1, gentamicin accumulates in lysosomes, induces alterations of their permeability, and triggers the mitochondrial pathway of apoptosis via activation of caspase-9 and -3 and changes in Bcl-2 family proteins. Early ROS production in lysosomes has been associated with gentamicin induced lysosomal membrane permeabilization. In order to better understand the multiple interconnected pathways of gentamicin-induced apoptosis and ensuing renal cell toxicity, we investigated the effect of gentamicin on p53 and p21 levels. We also studied the potential effect of gentamicin on proteasome by measuring the chymotrypsin-, trypsin- and caspase-like activities, and on endoplasmic reticulum by determining phopho-eIF2α, caspase-12 activation and GRP78 and 94. We observed an increase in p53 levels, which was dependent on ROS production. Accumulation of p53 resulted in accumulation of p21 and of phospho-eIF2α. These effects could be related to an impairment of proteasome as we demonstrated an inhibition of trypsin-and caspase-like activities. Moderate endoplasmic reticulum stress could also participate to cellular toxicity induced by gentamicin, with activation of caspase-12 without change in GRP74 and GRP98. All together, these data provide new mechanistic insights into the apoptosis induced by aminoglycoside antibiotics on renal cell lines. - Highlights: • Gentamicin induces apoptosis through p53 pathway. • Gentamicin inhibits proteosomal activity. • Gentamicin activates caspase-12.

  14. Evaluation of levels of antibiotic resistance in groundwater-derived E. coli isolates in the Midwest of Ireland and elucidation of potential predictors of resistance

    Science.gov (United States)

    O'Dwyer, Jean; Hynds, Paul; Pot, Matthieu; Adley, Catherine C.; Ryan, Michael P.

    2017-06-01

    Antibiotic-resistant (pathogenic and non-pathogenic) organisms and genes are now acknowledged as significant emerging aquatic contaminants with potentially adverse human and ecological health impacts, and thus require monitoring. This study is the first to investigate levels of resistance among Irish groundwater (private wells) samples; Escherichia coli isolates were examined against a panel of commonly prescribed human and veterinary therapeutic antibiotics, followed by determination of the causative factors of resistance. Overall, 42 confirmed E. coli isolates were recovered from a groundwater-sampling cohort. Resistance to the human panel of antibiotics was moderate; nine (21.4%) E. coli isolates demonstrated resistance to one or more human antibiotics. Conversely, extremely high levels of resistance to veterinary antibiotics were found, with all isolates presenting resistance to one or more veterinary antibiotics. Particularly high levels of resistance (93%) were found with respect to the aminoglycoside class of antibiotics. Results of statistical analysis indicate a significant association between the presence of human (multiple) antibiotic resistance ( p = 0.002-0.011) and both septic tank density and the presence of vulnerable sub-populations (<5 years). For the veterinary antibiotics, results point to a significant relationship ( p = <0.001) between livestock (cattle) density and the prevalence of multiple antibiotic resistant E. coli. Groundwater continues to be an important resource in Ireland, particularly in rural areas; thus, results of this preliminary study offer a valuable insight into the prevalence of antibiotic resistance in the hydrogeological environment and establish a need for further research with a larger geological diversity.

  15. Prevalence and Antimicrobial Resistance of Enterococcus Species: A Hospital-Based Study in China

    Directory of Open Access Journals (Sweden)

    Wei Jia

    2014-03-01

    Full Text Available Objective: to investigate the prevalence and antimicrobial resistance of Enterococcus species isolated from a university hospital, and explore the mechanisms underlying the antimicrobial resistance, so as to provide clinical evidence for the inappropriate clinical use of antimicrobial agents and the control and prevention of enterococcal infections. Methods: a total of 1,157 enterococcal strains isolated from various clinical specimens from January 2010 to December 2012 in the General Hospital of Ningxia Medical University were identified to species level with a VITEK-2 COMPACT fully automated microbiological system, and the antimicrobial susceptibility of Enterococcus species was determined using the Kirby-Bauer disc diffusion method. The multiple-drug resistant enterococcal isolates were screened from the clinical isolates of Enterococcus species from the burns department. The minimal inhibitory concentration (MIC of Enterococcus species to the three fluoroquinolones, including ciprofloxacin, gatifloxacin and levofloxacin was determined with the agar dilution method, and the changes in the MIC of Enterococcus species to the three fluoroquinolones following reserpine treatment were evaluated. The β-lactam, aminoglycoside, tetracycline, macrolide, glycopeptide resistance genes and the efflux pump emeA genes were detected in the enterococcal isolates using a polymerase chain reaction (PCR assay. Results: the 1,157 clinical isolates of Enterococcus species included 679 E. faecium isolates (58.7%, 382 E. faecalis isolates (33%, 26 E. casseliflavus isolates (2.2%, 24 E. avium isolates (2.1%, and 46 isolates of other Enterococcus species (4%. The prevalence of antimicrobial resistance varied significantly between E. faecium and E. faecalis, and ≤1.1% of these two Enterococcus species were found to be resistant to vancomycin, teicoplanin or linezolid. In addition, the Enterococcus species isolated from different departments of the hospital

  16. Screening of nasal carriage of methicillin-resistant Staphylococcus aureus during admission of patients to Frantz Fanon Hospital, Blida, Algeria

    Directory of Open Access Journals (Sweden)

    Mohamed Amine Ouidri

    2018-05-01

    Full Text Available A study was performed of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA strains isolated from nasal preoperative samples. Of 663 samples assessed, staphylococcus was detected in 143 (21.57%. The disc diffusion method (cefoxitin 30 μg, a screening test (oxacillin 6 μg/mL and a search for Protein Binding Additional Penicillin 2 (PLP2a allowed the detection and confirmation of resistance to methicillin for 36 strains, a rate of 5.43% of the total population studied. Eight MRSA carriers received care in the trauma service, 14 in cardiology, five in ear, nose and throat, four in neurosurgery and paediatrics, and one in SCI. Thirty-six methicillin-resistant of the nasal portage strains are in their great majority, 27 of 36, rather limited multi-R character (two to three families namely resistance: tetracyclines, fluoroquinolones, aminoglycosides, macrolides. One of the MRSA strains was found to have intermediate sensitivity to vancomycin. Keywords: Antibiotic resistance, Healthy volunteers, MRSA, Prevalence, Staphylococcus aureus

  17. Global developmental delay in guanidionacetate methyltransferase deficiency : differences in formal testing and clinical observation

    NARCIS (Netherlands)

    Verbruggen, Krijn T.; Knijff, Wilma A.; Soorani-Lunsing, Roelineke J.; Sijens, Paul E.; Verhoeven, Nanda M.; Salomons, Gajja S.; Goorhuis-Brouwer, Siena M.; van Spronsen, Francjan J.

    Guanidinoacetate N-methyltransferase (GAMT) deficiency is a defect in the biosynthesis of creatine (Cr). So far, reports have not focused on the description of developmental abilities in this disorder. Here, we present the result of formal testing of developmental abilities in a GAMT-deficient

  18. Antimicrobial resistance of bacterial pathogens in a Neonatal Intensive Care Unit

    Directory of Open Access Journals (Sweden)

    Farzana Ahmed

    2018-03-01

    Full Text Available The aim of this study was to identify the antimicrobial susceptibility pattern and relevant treatment options in a neonatal intensive care unit from January 2012 and June 2016. Out of the total 78 culture positive samples, Gram positive and Gram negative microorganisms were 26% and 74% respectively. Acinetobacter remained the predominant isolate (32.1% followed by Klebsiella species (18.0%. Most of the Gram positive isolates exhibited higher resistance to penicillin, cephalosporin, macrolides, gentamycin and quinolones. Gram positive isolates had sensitivity of 100% to linezolid, vancomycin, chloramphenicol followed by rifampicin (84%. In comparison to other commonly used antibiotics, sensitivity to these four medicines was statistically significant (p<0.05. Similarly, most of the Gram negative bacteria showed resistance to cephalosporin, aminoglycosides. About two-third cases showed resistant to meropenum, quinolones and combination preparation of piperacillin and tazobactam. Overall sensitivity among the Gram negative isolates was to polymixin B (100% and minocycline (97%, followed by colistin (83%. In comparison to other commonly used antibiotics, sensitivity to these three medicines was statistically significant (p<0.05.

  19. Structure of the human gene encoding the protein repair L-isoaspartyl (D-aspartyl) O-methyltransferase.

    Science.gov (United States)

    DeVry, C G; Tsai, W; Clarke, S

    1996-11-15

    The protein L-isoaspartyl/D-aspartyl O-methyltransferase (EC 2.1.1.77) catalyzes the first step in the repair of proteins damaged in the aging process by isomerization or racemization reactions at aspartyl and asparaginyl residues. A single gene has been localized to human chromosome 6 and multiple transcripts arising through alternative splicing have been identified. Restriction enzyme mapping, subcloning, and DNA sequence analysis of three overlapping clones from a human genomic library in bacteriophage P1 indicate that the gene spans approximately 60 kb and is composed of 8 exons interrupted by 7 introns. Analysis of intron/exon splice junctions reveals that all of the donor and acceptor splice sites are in agreement with the mammalian consensus splicing sequence. Determination of transcription initiation sites by primer extension analysis of poly(A)+ mRNA from human brain identifies multiple start sites, with a major site 159 nucleotides upstream from the ATG start codon. Sequence analysis of the 5'-untranslated region demonstrates several potential cis-acting DNA elements including SP1, ETF, AP1, AP2, ARE, XRE, CREB, MED-1, and half-palindromic ERE motifs. The promoter of this methyltransferase gene lacks an identifiable TATA box but is characterized by a CpG island which begins approximately 723 nucleotides upstream of the major transcriptional start site and extends through exon 1 and into the first intron. These features are characteristic of housekeeping genes and are consistent with the wide tissue distribution observed for this methyltransferase activity.

  20. The interplay between protein L-isoaspartyl methyltransferase activity and insulin-like signaling to extend lifespan in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Shilpi Khare

    Full Text Available The protein L-isoaspartyl-O-methyltransferase functions to initiate the repair of isomerized aspartyl and asparaginyl residues that spontaneously accumulate with age in a variety of organisms. Caenorhabditis elegans nematodes lacking the pcm-1 gene encoding this enzyme display a normal lifespan and phenotype under standard laboratory growth conditions. However, significant defects in development, egg laying, dauer survival, and autophagy have been observed in pcm-1 mutant nematodes when deprived of food and when exposed to oxidative stress. Interestingly, overexpression of this repair enzyme in both Drosophila and C. elegans extends adult lifespan under thermal stress. In this work, we show the involvement of the insulin/insulin-like growth factor-1 signaling (IIS pathway in PCM-1-dependent lifespan extension in C. elegans. We demonstrate that reducing the levels of the DAF-16 downstream transcriptional effector of the IIS pathway by RNA interference reduces the lifespan extension resulting from PCM-1 overexpression. Using quantitative real-time PCR analysis, we show the up-regulation of DAF-16-dependent stress response genes in the PCM-1 overexpressor animals compared to wild-type and pcm-1 mutant nematodes under mild thermal stress conditions. Additionally, similar to other long-lived C. elegans mutants in the IIS pathway, including daf-2 and age-1 mutants, PCM-1 overexpressor adult animals display increased resistance to severe thermal stress, whereas pcm-1 mutant animals survive less long under these conditions. Although we observe a higher accumulation of damaged proteins in pcm-1 mutant nematodes, the basal level of isoaspartyl residues detected in wild-type animals was not reduced by PCM-1 overexpression. Our results support a signaling role for the protein L-isoaspartyl methyltransferase in lifespan extension that involves the IIS pathway, but that may be independent of its function in overall protein repair.

  1. Functional characterization of a rice de novo DNA methyltransferase, OsDRM2, expressed in Escherichia coli and yeast

    Energy Technology Data Exchange (ETDEWEB)

    Pang, Jinsong, E-mail: pangjs542@nenu.edu.cn [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China); Dong, Mingyue; Li, Ning; Zhao, Yanli [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China); Liu, Bao, E-mail: baoliu@nenu.edu.cn [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China)

    2013-03-01

    Highlights: ► A rice de novo DNA methyltransferase OsDRM2 was cloned. ► In vitro methylation activity of OsDRM2 was characterized with Escherichia coli. ► Assays of OsDRM2 in vivo methylation were done with Saccharomyces cerevisiae. ► OsDRM2 methylation activity is not preferential to any type of cytosine context. ► The activity of OsDRM2 is independent of RdDM pathway. - Abstract: DNA methylation of cytosine nucleotides is an important epigenetic modification that occurs in most eukaryotic organisms and is established and maintained by various DNA methyltransferases together with their co-factors. There are two major categories of DNA methyltransferases: de novo and maintenance. Here, we report the isolation and functional characterization of a de novo methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length coding region of OsDRM2 was cloned and transformed into Escherichia coli and Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein, which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de novo activity of OsDRM2: (1) a 5′-CCGG-3′ containing DNA fragment that had been pre-treated with OsDRM2 protein expressed in E. coli was protected from digestion by the CG-methylation-sensitive isoschizomer HpaII; (2) methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae genomic DNA from transformants that had been introduced with OsDRM2 revealed CG and CHG methylation levels of 3.92–9.12%, and 2.88–6.93%, respectively, whereas the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These results were further supported by bisulfite sequencing of the 18S rRNA and EAF5 genes of the transformed S. cerevisiae, which exhibited different DNA methylation patterns, which were observed in the genomic DNA. Our findings establish that OsDRM2 is an active de novo DNA

  2. Functional characterization of a rice de novo DNA methyltransferase, OsDRM2, expressed in Escherichia coli and yeast

    International Nuclear Information System (INIS)

    Pang, Jinsong; Dong, Mingyue; Li, Ning; Zhao, Yanli; Liu, Bao

    2013-01-01

    Highlights: ► A rice de novo DNA methyltransferase OsDRM2 was cloned. ► In vitro methylation activity of OsDRM2 was characterized with Escherichia coli. ► Assays of OsDRM2 in vivo methylation were done with Saccharomyces cerevisiae. ► OsDRM2 methylation activity is not preferential to any type of cytosine context. ► The activity of OsDRM2 is independent of RdDM pathway. - Abstract: DNA methylation of cytosine nucleotides is an important epigenetic modification that occurs in most eukaryotic organisms and is established and maintained by various DNA methyltransferases together with their co-factors. There are two major categories of DNA methyltransferases: de novo and maintenance. Here, we report the isolation and functional characterization of a de novo methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length coding region of OsDRM2 was cloned and transformed into Escherichia coli and Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein, which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de novo activity of OsDRM2: (1) a 5′-CCGG-3′ containing DNA fragment that had been pre-treated with OsDRM2 protein expressed in E. coli was protected from digestion by the CG-methylation-sensitive isoschizomer HpaII; (2) methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae genomic DNA from transformants that had been introduced with OsDRM2 revealed CG and CHG methylation levels of 3.92–9.12%, and 2.88–6.93%, respectively, whereas the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These results were further supported by bisulfite sequencing of the 18S rRNA and EAF5 genes of the transformed S. cerevisiae, which exhibited different DNA methylation patterns, which were observed in the genomic DNA. Our findings establish that OsDRM2 is an active de novo DNA

  3. Resistance patterns of bacterial isolates to antimicrobials from 3 hospitals in the United Arab Emirates

    International Nuclear Information System (INIS)

    AlDhaheri, Ahmed S; AlNiyadi, Mohammed S; AlDhaheri Ahmed D; Bastaki, Salim M

    2009-01-01

    To compare the resistance pattern of common bacterial pathogens to commonly used drugs. Information and statistics of antimicrobial resistance for 1994 and 2005 were collected from the 3 hospital microbiology laboratories in the United Arab Emirates. The resistance patterns of Staphylococcus aureus, Escherichia coli, Klebsiella spp, and Pseudomonas aeruginosa to several front-line drugs were estimated. All laboratories used automatic machines (Vitek 2), which identifies and determines minimum inhibitory concentrations simultaneously. Increased resistance was observed for Staphylococcus aureus, (n=315, 2005) to erythromycin (approximately 6 fold, Al-Ain Hospital only), cloxacillin (Al-Ain Hospital), and gentamicin (more than 3-10 folds in all hospitals). Increased penicillin resistance was not observed. For the common Gram-negative organisms, there was a high resistance to ampicillin, gentamicin, ceftriaxone, ciprofloxacin, and imipenem, which seemed to increase for Escherichia coli, (by 4.2-200%, n=305, 2005); however, there was very little resistance to imipenem (0.4%) in Tawam Hospital. Variable resistance patterns were obtained for Pseudomonas aeruginosa (n=316, 2005) and Klebsiella spp,(n=316, 2005) against aminoglycosides, cephalosporins, ciprofloxacin, and norfloxacin. Overall, there was an obvious increase in resistance of bacteria and the prevalence rate to a number of drugs from 1-120 folds during the 11-year period. (author)

  4. Antimicrobial resistance among Enterobacteriaceae in South America: history, current dissemination status and associated socioeconomic factors.

    Science.gov (United States)

    Bonelli, Raquel Regina; Moreira, Beatriz Meurer; Picão, Renata Cristina

    2014-04-01

    South America exhibits some of the higher rates of antimicrobial resistance in Enterobactericeae worldwide. This continent includes 12 independent countries with huge socioeconomic differences, where the ample access to antimicrobials, including counterfeit ones, coexists with ineffective health systems and sanitation problems, favoring the emergence and dissemination of resistant strains. This work presents a literature review concerning the evolution and current status of antimicrobial resistance threats found among Enterobacteriaceae in South America. Resistance to β-lactams, fluoroquinolones and aminoglycosides was emphasized along with description of key epidemiological studies that highlight the success of specific resistance determinants in different parts of the continent. In addition, a discussion regarding political and socioeconomic factors possibly related to the dissemination of antimicrobial resistant strains in clinical settings and at the community is presented. Finally, in order to assess the possible sources of resistant bacteria, we compile the current knowledge about the occurrence of antimicrobial resistance in isolates in South American' food, food-producing animals and off-hospitals environments. By addressing that intensive intercontinental commerce and tourism neutralizes the protective effect of geographic barriers, we provide arguments reinforcing that globally integrated efforts are needed to decelerate the emergence and dissemination of antimicrobial resistant strains. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Genome-wide identification and comparative analysis of cytosine-5 DNA methyltransferases and demethylase families in wild and cultivated peanut

    Directory of Open Access Journals (Sweden)

    Pengfei eWang

    2016-02-01

    Full Text Available AbstractDNA methylation plays important roles in genome protection, regulation of gene expression and was associated with plants development. Plant DNA methylation pattern was mediated by cytosine-5 DNA methyltransferases and demethylase. Although the genomes of AA and BB wild peanuts have been fully sequence, these two gene families have not been studied. In this study we report the identification and analysis of putative cytosine-5 DNA methyltransferases (C5-MTases and demethylase in AA and BB wild peanuts. Cytosine-5 DNA methyltransferases in AA and BB wild peanuts could be classified in known MET, CMT and DRM2 groups based on their domain organization. This result was supported by the gene and protein structural characteristics and phylogenetic analysis. We found that some wild peanut DRM2 numbers didn’t contain UBA domain which was different from other plants such as Arabidopsis, maize, soybean. Five DNA demethylase were found in AA genome and five in BB genome. The selective pressure analysis showed that wild peanut C5-MTases gene mainly underwent purifying selection but many positive selection sites can be detected. Conversely, DNA demethylase genes mainly underwent positive selection during evolution. Additionally, the expression dynamic of cytosine-5 DNA methyltransferases and demethylase genes in different cultivated peanut tissues were analyzed. Expression result showed that cold, heat or drought stress could influence the expression level of C5-MTases and DNA demethylase genes in cultivated peanut. These results are useful for better understanding the complexity of these two gene families, and will facilitate epigenetic studies in peanut.

  6. Phenotypic and Genotypic Antibiotic Resistance of Salmonella from Chicken Carcasses Marketed at Ibague, Colombia

    Directory of Open Access Journals (Sweden)

    D Cortes Vélez

    Full Text Available ABSTRACT Salmonella enterica is responsible for alimentary toxic infections associated with the consumption of contaminated poultry products and the antimicrobial resistant patterns of Salmonella circulating in the Tolima region are currently unknown. To address this issue, both the phenotype and genotype antibiotic resistance patterns of 47 Salmonella isolated from raw chicken carcasses sold at the Ibague city were analyzed by the disc diffusion, microdilution and PCR assays. All 47 Salmonella isolates showed resistance to five or more antimicrobial agents. Resistance to Ampicillin (AMP, Amikacin (AMK, Gentamicin (GEN, Tobramycin (TOB, Cefazoline (CFZ, Cefoxitin (FOX, Nitrofurantoin (NIT, Trimethoprim-Sulfamethoxazole (SXT, Tetracycline (TET, Ciprofloxacin (CIP and Enrofloxacin (ENR was observed in 42.35% of Salmonella isolates. All tested S. Paratyphi B var Java isolates showed resistance to at least 12 antibiotics. S. Hvittingfoss showed resistance to 5 antibiotics, whereas S. Muenster showed resistance to seven antibiotics. Amplification of a number of antibiotic resistance genes showed that blaTEM (100% correlated well with resistance to Ampicilin and Cephalosporin, whereas aadB (87% correlated well with resistance to Aminoglycosides. It is concluded that Salmonella isolated from raw chicken meat marketed at Ibague showed MDR by both phenotypic and genotypic methods and they may represent an important threat to human health. Additional studies are needed to establish the relationship between antibiotic resistance in Salmonella from poultry products and clinical isolates.

  7. Mitochondrial 12S ribosomal RNA A1555G mutation associated with cardiomyopathy and hearing loss following high-dose chemotherapy and repeated aminoglycoside exposure

    DEFF Research Database (Denmark)

    Skou, Anne-Sofie; Tranebjærg, Lisbeth; Jensen, Tim

    2014-01-01

    A 19-month-old girl with the A1555G mitochondrial mutation in the 12S ribosomal RNA gene and acute myelogenous leukemia developed dilated cardiomyopathy and bilateral sensorineural hearing loss before undergoing allogeneic stem cell transplantation. She had received gentamicin during episodes of ...... of febrile neutropenia. Testing for the A1555G mutation is recommended in patients frequently treated with aminoglycosides....

  8. Crystal Structure of the Thermus thermophilus 16 S rRNA Methyltransferase RsmC in Complex with Cofactor and Substrate Guanosine

    Energy Technology Data Exchange (ETDEWEB)

    Demirci, H.; Gregory, S; Dahlberg, A; Jogl, G

    2008-01-01

    Post-transcriptional modification is a ubiquitous feature of ribosomal RNA in all kingdoms of life. Modified nucleotides are generally clustered in functionally important regions of the ribosome, but the functional contribution to protein synthesis is not well understood. Here we describe high resolution crystal structures for the N{sup 2}-guanine methyltransferase RsmC that modifies residue G1207 in 16 S rRNA near the decoding site of the 30 S ribosomal subunit. RsmC is a class I S-adenosyl-l-methionine-dependent methyltransferase composed of two methyltransferase domains. However, only one S-adenosyl-l-methionine molecule and one substrate molecule, guanosine, bind in the ternary complex. The N-terminal domain does not bind any cofactor. Two structures with bound S-adenosyl-l-methionine and S-adenosyl-l-homocysteine confirm that the cofactor binding mode is highly similar to other class I methyltransferases. Secondary structure elements of the N-terminal domain contribute to cofactor-binding interactions and restrict access to the cofactor-binding site. The orientation of guanosine in the active site reveals that G1207 has to disengage from its Watson-Crick base pairing interaction with C1051 in the 16 S rRNA and flip out into the active site prior to its modification. Inspection of the 30 S crystal structure indicates that access to G1207 by RsmC is incompatible with the native subunit structure, consistent with previous suggestions that this enzyme recognizes a subunit assembly intermediate.

  9. Characterization of a Vitis vinifera cv. Cabernet Sauvignon 3',5'-O-methyltransferase showing strong preference for anthocyanins and glycosylated flavonols.

    Science.gov (United States)

    Lücker, Joost; Martens, Stefan; Lund, Steven T

    2010-09-01

    At ripening initiation in red grapevine (Vitis vinifera) berries, the exocarp turns color from green to red and then to purple due to the accumulation and extent of methylation of anthocyanins. The accumulation of transcripts encoding an O-methyltransferase was recently shown to be closely correlated with the onset of ripening and the degree of blue/purple pigmentation in grapevine berries; however, the biochemical function of this gene has remained uncharacterized. In this study, an O-methyltransferase cDNA that showed a distinct expression pattern when compared to closely related sequences was expressed in Escherichia coli and enzyme assays were carried out with a broad array of anthocyanin and other flavonoid substrates. We demonstrate that this enzyme carries out 3',5'-O-methylation of anthocyanins and flavonol compounds in vitro, which are known to be present in grape berries, with a preference for glycosylated substrates. The highest relative specific activity for the enzyme was found with delphinidin 3-O-glucoside as substrate. The enzyme is not able to methylate flavan type skeletons with chiral centers, such as either catechins or dihydroquercetin. The enzyme showed negligible specific activity for caffeoyl-CoA, compared to flavonol and anthocyanin substrates. Phylogenetic analysis of the O-methyltransferase suggests that it may be a member of a distinct subclass of Type 2 bivalent metal-dependent S-adenosyl-methionine O-methyltransferases. (c) 2010. Published by Elsevier Ltd. All rights reserved.

  10. Human catechol-O-methyltransferase: Cloning and expression of the membrane-associated form

    International Nuclear Information System (INIS)

    Bertocci, B.; Miggiano, V.; Da Prada, M.; Dembic, Z.; Lahm, H.W.; Malherbe, P.

    1991-01-01

    A cDNA clone for human catechol-O-methyltransferase was isolated from a human hepatoma cell line (Hep G2) cDNA library by hybridization screening with a porcine cDNA probe. The cDNA clone was sequenced and found to have an insert of 1226 nucleotides. The deduced primary structure of hCOMT is composed of 271 amino acid residues with the predicted molecular mass of 30 kDa. At its N terminus it has a hydrophobic segment of 21 amino acid residues that may be responsible for insertion of hCOMT into the endoplasmic reticulum membrane. The primary structure of hCOMT exhibits high homology to the porcine partial cDNA sequence (93%). The deduced amino acid sequence contains two tryptic peptide sequences (T-22, T-33) found in porcine liver catechol-O-methyltransferase (CEMT). The coding region of hCOMT cDNA was placed under the control of the cytomegalovirus promoter to transfect human kidney 293 cells. The recombinant hCOMT was shown by immunoblot analysis to be mainly associated with the membrane fraction. RNA blot analysis revealed one COMT mRNA transcript of 1.4 kilobases in Hep G2 poly(A) + RNA

  11. An Iterative O-Methyltransferase Catalyzes 1,11-Dimethylation of Aspergillus fumigatus Fumaric Acid Amides.

    Science.gov (United States)

    Kalb, Daniel; Heinekamp, Thorsten; Schieferdecker, Sebastian; Nett, Markus; Brakhage, Axel A; Hoffmeister, Dirk

    2016-10-04

    S-adenosyl-l-methionine (SAM)-dependent methyltransfer is a common biosynthetic strategy to modify natural products. We investigated the previously uncharacterized Aspergillus fumigatus methyltransferase FtpM, which is encoded next to the bimodular fumaric acid amide synthetase FtpA. Structure elucidation of two new A. fumigatus natural products, the 1,11-dimethyl esters of fumaryl-l-tyrosine and fumaryl-l-phenylalanine, together with ftpM gene disruption suggested that FtpM catalyzes iterative methylation. Final evidence that a single enzyme repeatedly acts on fumaric acid amides came from an in vitro biochemical investigation with recombinantly produced FtpM. Size-exclusion chromatography indicated that this methyltransferase is active as a dimer. As ftpA and ftpM homologues are found clustered in other fungi, we expect our work will help to identify and annotate natural product biosynthesis genes in various species. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Characterization of novel methyltransferases METTL22 and FAM86A.1

    OpenAIRE

    Ali, Qamar

    2012-01-01

    Proteins are subjected to various post-translational modifications (PTMs) that affect their activity, interaction and localization. Methylation is one such PTM that is well known to play a regulatory role in heterochromatin and euchromatin formation through methyl marks on histone tails, and it has recently been shown that regulation through methylation is also applicable to non-histone proteins. A recent characterization of protein methyltransferase (MTase) METTL21D led to the discovery of a...

  13. Coproduction of Novel 16S rRNA Methylase RmtD and Metallo-β-Lactamase SPM-1 in a Panresistant Pseudomonas aeruginosa Isolate from Brazil▿

    OpenAIRE

    Doi, Yohei; de Oliveira Garcia, Doroti; Adams, Jennifer; Paterson, David L.

    2006-01-01

    Serious infections with Pseudomonas aeruginosa are frequently treated with the combination of a β-lactam antimicrobial and an aminoglycoside. P. aeruginosa strain PA0905 was isolated in 2005 from an inpatient in Brazil. It showed a panresistant phenotype that included resistance to β-lactams, aminoglycosides, and fluoroquinolones. The β-lactam resistance was conferred by the production of the metallo-β-lactamase SPM-1. No inhibitory zone was observed when a disk diffusion test was performed w...

  14. The Histone Methyltransferase Activity of MLL1 Is Dispensable for Hematopoiesis and Leukemogenesis

    Directory of Open Access Journals (Sweden)

    Bibhu P. Mishra

    2014-05-01

    Full Text Available Despite correlations between histone methyltransferase (HMT activity and gene regulation, direct evidence that HMT activity is responsible for gene activation is sparse. We address the role of the HMT activity for MLL1, a histone H3 lysine 4 (H3K4 methyltransferase critical for maintaining hematopoietic stem cells (HSCs. Here, we show that the SET domain, and thus HMT activity of MLL1, is dispensable for maintaining HSCs and supporting leukemogenesis driven by the MLL-AF9 fusion oncoprotein. Upon Mll1 deletion, histone H4 lysine 16 (H4K16 acetylation is selectively depleted at MLL1 target genes in conjunction with reduced transcription. Surprisingly, inhibition of SIRT1 is sufficient to prevent the loss of H4K16 acetylation and the reduction in MLL1 target gene expression. Thus, recruited MOF activity, and not the intrinsic HMT activity of MLL1, is central for the maintenance of HSC target genes. In addition, this work reveals a role for SIRT1 in opposing MLL1 function.

  15. The orphan nuclear receptor GCNF recruits DNA methyltransferase for Oct-3/4 silencing

    International Nuclear Information System (INIS)

    Sato, Noriko; Kondo, Mitsumasa; Arai, Ken-ichi

    2006-01-01

    Somatic DNA methylation patterns are determined in part by the de novo methylation that occurs after early embryonic demethylation. Oct-3/4, a pluripotency gene, is unmethylated in the blastocyst, but undergoes de novo methylation and silencing during gastrulation. Here we show that the transcriptional repressor GCNF recruits DNA methyltransferase to the Oct-3/4 promoter and facilitates its methylation. Although acetylation of histone H3 at lysine 9 (K9) and/or 14 (K14) and methylation of H3 at lysine 4 (K4) decrease during this period, as do Oct-3/4 transcript levels, H3K9 and H3K27 methylation levels remain constant, indicating that DNA methylation does not require repressive histone modifications. We found that GCNF interacts directly with Dnmt3 molecule(s) and verified that this interaction induces the methylation of the Oct-3/4 promoter. Our finding suggests a model in which differentiation-induced GCNF recruits de novo DNA methyltransferase and facilitates the silencing of a pluripotency gene

  16. 2′-O Methylation of Internal Adenosine by Flavivirus NS5 Methyltransferase

    Science.gov (United States)

    Dong, Hongping; Chang, David C.; Hua, Maggie Ho Chia; Lim, Siew Pheng; Chionh, Yok Hian; Hia, Fabian; Lee, Yie Hou; Kukkaro, Petra; Lok, Shee-Mei; Dedon, Peter C.; Shi, Pei-Yong

    2012-01-01

    RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2′-O methyltransferase activities that are required for the formation of 5′ type I cap (m7GpppAm) of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4) specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2′-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N6-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2′-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2′-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2′-O-methyladenosine. The 2′-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine) pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2′-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2′-O methylation of internal adenosine of

  17. YgdE is the 2'-O-ribose methyltransferase RlmM specific for nucleotide C2498 in bacterial 23S rRNA

    DEFF Research Database (Denmark)

    Purta, Elzbieta; O'Connor, Michelle; Bujnicki, Janusz M

    2009-01-01

    The rRNAs of Escherichia coli contain four 2'-O-methylated nucleotides. Similar to other bacterial species and in contrast with Archaea and Eukaryota, the E. coli rRNA modifications are catalysed by specific methyltransferases that find their nucleotide targets without being guided by small...... complementary RNAs. We show here that the ygdE gene encodes the methyltransferase that catalyses 2'-O-methylation at nucleotide C2498 in the peptidyl transferase loop of E. coli 23S rRNA. Analyses of rRNAs using MALDI mass spectrometry showed that inactivation of the ygdE gene leads to loss of methylation...... at nucleotide C2498. The loss of ygdE function causes a slight reduction in bacterial fitness. Methylation at C2498 was restored by complementing the knock-out strain with a recombinant copy of ygdE. The recombinant YgdE methyltransferase modifies C2498 in naked 23S rRNA, but not in assembled 50S subunits...

  18. Molecular Evolution of the Substrate Specificity of Chloroplastic Aldolases/Rubisco Lysine Methyltransferases in Plants.

    Science.gov (United States)

    Ma, Sheng; Martin-Laffon, Jacqueline; Mininno, Morgane; Gigarel, Océane; Brugière, Sabine; Bastien, Olivier; Tardif, Marianne; Ravanel, Stéphane; Alban, Claude

    2016-04-04

    Rubisco and fructose-1,6-bisphosphate aldolases (FBAs) are involved in CO2 fixation in chloroplasts. Both enzymes are trimethylated at a specific lysine residue by the chloroplastic protein methyltransferase LSMT. Genes coding LSMT are present in all plant genomes but the methylation status of the substrates varies in a species-specific manner. For example, chloroplastic FBAs are naturally trimethylated in both Pisum sativum and Arabidopsis thaliana, whereas the Rubisco large subunit is trimethylated only in the former species. The in vivo methylation status of aldolases and Rubisco matches the catalytic properties of AtLSMT and PsLSMT, which are able to trimethylate FBAs or FBAs and Rubisco, respectively. Here, we created chimera and site-directed mutants of monofunctional AtLSMT and bifunctional PsLSMT to identify the molecular determinants responsible for substrate specificity. Our results indicate that the His-Ala/Pro-Trp triad located in the central part of LSMT enzymes is the key motif to confer the capacity to trimethylate Rubisco. Two of the critical residues are located on a surface loop outside the methyltransferase catalytic site. We observed a strict correlation between the presence of the triad motif and the in vivo methylation status of Rubisco. The distribution of the motif into a phylogenetic tree further suggests that the ancestral function of LSMT was FBA trimethylation. In a recent event during higher plant evolution, this function evolved in ancestors of Fabaceae, Cucurbitaceae, and Rosaceae to include Rubisco as an additional substrate to the archetypal enzyme. Our study provides insight into mechanisms by which SET-domain protein methyltransferases evolve new substrate specificity. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

  19. Genome-based insights into the resistome and mobilome of multidrug-resistant Aeromonas sp. ARM81 isolated from wastewater.

    Science.gov (United States)

    Adamczuk, Marcin; Dziewit, Lukasz

    2017-01-01

    The draft genome of multidrug-resistant Aeromonas sp. ARM81 isolated from a wastewater treatment plant in Warsaw (Poland) was obtained. Sequence analysis revealed multiple genes conferring resistance to aminoglycosides, β-lactams or tetracycline. Three different β-lactamase genes were identified, including an extended-spectrum β-lactamase gene bla PER-1 . The antibiotic susceptibility was experimentally tested. Genome sequencing also allowed us to investigate the plasmidome and transposable mobilome of ARM81. Four plasmids, of which two carry phenotypic modules (i.e., genes encoding a zinc transporter ZitB and a putative glucosyltransferase), and 28 putative transposase genes were identified. The mobility of three insertion sequences (isoforms of previously identified elements ISAs12, ISKpn9 and ISAs26) was confirmed using trap plasmids.

  20. Coexistence of mitochondrial 12S rRNA C1494T and CO1/tRNASer(UCN) G7444A mutations in two Han Chinese pedigrees with aminoglycoside-induced and non-syndromic hearing loss

    International Nuclear Information System (INIS)

    Yuan Huijun; Chen Jing; Liu Xin; Cheng Jing; Wang Xinjian; Yang Li; Yang Shuzhi; Cao Juyang; Kang Dongyang; Dai Pu; Zha, Suoqiang; Han Dongyi; Young Wieyen; Guan Minxin

    2007-01-01

    Mutations in mitochondrial DNA are one of the important causes of hearing loss. We report here the clinical, genetic, and molecular characterization of two Han Chinese pedigrees with maternally transmitted aminoglycoside-induced and nonsyndromic bilateral hearing loss. Clinical evaluation revealed the wide range of severity, age-at-onset, and audiometric configuration of hearing impairment in matrilineal relatives in these families. The penetrances of hearing loss in these pedigrees were 20% and 18%, when aminoglycoside-induced deafness was included. When the effect of aminoglycosides was excluded, the penetrances of hearing loss in these seven pedigrees were 10% and 15%. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the presence of the deafness-associated 12S rRNA C1494T and CO1/tRNA Ser(UCN) G7444A mutations. Their distinct sets of mtDNA polymorphism belonged to Eastern Asian haplogroup C4a1, while other previously identified six Chinese mitochondrial genomes harboring the C1494T mutation belong to haplogroups D5a2, D, R, and F1, respectively. This suggested that the C1494T or G7444A mutation occurred sporadically and multiplied through evolution of the mitochondrial DNA (mtDNA). The absence of functionally significant mutations in tRNA and rRNAs or secondary LHON mutations in their mtDNA suggest that these mtDNA haplogroup-specific variants may not play an important role in the phenotypic expression of the 12S rRNA C1494T and CO1/tRNA Ser(UCN) G7444A mutations in those Chinese families. However, aminoglycosides and other nuclear modifier genes play a modifying role in the phenotypic manifestation of the C1494T mutation in these Chinese families

  1. Detection of meca gene from methicillin resistant staphylococcus aureus isolates of north sumatera

    Science.gov (United States)

    Septiani Nasution, Gabriella; Suryanto, Dwi; Lia Kusumawati, R.

    2018-03-01

    Methicillin Resistant Staphylococcus aureus (MRSA) is a major pathogen associated with hospital-acquired infections (nosocomial infections). MRSA is a type of S. aureus resistant to the sub-group of beta-lactam antibiotics such as penicillin, cephalosporin, monobactam, and carbapenem. MRSA is resistant because of genetic changes caused by exposure to irrational antibiotic therapy. This study aimed to detect mecA gene in North Sumatra isolates of MRSA and to determine the pattern of antibiotic resistance in S.aureus isolates classified as MRSA by Vitek 2 Compact in the Central Public Hospital Haji Adam Malik, Medan. Samples were 40 isolates of S. aureus classified as MRSA obtained from clinical microbiology specimens. DNA isolation of the isolates was conducted by a method of freeze-thaw cycling. Amplification of mecA gene was done by PCR technique using specific primer for the gene. PCR products were visualized using mini-gel electrophoresis. The results showed that all MRSA isolates showed to have 533 bp band of mecA. Antibiotics test of Vitek 2 Compact showed that despite all isolates were resistant to beta-lactam antibiotics groups; the isolates showed multidrug resistant to other common antibiotics, such as aminoglycosides, macrolides, and fluoroquinolones. However, they were still sensitive to vancomycin (82.5% isolates), linezolid (97.5% isolates), and tigecycline (100% isolates).

  2. Antibiotic resistance determinants in a Pseudomonas putida strain isolated from a hospital.

    Directory of Open Access Journals (Sweden)

    Lázaro Molina

    Full Text Available Environmental microbes harbor an enormous pool of antibiotic and biocide resistance genes that can impact the resistance profiles of animal and human pathogens via horizontal gene transfer. Pseudomonas putida strains are ubiquitous in soil and water but have been seldom isolated from humans. We have established a collection of P. putida strains isolated from in-patients in different hospitals in France. One of the isolated strains (HB3267 kills insects and is resistant to the majority of the antibiotics used in laboratories and hospitals, including aminoglycosides, ß-lactams, cationic peptides, chromoprotein enediyne antibiotics, dihydrofolate reductase inhibitors, fluoroquinolones and quinolones, glycopeptide antibiotics, macrolides, polyketides and sulfonamides. Similar to other P. putida clinical isolates the strain was sensitive to amikacin. To shed light on the broad pattern of antibiotic resistance, which is rarely found in clinical isolates of this species, the genome of this strain was sequenced and analysed. The study revealed that the determinants of multiple resistance are both chromosomally-borne as well as located on the pPC9 plasmid. Further analysis indicated that pPC9 has recruited antibiotic and biocide resistance genes from environmental microorganisms as well as from opportunistic and true human pathogens. The pPC9 plasmid is not self-transmissible, but can be mobilized by other bacterial plasmids making it capable of spreading antibiotic resistant determinants to new hosts.

  3. Enterobacteriaceae in gut of honey bee (Apis mellifera and the antibiotic resistance of the isolates

    Directory of Open Access Journals (Sweden)

    Jaroslav Gasper

    2017-11-01

    Full Text Available Bacterial species of Enterobacteriaceae and the antimicrobial resistance of the isolates were detected in Apis mellifera L. bees gut. Gut content was cultivated on Meat peptone and McConkey agars at 30 and 37 °C, then, the isolates were identified with MALDI TOF MS Biotyper. Isolated strains were tested for antibiotic resistance to penicillins, cephalosporins, carbapenems, fluoroquinolones and aminoglycosides. Altogether, 12 species representing Enterobacteriaceae family were isolated. Firmicutes and Candida  were represented by Bacillus megaterium and Issatchenkia orientalis  . Isolated Enterobacteriaceae  species were  Enterobacter cloacae, Hafnia alvei, Klebsiella oxytoca, Morganella morganii, Serratia marcescens, Ser. liquefaciens, Raoultella ornithinolytica, R. planticola, R. terrigena, Pantoea ananatis, P. agglomerans, Rahnella aquatilis. Enterobacter cloacae, Hafnia alvei, Klebsiella oxytoca, Morganella morganii, Serratia marcescens, Ser. liquefaciens isolates exhibited the antimicrobial resistance more frequently than Raoultella ornithinolytica, R. planticola, R. terrigena, Pantoea ananatis, P. agglomerans, Rahnella aquatilis. Microflora of gut of bees could serve as a source of resistant microorganisms.

  4. The Set1/COMPASS histone H3 methyltransferase helps regulate mitosis with the CDK1 and NIMA mitotic kinases in Aspergillus nidulans.

    Science.gov (United States)

    Govindaraghavan, Meera; Anglin, Sarah Lea; Osmani, Aysha H; Osmani, Stephen A

    2014-08-01

    Mitosis is promoted and regulated by reversible protein phosphorylation catalyzed by the essential NIMA and CDK1 kinases in the model filamentous fungus Aspergillus nidulans. Protein methylation mediated by the Set1/COMPASS methyltransferase complex has also been shown to regulate mitosis in budding yeast with the Aurora mitotic kinase. We uncover a genetic interaction between An-swd1, which encodes a subunit of the Set1 protein methyltransferase complex, with NIMA as partial inactivation of nimA is poorly tolerated in the absence of swd1. This genetic interaction is additionally seen without the Set1 methyltransferase catalytic subunit. Importantly partial inactivation of NIMT, a mitotic activator of the CDK1 kinase, also causes lethality in the absence of Set1 function, revealing a functional relationship between the Set1 complex and two pivotal mitotic kinases. The main target for Set1-mediated methylation is histone H3K4. Mutational analysis of histone H3 revealed that modifying the H3K4 target residue of Set1 methyltransferase activity phenocopied the lethality seen when either NIMA or CDK1 are partially functional. We probed the mechanistic basis of these genetic interactions and find that the Set1 complex performs functions with CDK1 for initiating mitosis and with NIMA during progression through mitosis. The studies uncover a joint requirement for the Set1 methyltransferase complex with the CDK1 and NIMA kinases for successful mitosis. The findings extend the roles of the Set1 complex to include the initiation of mitosis with CDK1 and mitotic progression with NIMA in addition to its previously identified interactions with Aurora and type 1 phosphatase in budding yeast. Copyright © 2014 by the Genetics Society of America.

  5. Chromosomal replication incompatibility in Dam methyltransferase deficient Escherichia coli cells

    DEFF Research Database (Denmark)

    Freiesleben, Ulrik Von

    1996-01-01

    Dam methyltransferase deficient Escherichia coli cells containing minichromosomes were constructed. Free plasmid DNA could not be detected in these cells and the minichromosomes were found to be integrated in multiple copies in the origin of replication (oriC) region of the host chromosome....... The absence of the initiation cascade in Dam- cells is proposed to account for this observation of apparent incompatibility between plasmid and chromosomal copies of oriC. Studies using oriC-pBR322 chimeric plasmids and their deletion derivatives indicated that the incompatibility determinant is an intact...

  6. Resistant gram-negative bacilli and antibiotic consumption in zarqa, jordan

    International Nuclear Information System (INIS)

    Bataineh, H.A.; Alrashed, K.M.

    2007-01-01

    To investigate the prevalence of antibiotic resistance among gram-negative bacteria in relation to antibiotic use in Prince Hashem Hospital (PHH), Jordan. One hundred consecutive gram-negative bacterial isolates from different sites were collected from patients admitted to the ICU at PHH. The susceptibilities of the strains to 12 antibiotics were performed and interpreted. The quantities and the numbers of the patients discharged on antibiotics and the quantities consumed were obtained from the hospital pharmacy records. The most common isolate was P. aeruginosa (n=21) The most common site of isolation was the respiratory tract (65%), The highest susceptibility was to piperacillin/ tazobactam(78%), and the lowest was to cefuroxime(34%). The aminoglycosides gentamicin and amikacin were active against 71% and 73% of the isolates respectively, Ciprofloxacin was active against 75% of the isolates. The most frequently used antibiotics were the third-generation cephalosporins ceftriaxone and ceftazidime, followed by imipenem and amikacin. Antibiotic resistance surveillance programs associated with registration of antibiotic consumption are necessary to promote optimal use of antibiotics. Rational prescribing of antibiotics should be encouraged through educational programs, surveillance and audit. Proper infection control measures should be practiced to prevent horizontal transfer of drug-resistant organisms. (author)

  7. CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC (III) METHYLTRANSFERASE (CYT19)

    Science.gov (United States)

    CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC(III) METHYLTRANSFERASE (cyt19)Stephen B. Waters1 , Felicia Walton1 , Miroslav Styblo1 , Karen Herbin-Davis2, and David J. Thomas2 1 School of Medicine, University of North Carolina at Chape...

  8. Kinetic analysis of Yersinia pestis DNA adenine methyltransferase activity using a hemimethylated molecular break light oligonucleotide.

    Directory of Open Access Journals (Sweden)

    Robert J Wood

    Full Text Available BACKGROUND: DNA adenine methylation plays an important role in several critical bacterial processes including mismatch repair, the timing of DNA replication and the transcriptional control of gene expression. The dependence of bacterial virulence on DNA adenine methyltransferase (Dam has led to the proposal that selective Dam inhibitors might function as broad spectrum antibiotics. METHODOLOGY/PRINCIPAL FINDINGS: Herein we report the expression and purification of Yersinia pestis Dam and the development of a continuous fluorescence based assay for DNA adenine methyltransferase activity that is suitable for determining the kinetic parameters of the enzyme and for high throughput screening against potential Dam inhibitors. The assay utilised a hemimethylated break light oligonucleotide substrate containing a GATC methylation site. When this substrate was fully methylated by Dam, it became a substrate for the restriction enzyme DpnI, resulting in separation of fluorophore (fluorescein and quencher (dabcyl and therefore an increase in fluorescence. The assays were monitored in real time using a fluorescence microplate reader in 96 well format and were used for the kinetic characterisation of Yersinia pestis Dam, its substrates and the known Dam inhibitor, S-adenosylhomocysteine. The assay has been validated for high throughput screening, giving a Z-factor of 0.71+/-0.07 indicating that it is a sensitive assay for the identification of inhibitors. CONCLUSIONS/SIGNIFICANCE: The assay is therefore suitable for high throughput screening for inhibitors of DNA adenine methyltransferases and the kinetic characterisation of the inhibition.

  9. Improved radioenzymatic assay for plasma norepinephrine using purified phenylethanolamine n-methyltransferase

    International Nuclear Information System (INIS)

    Bowsher, R.R.; Henry, D.P.

    1986-01-01

    Radioenzymatic assays have been developed for catecholamines using either catechol O-methyltransferase (COMT) or phenylethanolamine N-methyltransferase (PNMT). Assays using PNMT are specific for norepinephrine (NE) and require minimal manipulative effort but until now have been less sensitive than the more complex procedures using COMT. The authors report an improved purification scheme for bovine PNMT which has permitted development of an NE assay with dramatically improved sensitivity (0.5 pg), specificity and reproducibility (C.V. < 5%). PNMT was purified by sequential pH 5.0 treatment and dialysis and by column chromatographic procedures using DEAE-Sephacel, Sepharcryl S-200 and Phenyl-Boronate Agarose. Recovery of PNMT through the purification scheme was 50%, while blank recovery was <.001%. NE can be directly quantified in 25 ul of human plasma and an 80 tube assay can be completed within 4 h. The capillary to venous plasma NE gradient was examined in 8 normotensive male subjects. Capillary plasma (NE (211.2 +/- 61.3 pg/ml)) was lower than venous plasma NE (366.6 +/- 92.5 pg/ml) in all subjects (p < 0.005). This difference suggests that capillary (NE) may be a unique indicator of sympathetic nervous system activity in vivo. In conclusion, purification of PNMT has facilitated development of an improved radioenzymatic for NE with significantly improved sensitivity

  10. The Novel Kasugamycin 2′-N-Acetyltransferase Gene aac(2′)-IIa, Carried by the IncP Island, Confers Kasugamycin Resistance to Rice-Pathogenic Bacteria

    OpenAIRE

    Yoshii, Atsushi; Moriyama, Hiromitsu; Fukuhara, Toshiyuki

    2012-01-01

    Kasugamycin (KSM), a unique aminoglycoside antibiotic, has been used in agriculture for many years to control not only rice blast caused by the fungus Magnaporthe grisea but also rice bacterial grain and seedling rot or rice bacterial brown stripe caused by Burkholderia glumae or Acidovorax avenae subsp. avenae, respectively. Since both bacterial pathogens are seed-borne and cause serious injury to rice seedlings, the emergence of KSM-resistant B. glumae and A. avenae isolates highlights the ...

  11. Extended-Spectrum-Beta-Lactamases, AmpC Beta-Lactamases and Plasmid Mediated Quinolone Resistance in Klebsiella spp. from Companion Animals in Italy

    DEFF Research Database (Denmark)

    Donati, Valentina; Feltrin, Fabiola; Hendriksen, Rene S.

    2014-01-01

    also for the aac(6')-Ib-cr gene. All Klebsiella isolates showed multiresistance towards aminoglycosides, sulfonamides, tetracyclines, trimethoprim and amphenicols, mediated by strA/B, aadA2, aadB, ant (2")-Ia, aac(6')-Ib, sul, tet, dfr and cat genes in various combinations. The emergence in pets...... of multidrug-resistant Klebsiella with ESBL, AmpC and PMQR determinants, poses further and serious challenges in companion animal therapy and raise concerns for possible bidirectional transmission between pets and humans, especially at household level....

  12. Distinct effects of struvite and biochar amendment on the class 1 integron antibiotic resistance gene cassettes in phyllosphere and rhizosphere.

    Science.gov (United States)

    An, Xin-Li; Chen, Qing-Lin; Zhu, Dong; Su, Jian-Qiang

    2018-08-01

    Struvite recovered from wastewater is promising for recycling phosphorus into soil as fertilizers. However, struvite application may prompt the proliferation of antibiotic resistance in soil and plant. This study examined the impacts of struvite application and biochar amendment on integrons abundance and gene cassette contexts in rhizosphere soil and phyllosphere using quantitative PCR and clone library analysis. Microcosm experiments revealed that class 1 integron was the most prevalent in all samples, with higher concentration and higher relative abundance in rhizosphere than those in phyllosphere. The majority of resistance gene cassettes were associated with genes encoding resistance to aminoglycosides, beta-lactams and chloramphenicols. Struvite application significantly increased the genetic diversity of antibiotic resistance gene cassettes in both rhizosphere and phyllosphere. However, biochar amendment attenuated the increasing effect of struvite application exerting on the class 1 integron antibiotic resistance gene cassette pool in phyllosphere. These findings highlighted human activities to be the source of integron gene cassette pool and raised the possibility of using biochar amendment as an alternative mean for mitigating antibiotic resistance in environments. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. Tyrosine 87 is vital for the activity of human protein arginine methyltransferase 3 (PRMT3)

    Czech Academy of Sciences Publication Activity Database

    Handrková, H.; Petrák, J.; Halada, Petr; Pospíšilová, D.; Čmejla, R.

    2011-01-01

    Roč. 1814, č. 2 (2011), s. 277-282 ISSN 1570-9639 R&D Projects: GA MŠk LC07017 Institutional research plan: CEZ:AV0Z50200510 Keywords : DIAMOND-BLACKFAN ANEMIA * SUBSTRATE -SPECIFICITY * N-METHYLTRANSFERASE Subject RIV: CE - Biochemistry Impact factor: 3.635, year: 2011

  14. Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes

    International Nuclear Information System (INIS)

    Chen, Kai; Roberts, Gareth A.; Stephanou, Augoustinos S.; Cooper, Laurie P.; White, John H.; Dryden, David T.F.

    2010-01-01

    Research highlights: → Successful fusion of GFP to M.EcoKI DNA methyltransferase. → GFP located at C-terminal of sequence specificity subunit does not later enzyme activity. → FRET confirms structural model of M.EcoKI bound to DNA. -- Abstract: We describe the fusion of enhanced green fluorescent protein to the C-terminus of the HsdS DNA sequence-specificity subunit of the Type I DNA modification methyltransferase M.EcoKI. The fusion expresses well in vivo and assembles with the two HsdM modification subunits. The fusion protein functions as a sequence-specific DNA methyltransferase protecting DNA against digestion by the EcoKI restriction endonuclease. The purified enzyme shows Foerster resonance energy transfer to fluorescently-labelled DNA duplexes containing the target sequence and to fluorescently-labelled ocr protein, a DNA mimic that binds to the M.EcoKI enzyme. Distances determined from the energy transfer experiments corroborate the structural model of M.EcoKI.

  15. Development of fluorescent methods for DNA methyltransferase assay

    Science.gov (United States)

    Li, Yueying; Zou, Xiaoran; Ma, Fei; Tang, Bo; Zhang, Chun-yang

    2017-03-01

    DNA methylation modified by DNA methyltransferase (MTase) plays an important role in regulating gene transcription, cell growth and proliferation. The aberrant DNA MTase activity may lead to a variety of human diseases including cancers. Therefore, accurate and sensitive detection of DNA MTase activity is crucial to biomedical research, clinical diagnostics and therapy. However, conventional DNA MTase assays often suffer from labor-intensive operations and time-consuming procedures. Alternatively, fluorescent methods have significant advantages of simplicity and high sensitivity, and have been widely applied for DNA MTase assay. In this review, we summarize the recent advances in the development of fluorescent methods for DNA MTase assay. These emerging methods include amplification-free and the amplification-assisted assays. Moreover, we discuss the challenges and future directions of this area.

  16. Reduction of MLH1 and PMS2 confers temozolomide resistance and is associated with recurrence of glioblastoma.

    Science.gov (United States)

    Shinsato, Yoshinari; Furukawa, Tatsuhiko; Yunoue, Shunji; Yonezawa, Hajime; Minami, Kentarou; Nishizawa, Yukihiko; Ikeda, Ryuji; Kawahara, Kohichi; Yamamoto, Masatatsu; Hirano, Hirofumi; Tokimura, Hiroshi; Arita, Kazunori

    2013-12-01

    Although there is a relationship between DNA repair deficiency and temozolomide (TMZ) resistance in glioblastoma (GBM), it remains unclear which molecule is associated with GBM recurrence. We isolated three TMZ-resistant human GBM cell lines and examined the expression of O6-methylguanine-DNA methyltransferase (MGMT) and mismatch repair (MMR) components. We used immunohistochemical analysis to compare MutL homolog 1 (MLH1), postmeiotic segregation increased 2 (PMS2) and MGMT expression in primary and recurrent GBM specimens obtained from GBM patients during TMZ treatment. We found a reduction in MLH1 expression and a subsequent reduction in PMS2 protein levels in TMZ-resistant cells. Furthermore, MLH1 or PMS2 knockdown confered TMZ resistance. In recurrent GBM tumours, the expression of MLH1 and PMS2 was reduced when compared to primary tumours.

  17. Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity

    DEFF Research Database (Denmark)

    Pasini, Diego; Bracken, Adrian P; Jensen, Michael R

    2004-01-01

    SUZ12 is a recently identified Polycomb group (PcG) protein, which together with EZH2 and EED forms different Polycomb repressive complexes (PRC2/3). These complexes contain histone H3 lysine (K) 27/9 and histone H1 K26 methyltransferase activity specified by the EZH2 SET domain. Here we show...

  18. Crystallization and preliminary X-ray crystallographic studies of O-methyltransferase from Anabaena PCC 7120

    International Nuclear Information System (INIS)

    Li, Guoming; Tang, Zhenting; Meng, Geng; Dai, Kesheng; Zhao, Jindong; Zheng, Xiaofeng

    2009-01-01

    The O-methyltransferase (OMT) from the Anabaena PCC 7120 has been overexpressed in a soluble form in E. coli, purified and crystallized. The crystals belonged to space group C222 1 and diffracted to 2.4 Å resolution. O-Methyltransferase (OMT) is a ubiquitous enzyme that exists in bacteria, plants and humans and catalyzes a methyl-transfer reaction using S-adenosyl-l-methionine as a methyl donor and a wide range of phenolics as acceptors. To investigate the structure and function of OMTs, omt from Anabaena PCC 7120 was cloned into expression vector pET21a and expressed in a soluble form in Escherichia coli strain BL21 (DE3). The recombinant OMT protein was purified to homogeneity using a two-step strategy. Crystals of OMT that diffracted to a resolution of 2.4 Å were obtained using the hanging-drop vapour-diffusion method. The crystals belonged to space group C222 1 , with unit-cell parameters a = 131.620, b = 227.994, c = 150.777 Å, α = β = γ = 90°. There are eight molecules per asymmetric unit

  19. Structure and Mechanism of the Rebeccamycin Sugar 4'-O-Methyltransferase RebM

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Shanteri; McCoy, Jason G.; Zhang, Changsheng; Bingman, Craig A.; Phillips, Jr., George N.; Thorson, Jon S. (UW)

    2008-12-12

    The 2.65-{angstrom} crystal structure of the rebeccamycin 4'-O-methyltransferase RebM in complex with S-adenosyl-l-homocysteine revealed RebM to adopt a typical S-adenosylmethionine-binding fold of small molecule O-methyltransferases (O-MTases) and display a weak dimerization domain unique to MTases. Using this structure as a basis, the RebM substrate binding model implicated a predominance of nonspecific hydrophobic interactions consistent with the reported ability of RebM to methylate a wide range of indolocarbazole surrogates. This model also illuminated the three putative RebM catalytic residues (His{sup 140/141} and Asp{sup 166}) subsequently found to be highly conserved among sequence-related natural product O-MTases from GC-rich bacteria. Interrogation of these residues via site-directed mutagenesis in RebM demonstrated His{sup 140} and Asp{sup 166} to be most important for catalysis. This study reveals RebM to be a member of the general acid/base-dependent O-MTases and, as the first crystal structure for a sugar O-MTase, may also present a template toward the future engineering of natural product MTases for combinatorial applications.

  20. Structural insights into methyltransferase KsgA function in 30S ribosomal subunit biogenesis.

    Science.gov (United States)

    Boehringer, Daniel; O'Farrell, Heather C; Rife, Jason P; Ban, Nenad

    2012-03-23

    The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3'-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation.

  1. Structural Insights into Methyltransferase KsgA Function in 30S Ribosomal Subunit Biogenesis*

    Science.gov (United States)

    Boehringer, Daniel; O'Farrell, Heather C.; Rife, Jason P.; Ban, Nenad

    2012-01-01

    The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3′-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation. PMID:22308031

  2. Complete genome sequence of multidrug-resistant Staphylococcus cohnii ssp. urealyticus strain SNUDS-2 isolated from farmed duck, Republic of Korea.

    Science.gov (United States)

    Han, Jee Eun; Lee, Seungki; Jeong, Dae Gwin; Yoon, Sun-Woo; Kim, Doo-Jin; Lee, Moo-Seung; Kim, Hye Kwon; Park, Sung-Kyun; Kim, Ji Hyung; Park, Se Chang

    2017-09-01

    Staphylococcus cohnii has become increasingly recognized as a potential pathogen of clinically significant nosocomial and farm animal infections. This study was designed to determine the genome of a multidrug-resistant S. cohnii subsp. urealyticus strain SNUDS-2 isolated from a farmed duck in Korea. Genomic DNA was sequenced using the PacBio RS II system. The complete genome was annotated and the presence of antimicrobial resistance and virulence genes were identified. The annotated 2,625,703 bp genome contained various antimicrobial resistance genes conferring resistance to β-lactam, aminoglycosides, fluoroquinolones, phenicols and trimethoprim. The virulence-associated three synergistic hemolysins have been identified in the strain. To the best of our knowledge, this is the first complete genome of S. cohnii, and will provide important insights into the biodiversity of CoNS and valuable information for the control of this emerging pathogen. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

  3. The Histone Methyltransferase Inhibitor A-366 Uncovers a Role for G9a/GLP in the Epigenetics of Leukemia.

    Directory of Open Access Journals (Sweden)

    William N Pappano

    Full Text Available Histone methyltransferases are epigenetic regulators that modify key lysine and arginine residues on histones and are believed to play an important role in cancer development and maintenance. These epigenetic modifications are potentially reversible and as a result this class of enzymes has drawn great interest as potential therapeutic targets of small molecule inhibitors. Previous studies have suggested that the histone lysine methyltransferase G9a (EHMT2 is required to perpetuate malignant phenotypes through multiple mechanisms in a variety of cancer types. To further elucidate the enzymatic role of G9a in cancer, we describe herein the biological activities of a novel peptide-competitive histone methyltransferase inhibitor, A-366, that selectively inhibits G9a and the closely related GLP (EHMT1, but not other histone methyltransferases. A-366 has significantly less cytotoxic effects on the growth of tumor cell lines compared to other known G9a/GLP small molecule inhibitors despite equivalent cellular activity on methylation of H3K9me2. Additionally, the selectivity profile of A-366 has aided in the discovery of a potentially important role for G9a/GLP in maintenance of leukemia. Treatment of various leukemia cell lines in vitro resulted in marked differentiation and morphological changes of these tumor cell lines. Furthermore, treatment of a flank xenograft leukemia model with A-366 resulted in growth inhibition in vivo consistent with the profile of H3K9me2 reduction observed. In summary, A-366 is a novel and highly selective inhibitor of G9a/GLP that has enabled the discovery of a role for G9a/GLP enzymatic activity in the growth and differentiation status of leukemia cells.

  4. d-Tubocurarine and Berbamine: Alkaloids That Are Permeant Blockers of the Hair Cell's Mechano-Electrical Transducer Channel and Protect from Aminoglycoside Toxicity

    Directory of Open Access Journals (Sweden)

    Nerissa K. Kirkwood

    2017-09-01

    Full Text Available Aminoglycoside antibiotics are widely used for the treatment of life-threatening bacterial infections, but cause permanent hearing loss in a substantial proportion of treated patients. The sensory hair cells of the inner ear are damaged following entry of these antibiotics via the mechano-electrical transducer (MET channels located at the tips of the hair cell's stereocilia. d-Tubocurarine (dTC is a MET channel blocker that reduces the loading of gentamicin-Texas Red (GTTR into rat cochlear hair cells and protects them from gentamicin treatment. Berbamine is a structurally related alkaloid that reduces GTTR labeling of zebrafish lateral-line hair cells and protects them from aminoglycoside-induced cell death. Both compounds are thought to reduce aminoglycoside entry into hair cells through the MET channels. Here we show that dTC (≥6.25 μM or berbamine (≥1.55 μM protect zebrafish hair cells in vivo from neomycin (6.25 μM, 1 h. Protection of zebrafish hair cells against gentamicin (10 μM, 6 h was provided by ≥25 μM dTC or ≥12.5 μM berbamine. Hair cells in mouse cochlear cultures are protected from longer-term exposure to gentamicin (5 μM, 48 h by 20 μM berbamine or 25 μM dTC. Berbamine is, however, highly toxic to mouse cochlear hair cells at higher concentrations (≥30 μM whilst dTC is not. The absence of toxicity in the zebrafish assays prompts caution in extrapolating results from zebrafish neuromasts to mammalian cochlear hair cells. MET current recordings from mouse outer hair cells (OHCs show that both compounds are permeant open-channel blockers, rapidly and reversibly blocking the MET channel with half-blocking concentrations of 2.2 μM (dTC and 2.8 μM (berbamine in the presence of 1.3 mM Ca2+ at −104 mV. Berbamine, but not dTC, also blocks the hair cell's basolateral K+ current, IK,neo, and modeling studies indicate that berbamine permeates the MET channel more readily than dTC. These studies reveal key properties of

  5. The antibiotic resistance "mobilome": searching for the link between environment and clinic.

    Science.gov (United States)

    Perry, Julie A; Wright, Gerard D

    2013-01-01

    Antibiotic resistance is an ancient problem, owing to the co-evolution of antibiotic-producing and target organisms in the soil and other environments over millennia. The environmental "resistome" is the collection of all genes that directly or indirectly contribute to antibiotic resistance. Many of these resistance determinants originate in antibiotic-producing organisms (where they serve to mediate self-immunity), while others become resistance determinants only when mobilized and over-expressed in non-native hosts (like plasmid-encoded β-lactamases). The modern environmental resistome is under selective pressure from human activities such as agriculture, which may influence the composition of the local resistome and lead to gene transfer events. Beyond the environment, we are challenged in the clinic by the rise in both frequency and diversity of antibiotic resistant pathogens. We assume that clinical resistance originated in the environment, but few examples of direct gene exchange between the environmental resistome and the clinical resistome have been documented. Strong evidence exists to suggest that clinical aminoglycoside and vancomycin resistance enzymes, the extended-spectrum β-lactamase CTX-M and the quinolone resistance gene qnr have direct links to the environmental resistome. In this review, we highlight recent advances in our understanding of horizontal gene transfer of antibiotic resistance genes from the environment to the clinic. Improvements in sequencing technologies coupled with functional metagenomic studies have revealed previously underappreciated diversity in the environmental resistome, and also established novel genetic links to the clinic. Understanding mechanisms of gene exchange becomes vital in controlling the future dissemination of antibiotic resistance.

  6. Synthesis and antibacterial activity of 1-N-(1,3-dihydroxy-2-propyl)kanamycin B (UK-31,214).

    Science.gov (United States)

    Richardson, K; Brammer, K W; Jevons, S; Plews, R M; Wright, J R

    1979-10-01

    1-N-(1,3-Dihydroxy-2-propyl)kanamycin B was prepared and its in vitro activity against aminoglycoside-sensitive and aminoglycoside-resistant organisms was compared with that of kanamycin B and gentamicin. This kanamycin B derivative (code No. UK-31,214) demonstrated potent activity in all of these tests and gave good protection in experimental infections in mice.

  7. Synthesis and antibacterial activities of 1-N [(S)-omega-amino-2-hydroxyalkyl] kanamycin A derivatives.

    Science.gov (United States)

    Richardson, K; Jevons, S; Moore, J W; Ross, B C; Wright, J R

    1977-10-01

    Four 1-N-aminohydroxy-alkyl derivatives of kanamycin A were prepared and their in vitro activities against aminoglycoside-sensitive and aminoglycoside-resistant organisms were compared with amikacin. 1-N-[(S)-4-Amino-2-hydroxybutyl] kanamycin A (Fig. 1, compound 2, code no. UK-18,892) was equipotent to amikacin in all these tests and in mouse protection studies.

  8. Rapid detection of drug resistance and mutational patterns of extensively drug-resistant strains by a novel GenoType® MTBDRsl assay

    Directory of Open Access Journals (Sweden)

    A K Singh

    2013-01-01

    Full Text Available Background: The emergence of extensively drug-resistant tuberculosis (XDR-TB is a major concern in the India. The burden of XDR-TB is increasing due to inadequate monitoring, lack of proper diagnosis, and treatment. The GenoType ® Mycobacterium tuberculosis drug resistance second line (MTBDRsl assay is a novel line probe assay used for the rapid detection of mutational patterns conferring resistance to XDR-TB. Aim: The aim of this study was to study the rapid detection of drug resistance and mutational patterns of the XDR-TB by a novel GenoType ® MTBDRsl assay. Materials and Methods: We evaluated 98 multidrug-resistant (MDR M. tuberculosis isolates for second line drugs susceptibility testing by 1% proportion method (BacT/ALERT 3D system and GenoType ® MTBDRsl assay for rapid detection of conferring drug resistance to XDR-TB. Results: A total of seven (17.4% were identified as XDR-TB by using standard phenotypic method. The concordance between phenotypic and GenoType ® MTBDRsl assay was 91.7-100% for different antibiotics. The sensitivity and specificity of the MTBDRsl assay were 100% and 100% for aminoglycosides; 100% and 100% for fluoroquinolones; 91.7% and 100% for ethambutol. The most frequent mutations and patterns were gyrA MUT1 (A90V in seven (41.2% and gyrA + WT1-3 + MUT1 in four (23.5%; rrs MUT1 (A1401G in 11 (64.7%, and rrs WT1-2 + MUT1 in eight (47.1%; and embB MUT1B (M306V in 11 (64.7% strains. Conclusions: These data suggest that the GenoType ® MTBDRsl assay is rapid, novel test for detection of resistance to second line anti-tubercular drugs. This assay provides additional information about the frequency and mutational patterns responsible for XDR-TB resistance.

  9. The histone methyltransferase G9a as a therapeutic target to override gemcitabine resistance in pancreatic cancer

    Science.gov (United States)

    Pan, Mei-Ren; Hsu, Ming-Chuan; Luo, Chi-Wen; Chen, Li-Tzong; Shan, Yan-Shen; Hung, Wen-Chun

    2016-01-01

    Gemcitabine (GEM) resistance is a critical issue for pancreatic cancer treatment. The involvement of epigenetic modification in GEM resistance is still unclear. We established a GEM-resistant subline PANC-1-R from the parental PANC-1 pancreatic cancer cells and found the elevation of various chromatin-modifying enzymes including G9a in GEM-resistant cells. Ectopic expression of G9a in PANC-1 cells increased GEM resistance while inactivation of G9a in PANC-1-R cells reduced it. Challenge of PANC-1 cells with GEM increased the expression of stemness markers including CD133, nestin and Lgr5 and promoted sphere forming activity suggesting chemotherapy enriched cancer cells with stem-like properties. Inhibition of G9a in PANC-1-R cells reduced stemness and invasiveness and sensitized the cells to GEM. We revealed interleukin-8 (IL-8) is a downstream effector of G9a to increase GEM resistance. G9a-overexpressing PANC-1-R cells exhibited autocrine IL-8/CXCR1/2 stimulation to increase GEM resistance which could be decreased by anti-IL-8 antibody and G9a inhibitor. IL-8 released by cancer cells also activated pancreatic stellate cell (PSC) to increase GEM resistance. In orthotopic animal model, GEM could not suppress tumor growth of PANC-1-R cells and eventually promoted tumor metastasis. Combination with G9a inhibitor and GEM reduced tumor growth, metastasis, IL-8 expression and PSC activation in animals. Finally, we showed that overexpression of G9a correlated with poor survival and early recurrence in pancreatic cancer patients. Collectively, our results suggest G9a is a therapeutic target to override GEM resistance in the treatment of pancreatic cancer. PMID:27531902

  10. Identification of an S-adenosylmethionine (SAM) dependent arsenic methyltransferase in Danio rerio

    Energy Technology Data Exchange (ETDEWEB)

    Hamdi, Mohamad [Department of Biological Sciences, Oakland University, Rochester, MI 48309 (United States); Yoshinaga, Masafumi; Packianathan, Charles; Qin, Jie [Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, FL33199 (United States); Hallauer, Janell; McDermott, Joseph R. [Department of Biological Sciences, Oakland University, Rochester, MI 48309 (United States); Yang, Hung-Chi [Department of Medical Biotechnology and Laboratory Sciences, Chang-Gung University, Tao-Yuan, Kwei-San 333, Taiwan (China); Tsai, Kan-Jen [School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, Taiwan (China); Liu, Zijuan, E-mail: liu2345@oakland.edu [Department of Biological Sciences, Oakland University, Rochester, MI 48309 (United States)

    2012-07-15

    Arsenic methylation is an important cellular metabolic process that modulates arsenic toxicity and carcinogenicity. Biomethylation of arsenic produces a series of mono-, di- and tri-methylated arsenic metabolites that can be detected in tissues and excretions. Here we report that zebrafish exposed to arsenite (As{sup III}) produces organic arsenicals, including MMA{sup III}, MMA{sup V} and DMA{sup V} with characteristic tissue ratios, demonstrating that an arsenic methylation pathway exists in zebrafish. In mammals, cellular inorganic arsenic is methylated by a SAM-dependent arsenic methyltransferase, AS3MT. A zebrafish arsenic methyltransferase homolog, As3mt, was identified by sequence alignment. Western blotting analysis showed that As3mt was universally expressed in zebrafish tissues. Prominent expression in liver and intestine correlated with methylated arsenic metabolites detected in those tissues. As3mt was expressed in and purified from Escherichia coli for in vitro functional studies. Our results demonstrated that As3mt methylated As{sup III} to DMA{sup V} as an end product and produced MMA{sup III} and MMA{sup V} as intermediates. The activity of As3mt was inhibited by elevated concentrations of the substrate As{sup III} as well as the metalloid selenite, which is a well-known antagonistic micronutrient of arsenic toxicity. The activity As3mt was abolished by substitution of either Cys160 or Cys210, which corresponds to conserved cysteine residues in AS3MT homologs, suggesting that they are involved in catalysis. Expression in zebrafish of an enzyme that has a similar function to human and rodent orthologs in catalyzing intracellular arsenic biomethylation validates the applicability of zebrafish as a valuable vertebrate model for understanding arsenic-associated diseases in humans. -- Highlights: ► Zebrafish methylated As{sup III} to MMA{sup III}, MMA{sup V} and DMA{sup V}. ► A zebrafish arsenic methyltransferase (As3mt) was purified in E. coli.

  11. DNA repair methyltransferase (Mgmt) knockout mice are sensitive to the lethal effects of chemotherapeutic alkylating agents.

    NARCIS (Netherlands)

    B.J. Glassner (Brian); G. Weeda (Geert); J.M. Allan (James); J.L.M. Broekhof (Jose'); N.H.E. Carls (Nick); I. Donker (Ingrid); B.P. Engelward (Bevin); R.J. Hampson (Richard); R. Hersmus (Remko); M.J. Hickman (Mark); R.B. Roth (Richard); H.B. Warren (Henry); M.M. Wu (Mavis); J.H.J. Hoeijmakers (Jan); L.D. Samson (Leona)

    1999-01-01

    textabstractWe have generated mice deficient in O6-methylguanine DNA methyltransferase activity encoded by the murine Mgmt gene using homologous recombination to delete the region encoding the Mgmt active site cysteine. Tissues from Mgmt null mice displayed very low O6-methylguanine DNA

  12. Markov Networks of Collateral Resistance: National Antimicrobial Resistance Monitoring System Surveillance Results from Escherichia coli Isolates, 2004-2012.

    Directory of Open Access Journals (Sweden)

    William J Love

    2016-11-01

    Full Text Available Surveillance of antimicrobial resistance (AMR is an important component of public health. Antimicrobial drug use generates selective pressure that may lead to resistance against to the administered drug, and may also select for collateral resistances to other drugs. Analysis of AMR surveillance data has focused on resistance to individual drugs but joint distributions of resistance in bacterial populations are infrequently analyzed and reported. New methods are needed to characterize and communicate joint resistance distributions. Markov networks are a class of graphical models that define connections, or edges, between pairs of variables with non-zero partial correlations and are used here to describe AMR resistance relationships. The graphical least absolute shrinkage and selection operator is used to estimate sparse Markov networks from AMR surveillance data. The method is demonstrated using a subset of Escherichia coli isolates collected by the National Antimicrobial Resistance Monitoring System between 2004 and 2012 which included AMR results for 16 drugs from 14418 isolates. Of the 119 possible unique edges, 33 unique edges were identified at least once during the study period and graphical density ranged from 16.2% to 24.8%. Two frequent dense subgraphs were noted, one containing the five β-lactam drugs and the other containing both sulfonamides, three aminoglycosides, and tetracycline. Density did not appear to change over time (p = 0.71. Unweighted modularity did not appear to change over time (p = 0.18, but a significant decreasing trend was noted in the modularity of the weighted networks (p < 0.005 indicating relationships between drugs of different classes tended to increase in strength and frequency over time compared to relationships between drugs of the same class. The current method provides a novel method to study the joint resistance distribution, but additional work is required to unite the underlying biological and genetic

  13. Crystal structure of MboIIA methyltransferase.

    Science.gov (United States)

    Osipiuk, Jerzy; Walsh, Martin A; Joachimiak, Andrzej

    2003-09-15

    DNA methyltransferases (MTases) are sequence-specific enzymes which transfer a methyl group from S-adenosyl-L-methionine (AdoMet) to the amino group of either cytosine or adenine within a recognized DNA sequence. Methylation of a base in a specific DNA sequence protects DNA from nucleolytic cleavage by restriction enzymes recognizing the same DNA sequence. We have determined at 1.74 A resolution the crystal structure of a beta-class DNA MTase MboIIA (M.MboIIA) from the bacterium Moraxella bovis, the smallest DNA MTase determined to date. M.MboIIA methylates the 3' adenine of the pentanucleotide sequence 5'-GAAGA-3'. The protein crystallizes with two molecules in the asymmetric unit which we propose to resemble the dimer when M.MboIIA is not bound to DNA. The overall structure of the enzyme closely resembles that of M.RsrI. However, the cofactor-binding pocket in M.MboIIA forms a closed structure which is in contrast to the open-form structures of other known MTases.

  14. Whole genome sequencing for the molecular characterization of carbapenem-resistant Klebsiella pneumoniae strains isolated at the Italian ASST Fatebenefratelli Sacco Hospital, 2012-2014.

    Science.gov (United States)

    Rimoldi, Sara Giordana; Gentile, Bernardina; Pagani, Cristina; Di Gregorio, Annamaria; Anselmo, Anna; Palozzi, Anna Maria; Fortunato, Antonella; Pittiglio, Valentina; Ridolfo, Anna Lisa; Gismondo, Maria Rita; Rizzardini, Giuliano; Lista, Florigio

    2017-10-10

    The emergence of carbapenem-resistant Klebsiella pneumoniae strains is threatening antimicrobial treatment. Sixty-eight carbapenemase-producing K. pneumoniae strains isolated at Luigi Sacco University Hospital-ASST Fatebenefratelli Sacco (Milan, Italy) between 2012 and 2014 were characterised microbiologically and molecularly. They were tested for drug susceptibility and carbapenemase phenotypes, investigated by means of repetitive extra-genic palindromic polymerase chain reaction (REP-PCR), and fully sequenced by means of next-generation sequencing for the in silico analysis of multi-locus sequence typing (MLST), their resistome, virulome and plasmid content, and their core single nucleotide polymorphism (SNP) genotypes. All of the samples were resistant to carbapenems, other β-lactams and ciprofloxacin; many were resistant to aminoglycosides and tigecycline; and seven were resistant to colistin. Resistome analysis revealed the presence of blaKPC genes and, less frequently blaSHV, blaTEM, blaCTX-M and blaOXA, which are related to resistance to carbapenem and other β-lactams. Other genes conferring resistance to aminoglycoside, fluoroquinolone, phenicol, sulphonamide, tetracycline, trimethoprim and macrolide-lincosamide-streptogramin were also detected. Genes related to AcrAB-TolC efflux pump-dependent and pump-independent tigecycline resistance mechanisms were investigated, but it was not possible to clearly correlate the genomic features with tigecycline resistance because of the presence of a common mutation in susceptible, intermediate and resistant strains. Concerning colistin resistance, the mgrB gene was disrupted by an IS5-like element, and the mobile mcr-1 and mcr-2 genes were not detected in two cases. The virulome profile revealed type-3 fimbriae and iron uptake system genes, which are important during the colonisation stage in the mammalian host environment. The in silico detected plasmid replicons were classified as IncFIB(pQil), IncFIB(K), Col

  15. Environmental cycle of antibiotic resistance encoded genes: A systematic review

    Directory of Open Access Journals (Sweden)

    R. ghanbari

    2017-12-01

    Full Text Available Antibiotic-resistant bacteria and genes enter the environment in different ways. The release of these factors into the environment has increased concerns related to public health. The aim of the study was to evaluate the antibiotic resistance genes (ARGs in the environmental resources. In this systematic review, the data were extracted from valid sources of information including ScienceDirect, PubMed, Google Scholar and SID. Evaluation and selection of articles were conducted on the basis of the PRISMA checklist. A total of 39 articles were included in the study, which were chosen from a total of 1249 papers. The inclusion criterion was the identification of genes encoding antibiotic resistance against the eight important groups of antibiotics determined by using the PCR technique in the environmental sources including municipal and hospital wastewater treatment plants, animal and agricultural wastes, effluents from treatment plants, natural waters, sediments, and drinking waters. In this study, 113 genes encoding antibiotic resistance to eight groups of antibiotics (beta-lactams, aminoglycosides, tetracyclines, macrolides, sulfonamides, chloramphenicol, glycopeptides and quinolones were identified in various environments. Antibiotic resistance genes were found in all the investigated environments. The investigation of microorganisms carrying these genes shows that most of the bacteria especially gram-negative bacteria are effective in the acquisition and the dissemination of these pollutants in the environment. Discharging the raw wastewaters and effluents from wastewater treatments acts as major routes in the dissemination of ARGs into environment sources and can pose hazards to public health.

  16. The Antibiotic Resistance ‘Mobilome’: searching for the link between environment and clinic.

    Directory of Open Access Journals (Sweden)

    Julie ePerry

    2013-05-01

    Full Text Available Antibiotic resistance is an ancient problem, owing to the co-evolution of antibiotic-producing and target organisms in the soil and other environments over millennia. The environmental ‘resistome’ is the collection of all genes that directly or indirectly contribute to antibiotic resistance. Many of these resistance determinants originate in antibiotic-producing organisms (where they serve to mediate self-immunity, while others become resistance determinants only when mobilized and over-expressed in non-native hosts (like plasmid-based β-lactamases. The modern environmental resistome is under selective pressure from human activities such as agriculture, which may influence the composition of the local resistome and lead to gene transfer events. Beyond the environment, we are challenged in the clinic by the rise in both frequency and diversity of antibiotic resistant pathogens. We assume that clinical resistance originated in the environment, but few examples of direct gene exchange between the environmental resistome and the clinical resistome have been documented. Strong evidence exists to suggest that clinical aminoglycoside and vancomycin resistance enzymes, the extended-spectrum β-lactamase CTX-M and the quinolone resistance gene Qnr have direct links to the environmental resistome. In this review, we highlight recent advances in our understanding of horizontal gene transfer of antibiotic resistance genes from the environment to the clinic. Improvements in sequencing technologies coupled with functional metagenomic studies have revealed previously underappreciated diversity in the environmental resistome, and also established novel genetic links to the clinic. Understanding mechanisms of gene exchange becomes vital in controlling the future dissemination of antibiotic resistance.

  17. The antibiotic resistance “mobilome”: searching for the link between environment and clinic

    Science.gov (United States)

    Perry, Julie A.; Wright, Gerard D.

    2013-01-01

    Antibiotic resistance is an ancient problem, owing to the co-evolution of antibiotic-producing and target organisms in the soil and other environments over millennia. The environmental “resistome” is the collection of all genes that directly or indirectly contribute to antibiotic resistance. Many of these resistance determinants originate in antibiotic-producing organisms (where they serve to mediate self-immunity), while others become resistance determinants only when mobilized and over-expressed in non-native hosts (like plasmid-encoded β-lactamases). The modern environmental resistome is under selective pressure from human activities such as agriculture, which may influence the composition of the local resistome and lead to gene transfer events. Beyond the environment, we are challenged in the clinic by the rise in both frequency and diversity of antibiotic resistant pathogens. We assume that clinical resistance originated in the environment, but few examples of direct gene exchange between the environmental resistome and the clinical resistome have been documented. Strong evidence exists to suggest that clinical aminoglycoside and vancomycin resistance enzymes, the extended-spectrum β-lactamase CTX-M and the quinolone resistance gene qnr have direct links to the environmental resistome. In this review, we highlight recent advances in our understanding of horizontal gene transfer of antibiotic resistance genes from the environment to the clinic. Improvements in sequencing technologies coupled with functional metagenomic studies have revealed previously underappreciated diversity in the environmental resistome, and also established novel genetic links to the clinic. Understanding mechanisms of gene exchange becomes vital in controlling the future dissemination of antibiotic resistance. PMID:23755047

  18. Linezolid-resistant enterococci in Polish hospitals: species, clonality and determinants of linezolid resistance.

    Science.gov (United States)

    Gawryszewska, I; Żabicka, D; Hryniewicz, W; Sadowy, E

    2017-07-01

    The significant increase of the linezolid-resistant enterococci (LRE) has been observed in Polish hospitals since 2012 and our study aimed at elucidating the possible reasons for this phenomenon. Polish LRE isolates were analysed by multilocus-sequence typing (MLST) and multiple locus variable-number tandem repeat (VNTR) analysis (MLVA), polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP) to establish clonal relatedness and mechanism of linezolid resistance, respectively. Fifty analysed LRE (2008-2015) included mostly Enterococcus faecium (82%) and Enterococcus faecalis (16%). Enterococcus faecium belonged to the hospital-adapted lineages 17/18 and 78, while E. faecalis isolates represented ST6, a hospital-associated type, and ST116, found in both humans and food-production animals. The G2576T 23S rRNA mutation was the most frequent (94%) mechanism of linezolid/tedizolid resistance of LRE. None of the isolates carried the plasmid-associated gene of Cfr methyltransferase, whereas optrA, encoding the ABC-type drug transporter, was identified in two E. faecalis isolates. In these isolates, optrA was located on a plasmid, transferable to both E. faecium and E. faecalis, whose partial (36.3 kb) sequence was 100% identical to the pE394 plasmid, identified previously in China in both clinical and farm animal isolates. The optrA-E. faecium transconjugant displayed a significant growth deficiency, in contrast to the optrA-E. faecalis. Our study indicates the role of mutation acquisition by hospital-adapted clones of enterococci as a major driver of increasing resistance to linezolid and tedizolid. Transferability and apparent lack of a biological cost of resistance suggest that E. faecalis may be a natural reservoir of optrA, an emerging mechanism of oxazolidinone resistance.

  19. Identification and characterization of new molecular partners for the protein arginine methyltransferase 6 (PRMT6.

    Directory of Open Access Journals (Sweden)

    Alessandra Lo Sardo

    Full Text Available PRMT6 is a protein arginine methyltransferase that has been implicated in transcriptional regulation, DNA repair, and human immunodeficiency virus pathogenesis. Only few substrates of this enzyme are known and therefore its cellular role is not well understood. To identify in an unbiased manner substrates and potential regulators of PRMT6 we have used a yeast two-hybrid approach. We identified 36 new putative partners for PRMT6 and we validated the interaction in vivo for 7 of them. In addition, using invitro methylation assay we identified 4 new substrates for PRMT6, extending the involvement of this enzyme to other cellular processes beyond its well-established role in gene expression regulation. Holistic approaches create molecular connections that allow to test functional hypotheses. The assembly of PRMT6 protein network allowed us to formulate functional hypotheses which led to the discovery of new molecular partners for the architectural transcription factor HMGA1a, a known substrate for PRMT6, and to provide evidences for a modulatory role of HMGA1a on the methyltransferase activity of PRMT6.

  20. Two transcriptional activators of N-acetylserotonin O-methyltransferase 2 and melatonin biosynthesis in cassava.

    Science.gov (United States)

    Wei, Yunxie; Liu, Guoyin; Bai, Yujing; Xia, Feiyu; He, Chaozu; Shi, Haitao; Foyer, Christine

    2017-10-13

    Similar to the situation in animals, melatonin biosynthesis is regulated by four sequential enzymatic steps in plants. Although the melatonin synthesis genes have been identified in various plants, the upstream transcription factors of them remain unknown. In this study on cassava (Manihot esculenta), we found that MeWRKY79 and heat-shock transcription factor 20 (MeHsf20) targeted the W-box and the heat-stress elements (HSEs) in the promoter of N-acetylserotonin O-methyltransferase 2 (MeASMT2), respectively. The interaction between MeWRKY79, MeHsf20, and the MeASMT2 promoter was evidenced by the activation of promoter activity and chromatin immunoprecipitation (ChIP) in cassava protoplasts, and by an in vitro electrophoretic mobility shift assay (EMSA). The transcripts of MeWRKY79, MeHsf20, and MeASMT2 were all regulated by a 22-amino acid flagellin peptide (flg22) and by Xanthomonas axonopodis pv manihotis (Xam). In common with the phenotype of MeASMT2, transient expression of MeWRKY79 and MeHsf20 in Nicotiana benthamiana leaves conferred improved disease resistance. Through virus-induced gene silencing (VIGS) in cassava, we found that MeWRKY79- and MeHsf20-silenced plants showed lower transcripts of MeASMT2 and less accumulation of melatonin, which resulted in disease sensitivity that could be reversed by exogenous melatonin. Taken together, these results indicate that MeASMT2 is a target of MeWRKY79 and MeHsf20 in plant disease resistance. This study identifies novel upstream transcription factors of melatonin synthesis genes in cassava, thus extending our knowledge of the complex modulation of melatonin synthesis in plant defense. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  1. Thiopurines inhibit bovine viral diarrhea virus production in a thiopurine methyltransferase-dependent manner.

    Science.gov (United States)

    Hoover, Spencer; Striker, Rob

    2008-04-01

    The family Flaviviridae comprises positive-strand RNA viral pathogens of humans and livestock with few treatment options. We have previously shown that azathioprine (AZA) has in vitro activity against bovine viral diarrhea virus (BVDV). While the mechanism of inhibition is unknown, AZA and related thiopurine nucleoside analogues have been used as immunosuppressants for decades and both AZA metabolites and cellular genes involved in AZA metabolism have been extensively characterized. Here, we show that only certain riboside metabolites have antiviral activity and identify the most potent known antiviral AZA metabolite as 6-methylmercaptopurine riboside (6MMPr). The antiviral activity of 6MMPr is antagonized by adenosine, and is specific to BVDV and not to the related yellow fever virus. An essential step in the conversion of AZA to 6MMPr is the addition of a methyl group onto the sulfur atom attached to position six of the purine ring. Intracellularly, the methyl group is added by thiopurine methyltransferase (TPMT), an S-adenosyl methionine-dependent methyltransferase. Either chemically bypassing or inhibiting TPMT modulates antiviral activity of AZA metabolites. TPMT exists in several variants with varying levels of activity and since 6MMPr is a potent antiviral, the antiviral activity of AZA may be modulated by host genetics.

  2. The sensitivity of Bacillus subtilis to diverse antimicrobial compounds is influenced by Abh.

    Science.gov (United States)

    Murray, Ewan J; Stanley-Wall, Nicola R

    2010-12-01

    Abh is a transition state regulator of Bacillus subtilis that controls biofilm formation and the production of several diverse antimicrobial compounds. Using a high-throughput non-biased technique, we show for the first time that Abh influences the sensitivity of B. subtilis to diverse antimicrobial compounds. Following up on these findings with a combination of classical genetics and antibiotic susceptibility assays, we demonstrate that Abh influences cellular processes such as the remodelling of the cell wall. We present data demonstrating that the extracytoplasmic function sigma factor σ(X) controls resistance to β-lactam antibiotics by activating abh transcription. Downstream from Abh, activation of slrR expression by Abh is responsible for controlling the sensitivity of B. subtilis to such antibiotics due to the role that SlrR plays in regulating autolysin biosynthesis. The abh mutant additionally exhibits increased resistance to aminoglycoside antimicrobials. We confirm that aminoglycoside killing of B. subtilis is likely to be caused by oxidative damage but rule out the possibility that the increased resistance of the abh mutant to aminoglycosides is due to a general increase in resistance to oxidative stress.

  3. Epidemiology of infections caused by multiresistant gram-negatives: ESBLs, MBLs, panresistant strains.

    Science.gov (United States)

    Rossolini, Gian Maria; Mantengoli, Elisabetta; Docquier, Jean-Denis; Musmanno, Rosa Anna; Coratza, Grazietta

    2007-07-01

    Microbial drug resistance is a growing problem of global magnitude. In gram-negative pathogens, the most important resistance problems are encountered in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter, with increasing trends observed for all major anti-gram-negative agents (beta-lactams, fluoroquinolones and aminoglycosides). A matter of major concern is the emergence of new beta-lactamases capable of degrading the expanded-spectrum cephalosporins and/or carbapenems, such as the extended-spectrum beta-lactamases (ESBLs) and the carbapenemases. These beta-lactamase genes are often associated with resistance determinants to non-beta-lactam agents (e.g. aminoglycosides and fluoroquinolones), and strains producing ESBLs or carbapenemases often exhibit complex multidrug resistant phenotypes and sometimes are panresistant. The problem is worsened by the dearth of new agents active on multidrug-resistant Gram-negatives in the pipeline. The importance to develop better strategies to control resistance is underscored.

  4. The histone methyltransferase and putative oncoprotein MMSET is overexpressed in a large variety of human tumors

    DEFF Research Database (Denmark)

    Hudlebusch, Heidi Rye; Santoni-Rugiu, Eric; Simon, Ronald

    2011-01-01

    Multiple myeloma SET (Suppressor of variegation, Enhancer of zeste, and Trithorax) domain (MMSET) is a histone lysine methyltransferase deregulated in a subgroup of multiple myelomas with the t(4;14)(p16;q32) translocation and poor prognosis. With the aim of understanding, if MMSET can be involved...

  5. No up-regulation of the phosphatidylethanolamine N-methyltransferase pathway and choline production by sex hormones in cats

    NARCIS (Netherlands)

    Valtolina, Chiara; Vaandrager, Arie B; Favier, Robert P; Robben, Joris H; Tuohetahuntila, Maidina; Kummeling, Anne; Jeusette, Isabelle; Rothuizen, Jan

    2015-01-01

    BACKGROUND: Feline hepatic lipidosis (FHL) is a common cholestatic disease affecting cats of any breed, age and sex. Both choline deficiency and low hepatic phosphatidylethanolamine N-methyltransferase (PEMT) activity are associated with hepatic lipidosis (HL) in humans, mice and rats. The PEMT

  6. The frequency of resistance to antibiotics of most frequently isolated bacteria from blood cultures during the period 1997-2002

    Directory of Open Access Journals (Sweden)

    Mirović Veljko

    2004-01-01

    Full Text Available The aim of this study was to determine the frequency of resistance to antibiotics of the most frequently isolated bacteria from blood cultures of hospitalized patients during the period 1997-2002. The resistance to antibiotics was determined by disk diffusion method according to National Committee for Clinical Laboratory Standards procedures. The majority of staphylococci isolates were resistant to methicillin, and the proportion of methicillin-resistant Staphylococcus aureus was stable (76.8-81.6%, during the follow-up period. None of the staphylococci isolates were resistant to vancomycin, but there was a very high incidence of high-level resistance of enterococci to aminoglycosides (47.2-72.2%. In 1998, only one strain among enterococci was resistant to vancomycin (Enterococcus faecium, VanA fenotype. Enterococcus spp isolates expressed variable frequency of resistance to ampicillin (15-40.1% during the follow-up period. Among Enterobacteriaceae there were no isolates resistant to imipenem, but dramatic increase of the resistance to ceftriaxone was found from 35.9% in 1997 to 95.9% in 2002 (p<0.001. Extended spectrum beta-lactamases production was found in all the species of enterobacteria isolates. Resistance to imipenem was observed in Acinetobacter spp isolates in 2002 for the first time. Pseudomonas spp isolates expressed high and very variable resistance to all antibiotics tested during the follow-up period.

  7. Trend and seasonality of community-acquired Escherichia coli antimicrobial resistance and its dynamic relationship with antimicrobial use assessed by ARIMA models.

    Science.gov (United States)

    Asencio Egea, María Ángeles; Huertas Vaquero, María; Carranza González, Rafael; Herráez Carrera, Óscar; Redondo González, Olga; Arias Arias, Ángel

    2017-12-04

    We studied the trend and seasonality of community-acquired Escherichia coli resistance and quantified its correlation with the previous use of certain antibiotics. A time series study of resistant community-acquired E. coli isolates and their association with antibiotic use was conducted in a Primary Health Care Area from 2008 to 2012. A Poisson regression model was constructed to estimate the trend and seasonality of E. coli resistance. A significant increasing trend in mean E. coli resistance to cephalosporins, aminoglycosides and nitrofurantoin was observed. Seasonal resistance to ciprofloxacin and amoxicillin-clavulanic acid was significantly higher in autumn-winter. There was a delay of 7, 10 and 12 months between the use of cotrimoxazole (P<0.038), fosfomycin (P<0.024) and amoxicillin-clavulanic acid (P<0.015), respectively, and the occurrence of E. coli resistance. An average delay of 10 months between the previous use of amoxicillin-clavulanic acid, cotrimoxazole and fosfomycin and the appearance of resistant community-acquired E. coli strains was detected. Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  8. Crystallization of the novel S-adenosyl-l-methionine-dependent C-methyltransferase CouO from Streptomyces rishiriensis and preliminary diffraction data analysis

    International Nuclear Information System (INIS)

    Lyskowski, Andrzej; Tengg, Martin; Steinkellner, Georg; Schwab, Helmut; Gruber-Khadjawi, Mandana; Gruber, Karl

    2012-01-01

    Recombinant Q9F8T9 protein from Streptomyces rishiriensis (CouO), an S-adenosyl-l-methionine-dependent C-methyltransferase, has been successfully cloned, expressed and purified. Recombinant Q9F8T9 protein from Streptomyces rishiriensis (CouO), an S-adenosyl-l-methionine-dependent C-methyltransferase, has been successfully cloned, expressed and purified. CouO was crystallized from a single condition in the Morpheus crystallization screen. A vitrified crystal diffracted to 2.05 Å resolution and belonged to space group P2 1 , with unit-cell parameters a = 33.02, b = 82.87, c = 76.77 Å, β = 96.93°

  9. An easy-to-perform photometric assay for methyltransferase activity measurements.

    Science.gov (United States)

    Schäberle, Till F; Siba, Christian; Höver, Thomas; König, Gabriele M

    2013-01-01

    Methyltransferases (MTs) catalyze the transfer of a methyl group from S-adenosylmethionine (SAM) to a suitable substrate. Such methylations are important modifications in secondary metabolisms, especially on natural products produced by polyketide synthases and nonribosomal peptide synthetases, many of which are of special interest due to their prominent pharmacological activities (e.g., lovastatin, cyclosporin). To gain basic biochemical knowledge on the methylation process, it is of immense relevance to simplify methods concerning experimental problems caused by a large variety in substrates. Here, we present a photometric method to analyze MT activity by measuring SAM consumption in a coupled enzyme assay. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Diversity of fecal coliforms and their antimicrobial resistance patterns in wastewater treatment model plant.

    Science.gov (United States)

    Luczkiewicz, A; Fudala-Ksiazek, S; Jankowska, K; Quant, B; Olańczuk-Neyman, K

    2010-01-01

    The occurrence of resistance patterns among wastewater fecal coliforms was determined in the study. Susceptibility of the isolates was tested against 19 antimicrobial agents: aminoglycosides, aztreonam, carbapenems, cephalosporines, beta-lactam/beta-lactamase inhibitors, penicillines, tetracycline, trimethoprim/sulfamethoxazole, and fluoroquinolones. Additionally the removal of resistant isolates was evaluated in the laboratory-scale wastewater treatment model plant (M-WWTP), continuously supplied with the wastewater obtained from the full-scale WWTP. Number of fecal coliforms in raw (after mechanical treatment) and treated wastewater, as well as in aerobic chamber effluent was determined using selective medium. The selected strains were identified and examined for antibiotic resistance using Phoenix Automated Microbiology System (BD Biosciences, USA). The strains were identified as Escherichia coli (n=222), Klebsiella pneumoniae ssp. ozaenae (n=9), and Pantoea agglomerans (n=1). The isolate of P. agglomerans as well as 48% of E. coli isolates were sensitive to all antimicrobials tested. The most frequent resistance patterns were found for ampicillin: 100% of K. pneumoniae ssp. ozaenae and 41% of E. coli isolates. Among E. coli isolates 12% was regarded as multiple antimicrobial resistant (MAR). In the studied M-WWTP, the applied activated sludge processes reduced considerably the number of fecal coliforms, but increased the ratio of antimicrobial-resistant E. coli isolates to sensitive ones, especially among strains with MAR patterns.

  11. Simultaneous analysis of aminoglycosides with many other classes of drug residues in bovine tissues by ultrahigh-performance liquid chromatography-tandem mass spectrometry using an ion-pairing reagent added to final extracts.

    Science.gov (United States)

    Lehotay, Steven J; Lightfield, Alan R

    2018-01-01

    The way to maximize scope of analysis, sample throughput, and laboratory efficiency in the monitoring of veterinary drug residues in food animals is to determine as many analytes as possible as fast as possible in as few methods as possible. Capital and overhead expenses are also reduced by using fewer instruments in the overall monitoring scheme. Traditionally, the highly polar aminoglycoside antibiotics require different chromatographic conditions from other classes of drugs, but in this work, we demonstrate that an ion-pairing reagent (sodium 1-heptanesulfonate) added to the combined final extracts from two sample preparation methods attains good separation of 174 targeted drugs, including 9 aminoglycosides, in the same 10.5-min ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The full method was validated in bovine kidney, liver, and muscle tissues according to US regulatory protocols, and 137-146 (79-84%) of the drugs gave between 70 and 120% average recoveries with ≤ 25% RSDs in the different types of tissues spiked at 0.5, 1, and 2 times the regulatory levels of interest (10-1000 ng/g depending on the drug). This method increases sample throughput and the possible number of drugs monitored in the US National Residue Program, and requires only one UHPLC-MS/MS method and instrument for analysis rather than two by the previous scheme. Graphical abstract Outline of the streamlined approach to monitor 174 veterinary drugs, including aminoglycosides, in bovine tissues by combining two extracts of the same sample with an ion-pairing reagent for analysis by UHPLC-MS/MS.

  12. Conversion of nicotinic acid to trigonelline is catalyzed by N-methyltransferase belonged to motif B′ methyltransferase family in Coffea arabica

    International Nuclear Information System (INIS)

    Mizuno, Kouichi; Matsuzaki, Masahiro; Kanazawa, Shiho; Tokiwano, Tetsuo; Yoshizawa, Yuko; Kato, Misako

    2014-01-01

    Graphical abstract: Trigonelline synthase catalyzes the conversion of nicotinic acid to trigonelline. We isolated and characterized trigonelline synthase gene(s) from Coffea arabica. - Highlights: • Trigonelline is a major compound in coffee been same as caffeine is. • We isolated and characterized trigonelline synthase gene. • Coffee trigonelline synthases are highly homologous with coffee caffeine synthases. • This study contributes the fully understanding of pyridine alkaloid metabolism. - Abstract: Trigonelline (N-methylnicotinate), a member of the pyridine alkaloids, accumulates in coffee beans along with caffeine. The biosynthetic pathway of trigonelline is not fully elucidated. While it is quite likely that the production of trigonelline from nicotinate is catalyzed by N-methyltransferase, as is caffeine synthase (CS), the enzyme(s) and gene(s) involved in N-methylation have not yet been characterized. It should be noted that, similar to caffeine, trigonelline accumulation is initiated during the development of coffee fruits. Interestingly, the expression profiles for two genes homologous to caffeine synthases were similar to the accumulation profile of trigonelline. We presumed that these two CS-homologous genes encoded trigonelline synthases. These genes were then expressed in Escherichiacoli, and the resulting recombinant enzymes that were obtained were characterized. Consequently, using the N-methyltransferase assay with S-adenosyl[methyl- 14 C]methionine, it was confirmed that these recombinant enzymes catalyzed the conversion of nicotinate to trigonelline, coffee trigonelline synthases (termed CTgS1 and CTgS2) were highly identical (over 95% identity) to each other. The sequence homology between the CTgSs and coffee CCS1 was 82%. The pH-dependent activity curve of CTgS1 and CTgS2 revealed optimum activity at pH 7.5. Nicotinate was the specific methyl acceptor for CTgSs, and no activity was detected with any other nicotinate derivatives, or with

  13. Conversion of nicotinic acid to trigonelline is catalyzed by N-methyltransferase belonged to motif B′ methyltransferase family in Coffea arabica

    Energy Technology Data Exchange (ETDEWEB)

    Mizuno, Kouichi, E-mail: koumno@akita-pu.ac.jp [Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195 (Japan); Matsuzaki, Masahiro [Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195 (Japan); Kanazawa, Shiho [Graduate School of Humanities and Sciences, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo 112-8610 (Japan); Tokiwano, Tetsuo; Yoshizawa, Yuko [Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195 (Japan); Kato, Misako [Graduate School of Humanities and Sciences, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo 112-8610 (Japan)

    2014-10-03

    Graphical abstract: Trigonelline synthase catalyzes the conversion of nicotinic acid to trigonelline. We isolated and characterized trigonelline synthase gene(s) from Coffea arabica. - Highlights: • Trigonelline is a major compound in coffee been same as caffeine is. • We isolated and characterized trigonelline synthase gene. • Coffee trigonelline synthases are highly homologous with coffee caffeine synthases. • This study contributes the fully understanding of pyridine alkaloid metabolism. - Abstract: Trigonelline (N-methylnicotinate), a member of the pyridine alkaloids, accumulates in coffee beans along with caffeine. The biosynthetic pathway of trigonelline is not fully elucidated. While it is quite likely that the production of trigonelline from nicotinate is catalyzed by N-methyltransferase, as is caffeine synthase (CS), the enzyme(s) and gene(s) involved in N-methylation have not yet been characterized. It should be noted that, similar to caffeine, trigonelline accumulation is initiated during the development of coffee fruits. Interestingly, the expression profiles for two genes homologous to caffeine synthases were similar to the accumulation profile of trigonelline. We presumed that these two CS-homologous genes encoded trigonelline synthases. These genes were then expressed in Escherichiacoli, and the resulting recombinant enzymes that were obtained were characterized. Consequently, using the N-methyltransferase assay with S-adenosyl[methyl-{sup 14}C]methionine, it was confirmed that these recombinant enzymes catalyzed the conversion of nicotinate to trigonelline, coffee trigonelline synthases (termed CTgS1 and CTgS2) were highly identical (over 95% identity) to each other. The sequence homology between the CTgSs and coffee CCS1 was 82%. The pH-dependent activity curve of CTgS1 and CTgS2 revealed optimum activity at pH 7.5. Nicotinate was the specific methyl acceptor for CTgSs, and no activity was detected with any other nicotinate derivatives, or

  14. Protein Arginine Methyltransferase 5 Inhibition Upregulates Foxp3+ Regulatory T Cells Frequency and Function during the Ulcerative Colitis

    Directory of Open Access Journals (Sweden)

    Yingxia Zheng

    2017-05-01

    Full Text Available Ulcerative colitis (UC pathogenesis is related to imbalance of immune responses, and the equilibrium between inflammatory T cells and Foxp3+ regulatory T cells (Tregs plays an important role in the intestinal homeostasis. Protein arginine methyltransferases (PRMTs regulate chromatin remodeling and gene expression. Here, we investigated whether inhibition of PRMTs affects colitis pathogenesis in mice and inflammatory bowel disease patients and further explored the underlying mechanisms. In this study, we found that protein arginine N-methyltransferase inhibitor 1 (AMI-1 treatments increased Tregs frequency, function, and reduced colitis incidence. Adoptive transfer of AMI-1-treated Tregs could reduce the colitis incidence. Colitis was associated with increased local PRMT5 expression, which was inhibited by AMI-1 treatment. Additionally, PRMT5 knockdown T cells produced a better response to TGFβ and promoted Tregs differentiation through decreased DNA methyltransferase 1 (DNMT1 expression. PRMT5 also enhanced H3K27me3 and DNMT1 binding to Foxp3 promoter, which restricted Tregs differentiation. Furthermore, PRMT5 knockdown led to decreased Foxp3 promoter methylation during Tregs induction. PRMT5 expression had a negative relationship with Tregs in UC patients, knockdown of PRMT5 expression increased Tregs frequency and decreased TNFα, IL-6, and IL-13 levels. Our study outlines a novel regulation of PRMT5 on Tregs development and function. Strategies to decrease PRMT5 expression might have therapeutic potential to control UC.

  15. Comparative Study of Erythrocyte Sedimentation Rate after Aminoglycoside and Aminocyclitol Treatment in Goats (Capra hircus

    Directory of Open Access Journals (Sweden)

    Toncho DINEV

    2016-07-01

    Full Text Available The aim of the present study was to follow up the erythrocyte sedimentation rate (ESR in healthy female goats during and after 5-day parenteral treatment with amikacin (10 mg/kg, tobramycin (5 mg/kg, apramycin (20 mg/kg, gentamicin (4 mg/kg, kanamycin (10 mg/kg and spectinomycin (20 mg/kg. Gentamicin and tobramycin caused an initial increase followed by a significant decrease of ESR on the 5th day for gentamicin and the 10th day for tobramycin, respectively, followed by recovery after the treatment. Reversely, amikacin and especially spectinomycin produced an increase of ESR without recovery several days post treatment. Kanamycin caused decrease of ESR on the 5th day without recovery in the subsequent days. Only apramycin did not give rise to increasing of ESR. In conclusion the aminoglycosides, especially tobramycin and gentamicin, caused more severe alterations of ESR than the aminocyclitols.

  16. Molecular cloning, characterization and expression of the caffeic acid O-methyltransferase (COMT) ortholog from kenaf (Hibiscus cannabinus)

    Science.gov (United States)

    We cloned the full-length of the gene putatively encoding caffeic acid O-methyltransferase (COMT) from kenaf (Hibiscus cannabinus L.) using degenerate primers and the RACE (rapid amplification of cDNA ends) method. Kenaf is an herbaceous and rapidly growing dicotyledonous plant with great potential ...

  17. Virulence and genomic feature of multidrug resistant Campylobacter jejuni isolated from broiler chicken

    Directory of Open Access Journals (Sweden)

    Haihong Hao

    2016-10-01

    Full Text Available The aim of this study was to reveal the molecular mechanism involved in multidrug resistance and virulence of Campylobacter jejuni isolated from broiler chickens. The virulence of six multidrug resistant C. jejuni was determined by in vitro and in vivo methods. The de novo whole genome sequencing technology and molecular biology methods were used to analyze the genomic features associated with the multidrug resistance and virulence of a selected isolate (C. jejuni 1655. The comparative genomic analyses revealed a large number of single nucleotide polymorphisms, deletions, rearrangements, and inversions in C. jejuni 1655 compared to reference C. jejuni genomes. The co-emergence of Thr-86-Ile mutation in gyrA gene, A2075G mutation in 23S rRNA gene, tetO, aphA and aadE genes and pTet plasmid in C. jejuni 1655 contributed its multidrug resistance to fluoroquinolones, macrolides, tetracycline and aminoglycosides. The combination of multiple virulence genes may work together to confer the relative higher virulence in C. jejuni 1655. The co-existence of mobile gene elements (e.g. pTet and CRISPR-Cas system in C. jejuni 1655 may play an important role in the gene transfer and immune defense. The present study provides basic information of phenotypic and genomic features of C. jejuni 1655, a strain recently isolated from a chicken displaying multidrug resistance and relatively high level of virulence.

  18. Comparative Genomics of Two ST 195 Carbapenem-Resistant Acinetobacter baumannii with Different Susceptibility to Polymyxin Revealed Underlying Resistance Mechanism

    Science.gov (United States)

    Lean, Soo-Sum; Yeo, Chew Chieng; Suhaili, Zarizal; Thong, Kwai-Lin

    2016-01-01

    Acinetobacter baumannii is a Gram-negative nosocomial pathogen of importance due to its uncanny ability to acquire resistance to most antimicrobials. These include carbapenems, which are the drugs of choice for treating A. baumannii infections, and polymyxins, the drugs of last resort. Whole genome sequencing was performed on two clinical carbapenem-resistant A. baumannii AC29 and AC30 strains which had an indistinguishable ApaI pulsotype but different susceptibilities to polymyxin. Both genomes consisted of an approximately 3.8 Mbp circular chromosome each and several plasmids. AC29 (susceptible to polymyxin) and AC30 (resistant to polymyxin) belonged to the ST195 lineage and are phylogenetically clustered under the International Clone II (IC-II) group. An AbaR4-type resistance island (RI) interrupted the comM gene in the chromosomes of both strains and contained the blaOXA−23 carbapenemase gene and determinants for tetracycline and streptomycin resistance. AC29 harbored another copy of blaOXA−23 in a large (~74 kb) conjugative plasmid, pAC29b, but this gene was absent in a similar plasmid (pAC30c) found in AC30. A 7 kb Tn1548::armA RI which encodes determinants for aminoglycoside and macrolide resistance, is chromosomally-located in AC29 but found in a 16 kb plasmid in AC30, pAC30b. Analysis of known determinants for polymyxin resistance in AC30 showed mutations in the pmrA gene encoding the response regulator of the two-component pmrAB signal transduction system as well as in the lpxD, lpxC, and lpsB genes that encode enzymes involved in the biosynthesis of lipopolysaccharide (LPS). Experimental evidence indicated that impairment of LPS along with overexpression of pmrAB may have contributed to the development of polymyxin resistance in AC30. Cloning of a novel variant of the blaAmpC gene from AC29 and AC30, and its subsequent expression in E. coli also indicated its likely function as an extended-spectrum cephalosporinase. PMID:26779129

  19. Paradoxical elevated thiopurine S-methyltransferase activity after pancytopenia during azathioprine therapy: potential influence of red blood cell age

    NARCIS (Netherlands)

    de Boer, Nanne K. H.; van Bodegraven, Adriaan A.; de Graaf, Peer; van der Hulst, Rene W. M.; Zoetekouw, Lida; van Kuilenburg, André B. P.

    2008-01-01

    There is an increased risk of developing bone marrow depression and infections during azathioprine therapy for inflammatory bowel disease. Patients with low or absent thiopurine S-methyltransferase (TPMT) activity have an increased risk of developing myelotoxicity. We describe a patient who

  20. Impaired Homocysteine Transmethylation and Protein-Methyltransferase Activity Reduce Expression of Selenoprotein P: Implications for Obesity and Metabolic Syndrome

    Science.gov (United States)

    Obesity causes Metabolic Syndrome and Type-II Diabetes, disrupting hepatic function, methionine (Met)/homocysteine (Hcy) transmethylation and methyltransferase (PRMT) activities. Selenoprotein P (SEPP1), exported from the liver, is the predominate form of plasma selenium (Se) and the physiological S...

  1. Polymorphisms in O-methyltransferase genes are associated with stover cell wall digestibility in European maize (Zea mays L.)

    DEFF Research Database (Denmark)

    Brenner, Everton A; Zein, Imad; Chen, Yongsheng

    2010-01-01

    Background OMT (O-methyltransferase) genes are involved in lignin biosynthesis, which relates to stover cell wall digestibility. Reduced lignin content is an important determinant of both forage quality and ethanol conversion efficiency of maize stover. Results Variation in genomic sequences codi...

  2. Alternative Evolutionary Paths to Bacterial Antibiotic Resistance Cause Distinct Collateral Effects.

    Science.gov (United States)

    Barbosa, Camilo; Trebosc, Vincent; Kemmer, Christian; Rosenstiel, Philip; Beardmore, Robert; Schulenburg, Hinrich; Jansen, Gunther

    2017-09-01

    When bacteria evolve resistance against a particular antibiotic, they may simultaneously gain increased sensitivity against a second one. Such collateral sensitivity may be exploited to develop novel, sustainable antibiotic treatment strategies aimed at containing the current, dramatic spread of drug resistance. To date, the presence and molecular basis of collateral sensitivity has only been studied in few bacterial species and is unknown for opportunistic human pathogens such as Pseudomonas aeruginosa. In the present study, we assessed patterns of collateral effects by experimentally evolving 160 independent populations of P. aeruginosa to high levels of resistance against eight commonly used antibiotics. The bacteria evolved resistance rapidly and expressed both collateral sensitivity and cross-resistance. The pattern of such collateral effects differed to those previously reported for other bacterial species, suggesting interspecific differences in the underlying evolutionary trade-offs. Intriguingly, we also identified contrasting patterns of collateral sensitivity and cross-resistance among the replicate populations adapted to the same drug. Whole-genome sequencing of 81 independently evolved populations revealed distinct evolutionary paths of resistance to the selective drug, which determined whether bacteria became cross-resistant or collaterally sensitive towards others. Based on genomic and functional genetic analysis, we demonstrate that collateral sensitivity can result from resistance mutations in regulatory genes such as nalC or mexZ, which mediate aminoglycoside sensitivity in β-lactam-adapted populations, or the two-component regulatory system gene pmrB, which enhances penicillin sensitivity in gentamicin-resistant populations. Our findings highlight substantial variation in the evolved collateral effects among replicates, which in turn determine their potential in antibiotic therapy. © The Author 2017. Published by Oxford University Press on

  3. Jasmonic acid carboxyl methyltransferase regulates development and herbivory-induced defense response in rice.

    Science.gov (United States)

    Qi, Jinfeng; Li, Jiancai; Han, Xiu; Li, Ran; Wu, Jianqiang; Yu, Haixin; Hu, Lingfei; Xiao, Yutao; Lu, Jing; Lou, Yonggen

    2016-06-01

    Jasmonic acid (JA) and related metabolites play a key role in plant defense and growth. JA carboxyl methyltransferase (JMT) may be involved in plant defense and development by methylating JA to methyl jasmonate (MeJA) and thus influencing the concentrations of JA and related metabolites. However, no JMT gene has been well characterized in monocotyledon defense and development at the molecular level. After we cloned a rice JMT gene, OsJMT1, whose encoding protein was localized in the cytosol, we found that the recombinant OsJMT1 protein catalyzed JA to MeJA. OsJMT1 is up-regulated in response to infestation with the brown planthopper (BPH; Nilaparvata lugens). Plants in which OsJMT1 had been overexpressed (oe-JMT plants) showed reduced height and yield. These oe-JMT plants also exhibited increased MeJA levels but reduced levels of herbivore-induced JA and jasmonoyl-isoleucine (JA-Ile). The oe-JMT plants were more attractive to BPH female adults but showed increased resistance to BPH nymphs, probably owing to the different responses of BPH female adults and nymphs to the changes in levels of H2 O2 and MeJA in oe-JMT plants. These results indicate that OsJMT1, by altering levels of JA and related metabolites, plays a role in regulating plant development and herbivore-induced defense responses in rice. © 2015 Institute of Botany, Chinese Academy of Sciences.

  4. Isolation of DNA methyltransferase from plants

    International Nuclear Information System (INIS)

    Ehrlich, K.; Malbroue, C.

    1987-01-01

    DNA methyltransferases (DMT) were isolated from nuclei of cauliflower, soybean, and pea by extraction with 0.35 M NaCl. Assays were performed on hemimethylated Micrococcus luteus DNA or on M. luteus DNA to test for maintenance or de novo methylase activity, respectively. Fully methylated DNA was used as a substrate to determine background levels of methylation. Based on these tests, yields of maintenance DMT activity in the crude extract from pea hypocotyl, soybean hypocotyl, and cauliflower inflorescence were 2.8, 0.9, and 1.6 units per g wet tissue (one unit equals 1 pmol of methyl from [ 3 H]AdoMet incorporated into acid precipitable material per h at 30 0 ). Two peaks of DMT activity were detected in the soybean nuclear extract following phosphocellulose chromatography. One eluted at 0.4 M and the other at 0.8 M KCl. With both fractions maintenance activity was approximately 2 times that of the de novo activity. Using gel filtration the DMT eluted at 220,000 Daltons. The optimal pH for activity was between 6.5 and 7.0, and the optimal temperature was 30 0

  5. Molecular characterization of multidrug-resistant Klebsiella pneumoniae isolates

    Directory of Open Access Journals (Sweden)

    Xiang-hua Hou

    2015-09-01

    Full Text Available Klebsiella pneumoniae is an important cause of healthcare-associated infections worldwide. Selective pressure, the extensive use of antibiotics, and the conjugational transmission of antibiotic resistance genes across bacterial species and genera facilitate the emergence of multidrug-resistant (MDR K. pneumoniae. Here, we examined the occurrence, phenotypes and genetic features of MDR K. pneumoniae isolated from patients in intensive care units (ICUs at the First Affiliated Hospital of Xiamen University in Xiamen, China, from January to December 2011. Thirty-eight MDR K. pneumoniae strains were collected. These MDR K. pneumoniae isolates possessed at least seven antibiotic resistance determinants, which contribute to the high-level resistance of these bacteria to aminoglycosides, macrolides, quinolones and β-lactams. Among these isolates, 24 strains were extended-spectrum β-lactamase (ESBL producers, 2 strains were AmpC producers, and 12 strains were both ESBL and AmpC producers. The 38 MDR isolates also contained class I (28/38 and class II integrons (10/38. All 28 class I-positive isolates contained aacC1, aacC4, orfX, orfX’ and aadA1 genes. β-lactam resistance was conferred through blaSHV (22/38, blaTEM (10/38, and blaCTX-M (7/38. The highly conserved blaKPC-2 (37/38 and blaOXA-23(1/38 alleles were responsible for carbapenem resistance, and a gyrAsite mutation (27/38 and the plasmid-mediated qnrB gene (13/38 were responsible for quinolone resistance. Repetitive-sequence-based PCR (REP-PCR fingerprinting of these MDR strains revealed the presence of five groups and sixteen patterns. The MDR strains from unrelated groups showed different drug resistance patterns; however, some homologous strains also showed different drug resistance profiles. Therefore, REP-PCR-based analyses can provide information to evaluate the epidemic status of nosocomial infection caused by MDR K. pneumoniae; however, this test lacks the power to discriminate some

  6. Role of integrons, plasmids and SXT elements in multidrug resistance of Vibrio cholerae and Providencia vermicola obtained from a clinical isolate of diarrhea

    Directory of Open Access Journals (Sweden)

    Neha eRajpara

    2015-02-01

    Full Text Available The isolates of Vibrio cholerae and Providencia vermicola obtained from a diarrhoeal patient were investigated for genetic elements governing their drug resistance phenotypes. Out of fourteen antibiotics tested, V. cholerae Vc IDH02365 isolate showed resistance to nine antibiotics, while P. vermicola Pv NBA2365 was found to be resistant to all the antibiotics except polymyxin B. Though SXT integrase was depicted in both the bacteria, class 1 integron was found to be associated only with Pv NBA2365. Integrons in Pv NBA2365 conferred resistance to β-lactams, aminoglycosides and trimethoprim. Pv NBA2365 carried two transformable plasmids imparting distinct antibiotic resistance traits to their Escherichia coli transformants. In rabbit ileal loop assays, Pv NBA2365 did not show any fluid accumulation in contrast with Vc IDH02365 that showed high fluid accumulation. To the best of our knowledge, this is the first report of a highly drug resistant P.vermicola and additionally co-existence of multidrug resistant V. cholerae and P. vermicola. Both the microbes appeared to possess a wide array of mobile genetic elements for a large spectrum of antimicrobial agents, some of which are being used in the treatment of acute diarrhoea.

  7. Activation of the SOS response increases the frequency of small colony variants

    DEFF Research Database (Denmark)

    Vestergaard, Martin; Paulander, Wilhelm Erik Axel; Ingmer, Hanne

    2015-01-01

    BACKGROUND: In Staphylococcus aureus sub-populations of slow-growing cells forming small colony variants (SCVs) are associated with persistent and recurrent infections that are difficult to eradicate with antibiotic therapies. In SCVs that are resistant towards aminoglycosides, mutations have been...... with different mechanism of action influence the formation of SCVs that are resistant to otherwise lethal concentrations of the aminoglycoside, gentamicin. We found that exposure of S. aureus to fluoroquinolones and mitomycin C increased the frequency of gentamicin resistant SCVs, while other antibiotic classes...... failed to do so. The higher proportion of SCVs in cultures exposed to fluoroquinolones and mitomycin C compared to un-exposed cultures correlate with an increased mutation rate monitored by rifampicin resistance and followed induction of the SOS DNA damage response. CONCLUSION: Our observations suggest...

  8. Identification and Characterization of a Novel Human Methyltransferase Modulating Hsp70 Protein Function through Lysine Methylation*

    Science.gov (United States)

    Jakobsson, Magnus E.; Moen, Anders; Bousset, Luc; Egge-Jacobsen, Wolfgang; Kernstock, Stefan; Melki, Ronald; Falnes, Pål Ø.

    2013-01-01

    Hsp70 proteins constitute an evolutionarily conserved protein family of ATP-dependent molecular chaperones involved in a wide range of biological processes. Mammalian Hsp70 proteins are subject to various post-translational modifications, including methylation, but for most of these, a functional role has not been attributed. In this study, we identified the methyltransferase METTL21A as the enzyme responsible for trimethylation of a conserved lysine residue found in several human Hsp70 (HSPA) proteins. This enzyme, denoted by us as HSPA lysine (K) methyltransferase (HSPA-KMT), was found to catalyze trimethylation of various Hsp70 family members both in vitro and in vivo, and the reaction was stimulated by ATP. Furthermore, we show that HSPA-KMT exclusively methylates 70-kDa proteins in mammalian protein extracts, demonstrating that it is a highly specific enzyme. Finally, we show that trimethylation of HSPA8 (Hsc70) has functional consequences, as it alters the affinity of the chaperone for both the monomeric and fibrillar forms of the Parkinson disease-associated protein α-synuclein. PMID:23921388

  9. The putative protein methyltransferase LAE1 controls cellulase gene expression in Trichoderma reesei

    Science.gov (United States)

    Seiboth, Bernhard; Karimi, Razieh Aghcheh; Phatale, Pallavi A; Linke, Rita; Hartl, Lukas; Sauer, Dominik G; Smith, Kristina M; Baker, Scott E; Freitag, Michael; Kubicek, Christian P

    2012-01-01

    Summary Trichoderma reesei is an industrial producer of enzymes that degrade lignocellulosic polysaccharides to soluble monomers, which can be fermented to biofuels. Here we show that the expression of genes for lignocellulose degradation are controlled by the orthologous T. reesei protein methyltransferase LAE1. In a lae1 deletion mutant we observed a complete loss of expression of all seven cellulases, auxiliary factors for cellulose degradation, β-glucosidases and xylanases were no longer expressed. Conversely, enhanced expression of lae1 resulted in significantly increased cellulase gene transcription. Lae1-modulated cellulase gene expression was dependent on the function of the general cellulase regulator XYR1, but also xyr1 expression was LAE1-dependent. LAE1 was also essential for conidiation of T. reesei. Chromatin immunoprecipitation followed by high-throughput sequencing (‘ChIP-seq’) showed that lae1 expression was not obviously correlated with H3K4 di- or trimethylation (indicative of active transcription) or H3K9 trimethylation (typical for heterochromatin regions) in CAZyme coding regions, suggesting that LAE1 does not affect CAZyme gene expression by directly modulating H3K4 or H3K9 methylation. Our data demonstrate that the putative protein methyltransferase LAE1 is essential for cellulase gene expression in T. reesei through mechanisms that remain to be identified. PMID:22554051

  10. Characterization of antimicrobial resistance in Salmonella enterica strains isolated from Brazilian poultry production.

    Science.gov (United States)

    Mattiello, Samara P; Drescher, Guilherme; Barth, Valdir C; Ferreira, Carlos A S; Oliveira, Sílvia D

    2015-11-01

    Antimicrobial resistance profiles and presence of resistance determinants and integrons were evaluated in Salmonella enterica strains from Brazilian poultry. The analysis of 203 isolates showed that those from the poultry environment (88 isolates) were significantly more resistant to antimicrobials than isolates from other sources, particularly those isolated from poultry by-product meal (106 isolates). Thirty-seven isolates were resistant to at least three antimicrobial classes. Class 1 integrons were detected in 26 isolates, and the analysis of the variable region between the 5' conserved segment (CS) and 3' CS of each class 1 integron-positive isolate showed that 13 contained a typical 3' CS and 14 contained an atypical 3' CS. One Salmonella Senftenberg isolate harbored two class 1 integrons, showing both typical and atypical 3' CSs. The highest percentage of resistance was found to sulfonamides, and sul genes were detected in the majority of the resistant isolates. Aminoglycoside resistance was detected in 50 isolates, and aadA and aadB were present in 28 and 32 isolates, respectively. In addition, strA and strB were detected in 78.1 and 65.6% isolates resistant to streptomycin, respectively. Twenty-one isolates presented reduced susceptibility to β-lactams and harbored bla(TEM), bla(CMY), and/or bla(CTX-M). Forty isolates showed reduced susceptibility to tetracycline, and most presented tet genes. These results highlight the importance of the environment as a reservoir of resistant Salmonella, which may enable the persistence of resistance determinants in the poultry production chain, contributing, therefore, to the debate regarding the impacts that antimicrobial use in animal production may exert in human health.

  11. Inhibition of Y-box binding protein-1 slows the growth of glioblastoma multiforme and sensitizes to temozolomide independent O6-methylguanine-DNA methyltransferase.

    Science.gov (United States)

    Gao, Yuanyuan; Fotovati, Abbas; Lee, Cathy; Wang, Michelle; Cote, Gilbert; Guns, Emma; Toyota, Brian; Faury, Damien; Jabado, Nada; Dunn, Sandra E

    2009-12-01

    Glioblastoma multiforme (GBM) is an aggressive type of brain tumor where 5 years. In adults, GBM is the most common type of brain tumor. It is rarer in children, where it constitutes approximately 15% of all brain tumors diagnosed. These tumors are often invasive, making surgical resection difficult. Further, they can be refractory to current therapies such as temozolomide. The current dogma is that temozolomide resistance rests on the expression of O6-methylguanine-DNA methyltransferase (MGMT) because it cleaves methylated DNA adducts formed by the drug. Our laboratory recently reported that another drug resistance gene known as the Y-box binding protein-1 (YB-1) is highly expressed in primary GBM but not in normal brain tissues based on the evaluation of primary tumors. We therefore questioned whether GBM depend on YB-1 for growth and/or response to temozolomide. Herein, we report that YB-1 inhibition reduced tumor cell invasion and growth in monolayer as well as in soft agar. Moreover, blocking this protein ultimately delayed tumor onset in mice. Importantly, inhibiting YB-1 enhanced temozolomide sensitivity in a manner that was independent of MGMT in models of adult and pediatric GBM. In conclusion, inhibiting YB-1 may be a novel way to improve the treatment of GBM.

  12. Resistance to Linezolid Caused by Modifications at Its Binding Site on the Ribosome

    Science.gov (United States)

    Long, Katherine S.

    2012-01-01

    Linezolid is an oxazolidinone antibiotic in clinical use for the treatment of serious infections of resistant Gram-positive bacteria. It inhibits protein synthesis by binding to the peptidyl transferase center on the ribosome. Almost all known resistance mechanisms involve small alterations to the linezolid binding site, so this review will therefore focus on the various changes that can adversely affect drug binding and confer resistance. High-resolution structures of linezolid bound to the 50S ribosomal subunit show that it binds in a deep cleft that is surrounded by 23S rRNA nucleotides. Mutation of 23S rRNA has for some time been established as a linezolid resistance mechanism. Although ribosomal proteins L3 and L4 are located further away from the bound drug, mutations in specific regions of these proteins are increasingly being associated with linezolid resistance. However, very little evidence has been presented to confirm this. Furthermore, recent findings on the Cfr methyltransferase underscore the modification of 23S rRNA as a highly effective and transferable form of linezolid resistance. On a positive note, detailed knowledge of the linezolid binding site has facilitated the design of a new generation of oxazolidinones that show improved properties against the known resistance mechanisms. PMID:22143525

  13. Exosomal DNMT1 mediates cisplatin resistance in ovarian cancer.

    Science.gov (United States)

    Cao, Ya-Lei; Zhuang, Ting; Xing, Bao-Heng; Li, Na; Li, Qin

    2017-08-01

    Ovarian cancer is the most common malignancy in women. Owing to late syndromic presentation and lack of efficient early detection, most cases are diagnosed at advanced stages. Surgery and platinum-based chemotherapy are still the standard care currently. However, resistance invoked often compromises the clinical value of the latter. Expression of DNA methyltransferase 1 (DNMT1) was analysed by gene array. Protein was determined by immunoblotting. Exosome was isolated with commercial kit. Cell proliferation was measured by CCK8 method. Annexin V-PI double staining was performed for apoptosis evaluation. Xenograft model was established and administrated with exosome. Tumour growth and overall survival were monitored. We demonstrated the upregulation of DNMT1 in both tumour and derived cell line. DNMT1 transcripts were highly enriched in exosomes from conditioned medium of ovarian cells. Co-incubation with exosomes stimulated endogenous expression and rendered host cell the resistance to cytotoxicity of cisplatin. In vivo administration of DNMT1-containing exosomes exacerbated xenograft progression and reduced overall survival significantly. Moreover, treatment with exosome inhibitor GW4869 almost completely restored sensitivity in resistant cells. Our data elucidated an unappreciated mechanism of exosomal DNMT1 in cisplatin resistance in ovarian cancer, also indicating the potential of the combination of exosome inhibitor with cisplatin in resistant patients. Copyright © 2017 John Wiley & Sons, Ltd.

  14. Changing prevalence and resistance patterns in children with drug-resistant tuberculosis in Mumbai.

    Science.gov (United States)

    Shah, Ira; Shah, Forum

    2017-05-01

    The prevalence of drug-resistant (DR) tuberculosis (TB) in children is increasing. Although, in India, multi-drug-resistant (MDR) TB rates have been relatively stable, the number of children with pre-extensively drug-resistant and extensively drug-resistant (XDR) TB is increasing. To determine whether the prevalence of DR TB in children in Mumbai is changing and to study the evolving patterns of resistance. A retrospective study was undertaken in 1311 paediatric patients referred between April 2007 and March 2013 to the Paediatric TB clinic at B. J. Wadia Hospital for Children, Mumbai. Children were defined as having DR TB on the basis of drug susceptibility testing (DST) of Mycobacterium tuberculosis grown on culture of body fluids (in the case of extra pulmonary TB) or from gastric lavage/bronchi-alveolar lavage/sputum in patients with pulmonary TB or from DST of the contacts. The prevalence of DR TB was calculated and the type of DR was evaluated yearly and in the pre-2010 and post-2010 eras. The overall prevalence of DR TB was 86 (6.6%) with an increase from 23 (5.6%) patients pre-2010 to 63 (7%) post-2010 (P = 0.40). Nine (10.4%) patients were diagnosed on the basis of contact with a parent with DR TB. Overall fluoroquinolone resistance increased from 9 (39.1%) pre-2010 to 59 (93.7%) post-2010 (P = 0.0001): moxifloxacin resistance increased from 2 (8.7%) to 29 (46%) (P = 0.0018) and ofloxacin resistance increased from 7 (30.4%) to 30 (47.6%) (P = 0.14). Ethionamide resistance also increased from 6 (26.1%) to 31 (49.2%) (P = 0.04), aminoglycoside resistance was one (4.3%) pre-2010 and 12 (19%) post-2010 (P = 0.17) and resistance remained virtually the same for both amikacin [0 pre-2010 and 6 (9.5%) after 2010] and kanamycin [one (4.3%) pre- and 6 (9.5%) post-2010]. Of the first-line drugs, resistance remained the same for isoniazid [23 (100%) to 61 (96.8%)], rifampicin [22 (95.7%) to 51 (80.9%),P = 0.17], pyrazinamide [15 (65.2%) to

  15. Antimicrobial Chemicals Are Associated with Elevated Antibiotic Resistance Genes in the Indoor Dust Microbiome.

    Science.gov (United States)

    Hartmann, Erica M; Hickey, Roxana; Hsu, Tiffany; Betancourt Román, Clarisse M; Chen, Jing; Schwager, Randall; Kline, Jeff; Brown, G Z; Halden, Rolf U; Huttenhower, Curtis; Green, Jessica L

    2016-09-20

    Antibiotic resistance is increasingly widespread, largely due to human influence. Here, we explore the relationship between antibiotic resistance genes and the antimicrobial chemicals triclosan, triclocarban, and methyl-, ethyl-, propyl-, and butylparaben in the dust microbiome. Dust samples from a mixed-use athletic and educational facility were subjected to microbial and chemical analyses using a combination of 16S rRNA amplicon sequencing, shotgun metagenome sequencing, and liquid chromatography tandem mass spectrometry. The dust resistome was characterized by identifying antibiotic resistance genes annotated in the Comprehensive Antibiotic Resistance Database (CARD) from the metagenomes of each sample using the Short, Better Representative Extract Data set (ShortBRED). The three most highly abundant antibiotic resistance genes were tet(W), blaSRT-1, and erm(B). The complete dust resistome was then compared against the measured concentrations of antimicrobial chemicals, which for triclosan ranged from 0.5 to 1970 ng/g dust. We observed six significant positive associations between the concentration of an antimicrobial chemical and the relative abundance of an antibiotic resistance gene, including one between the ubiquitous antimicrobial triclosan and erm(X), a 23S rRNA methyltransferase implicated in resistance to several antibiotics. This study is the first to look for an association between antibiotic resistance genes and antimicrobial chemicals in dust.

  16. Antibiotic resistance marker genes as environmental pollutants in GMO-pristine agricultural soils in Austria.

    Science.gov (United States)

    Woegerbauer, Markus; Zeinzinger, Josef; Gottsberger, Richard Alexander; Pascher, Kathrin; Hufnagl, Peter; Indra, Alexander; Fuchs, Reinhard; Hofrichter, Johannes; Kopacka, Ian; Korschineck, Irina; Schleicher, Corina; Schwarz, Michael; Steinwider, Johann; Springer, Burkhard; Allerberger, Franz; Nielsen, Kaare M; Fuchs, Klemens

    2015-11-01

    Antibiotic resistance genes may be considered as environmental pollutants if anthropogenic emission and manipulations increase their prevalence above usually occurring background levels. The prevalence of aph(3')-IIa/nptII and aph(3')-IIIa/nptIII - frequent marker genes in plant biotechnology conferring resistance to certain aminoglycosides - was determined in Austrian soils from 100 maize and potato fields not yet exposed to but eligible for GMO crop cultivation. Total soil DNA extracts were analysed by nptII/nptIII-specific TaqMan real time PCR. Of all fields 6% were positive for nptII (median: 150 copies/g soil; range: 31-856) and 85% for nptIII (1190 copies/g soil; 13-61600). The copy-number deduced prevalence of nptIII carriers was 14-fold higher compared to nptII. Of the cultivable kanamycin-resistant soil bacteria 1.8% (95% confidence interval: 0-3.3%) were positive for nptIII, none for nptII (0-0.8%). The nptII-load of the studied soils was low rendering nptII a typical candidate as environmental pollutant upon anthropogenic release into these ecosystems. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. The Endospore-Forming Pathogen Bacillus cereus Exploits a Small Colony Variant-Based Diversification Strategy in Response to Aminoglycoside Exposure.

    Science.gov (United States)

    Frenzel, Elrike; Kranzler, Markus; Stark, Timo D; Hofmann, Thomas; Ehling-Schulz, Monika

    2015-12-08

    Bacillus cereus is among the microorganisms most often isolated from cases of food spoilage and causes gastrointestinal diseases as well as nongastrointestinal infections elicited by the emetic toxin cereulide, enterotoxins, and a panel of tissue-destructive virulence factors. This opportunistic pathogen is increasingly associated with rapidly fatal clinical infections especially linked to neonates and immunocompromised individuals. Fatality results from either the misdiagnosis of B. cereus as a contaminant of the clinical specimen or from failure of antibiotic therapy. Here we report for the first time that exposure to aminoglycoside antibiotics induces a phenotype switching of emetic B. cereus subpopulations to a slow-growing small colony variant (SCV) state. Along with altered antibiotic resistance, SCVs showed distinct phenotypic and metabolic properties, bearing the risk of antibiotic treatment failure and of clinical misdiagnosis by standard identification tests used in routine diagnostic. The SCV subpopulation is characterized by enhanced production of the toxin cereulide, but it does not secrete tissue-destructive and immune system-affecting enzymes such as sphingomyelinase and phospholipase. SCVs showed significantly prolonged persistence and decreased virulence in the Galleria mellonella model for bacterial infections, indicating diversification concerning their ecological lifestyle. Importantly, diversification into coexisting wild-type and SCV subpopulations also emerged during amikacin pressure during in vivo infection experiments. This study shows for the first time that pathogenic spore-forming B. cereus strains are able to switch to a so far unreported slow-growing lifestyle, which differs substantially in terms of developmental, phenotypic, metabolic, and virulence traits from the wild-type populations. This underpins the necessity of molecular-based differential diagnostics and a well-chosen therapeutic treatment strategy in clinical

  18. Hair cell regeneration in the bullfrog vestibular otolith organs following aminoglycoside toxicity

    Science.gov (United States)

    Baird, Richard A.; Torres, M. A.; Schuff, N. R.

    1994-01-01

    Adult bullfrogs were given single intraotic injections of the aminoglycoside antibiotic gentamicin sulfate and sacrificed at postinjection times ranging from 0.5 to 9 days. The saccular and utricular maculae of normal and injected animals were examined in wholemount and cross-section. Intraotic 200 (mu) M gentamicin concentrations resulted in the uniform destruction of the hair bundles and, at later times, the cell bodies of saccular hair cells. In the utriculus, striolar hair cells were selectively damaged while extrastriolar hair cells were relatively unaffected. Regenerating hair cells, identified in sectioned material by their small cell bodies and short, well-formed hair bundles, were seen in the saccular and utricular maculae as early as 24-48 h postinjection. Immature versions of mature hair cell types in both otolith organs were recognized by the presence of absence of a bulbed kinocilia and the relative lengths of their kinocilia and longest sterocilia. Utricular hair cell types with kinocilia longer than their longest stereocilia were observed at earlier times than hair cell types with shorter kinocilia. In the same sacculus, the hair bundles of gentamicin-treated animals, even at 9 days postinjection, were significantly smaller than those of normal animals. The hair bundles of utricular hair cells, on the other hand, reached full maturity within the same time period.

  19. Crystal structure of Mycobacterium tuberculosis O-6-methylguanine-DNA methyltransferase protein clusters assembled on to damaged DNA

    Czech Academy of Sciences Publication Activity Database

    Miggiano, R.; Perugino, G.; Ciaramella, M.; Serpe, M.; Rejman, Dominik; Páv, Ondřej; Pohl, Radek; Garavaglia, S.; Lahiri, S.; Rizzi, M.; Rossi, F.

    2016-01-01

    Roč. 473, č. 2 (2016), s. 123-133 ISSN 0264-6021 EU Projects: European Commission(XE) 241587 - SYSTEMTB Institutional support: RVO:61388963 Keywords : DNA repair * DNA-binding protein * Mycobacterium tuberculosis * O-6-methylguanine-DNA methyltransferase * co-operativity * crystal structure Subject RIV: CE - Biochemistry Impact factor: 3.797, year: 2016

  20. RpoS plays a central role in the SOS induction by sub-lethal aminoglycoside concentrations in Vibrio cholerae.

    Science.gov (United States)

    Baharoglu, Zeynep; Krin, Evelyne; Mazel, Didier

    2013-01-01

    Bacteria encounter sub-inhibitory concentrations of antibiotics in various niches, where these low doses play a key role for antibiotic resistance selection. However, the physiological effects of these sub-lethal concentrations and their observed connection to the cellular mechanisms generating genetic diversification are still poorly understood. It is known that, unlike for the model bacterium Escherichia coli, sub-minimal inhibitory concentrations (sub-MIC) of aminoglycosides (AGs) induce the SOS response in Vibrio cholerae. SOS is induced upon DNA damage, and since AGs do not directly target DNA, we addressed two issues in this study: how sub-MIC AGs induce SOS in V. cholerae and why they do not do so in E. coli. We found that when bacteria are grown with tobramycin at a concentration 100-fold below the MIC, intracellular reactive oxygen species strongly increase in V. cholerae but not in E. coli. Using flow cytometry and gfp fusions with the SOS regulated promoter of intIA, we followed AG-dependent SOS induction. Testing the different mutation repair pathways, we found that over-expression of the base excision repair (BER) pathway protein MutY relieved this SOS induction in V. cholerae, suggesting a role for oxidized guanine in AG-mediated indirect DNA damage. As a corollary, we established that a BER pathway deficient E. coli strain induces SOS in response to sub-MIC AGs. We finally demonstrate that the RpoS general stress regulator prevents oxidative stress-mediated DNA damage formation in E. coli. We further show that AG-mediated SOS induction is conserved among the distantly related Gram negative pathogens Klebsiella pneumoniae and Photorhabdus luminescens, suggesting that E. coli is more of an exception than a paradigm for the physiological response to antibiotics sub-MIC.

  1. Identification and characterization of the cytosine-5 DNA methyltransferase gene family in Salvia miltiorrhiza

    OpenAIRE

    Jiang Li; Caili Li; Shanfa Lu

    2018-01-01

    Cytosine DNA methylation is highly conserved epigenetic modification involved in a wide range of biological processes in eukaryotes. It was established and maintained by cytosine-5 DNA methyltransferases (C5-MTases) in plants. Through genome-wide identification, eight putative SmC5-MTase genes were identified from the genome of Salvia miltiorrhiza, a well-known traditional Chinese medicine material and an emerging model medicinal plant. Based on conserved domains and phylogenetic analysis, ei...

  2. Antimicrobial resistance patterns, clinical features, and risk factors for septic shock and death of nosocomial E coli bacteremia in adult patients with hematological disease: A monocenter retrospective study in China.

    Science.gov (United States)

    Ma, Jie; Li, Ning; Liu, Yajie; Wang, Chong; Liu, Xiaoyan; Chen, Shengmei; Xie, Xinsheng; Gan, Silin; Wang, Meng; Cao, Weijie; Wang, Fang; Liu, Yanfan; Wan, Dingming; Sun, Ling; Sun, Hui

    2017-05-01

    aminoglycoside, especially amikacin, will be helpful to increase the antimicrobial coverage against ESBL-EC while combining tigecycline with aminoglycoside should be considered for seriously carbapenem-resistant infectious patients.

  3. Carrier frequency of guanidinoacetate methyltransferase deficiency in the general population by functional characterization of missense variants in the GAMT gene

    NARCIS (Netherlands)

    Desroches, C.L.; Patel, J.; Wang, P.X.; Minassian, B.; Marshall, C.R.; Salomons, G.; Mercimek-Mahmutoglu, S.

    2015-01-01

    Guanidinoacetate methyltransferase (GAMT) deficiency is a neurodegenerative disease. Although no symptomatic patients on treatment achieved normal neurodevelopment, three asymptomatic newborns were reported with normal neurodevelopmental outcome on neonatal treatment. GAMT deficiency is therefore a

  4. Thirteen new patients with guanidinoacetate methyltransferase deficiency and functional characterization of nineteen novel missense variants in the GAMT gene

    DEFF Research Database (Denmark)

    Mercimek-Mahmutoglu, Saadet; Ndika, Joseph; Kanhai, Warsha

    2014-01-01

    Guanidinoacetate methyltransferase deficiency (GAMT-D) is an autosomal recessively inherited disorder of creatine biosynthesis. Creatine deficiency on cranial proton magnetic resonance spectroscopy, and elevated guanidinoacetate levels in body fluids are the biomarkers of GAMT-D. In 74 patients 5...

  5. Trends in Drug Resistance of Acinetobacter baumannii over a 10-year Period: Nationwide Data from the China Surveillance of Antimicrobial Resistance Program.

    Science.gov (United States)

    Gao, Lei; Lyu, Yuan; Li, Yun

    2017-03-20

    Acinetobacter baumannii has emerged as an important pathogen causing a variety of infections. Using data from the China Surveillance of Antimicrobial Resistance Program conducted biennially, we investigated the secular changes in the resistance of 2917 isolates of A. baumannii from 2004 to 2014 to differ antimicrobial agents. Pathogen samples were collected from 17 to 20 hospitals located in the eastern, central, and western regions of China. Minimum inhibitory concentrations (MICs) were determined by a 2-fold agar dilution method, and antimicrobial susceptibility was established using the 2014 Clinical Laboratory Standards Institute-approved breakpoints. Isolates not susceptible to all the tested aminoglycosides, fluoroquinolones, β-lactams, β-lactam/β-lactam inhibitors and carbapenems were defined as extensively drug resistant. The rates of nonsusceptibility to common antimicrobial agents remained high (>65%) over the years with some fluctuations to certain agents. The prevalence of imipenem-resistant A. baumannii (IRAB) increased from 13.3% in 2004 to 70.5% in 2014 and that of extensively drug-resistant A. baumannii (XDRAB) increased from 11.1% in 2004 to 60.4% in 2014. The activity of tigecycline was stable with MIC90 ≤4 mg/L against A. baumannii from 2009 to 2014. Susceptibility to colistin remained high (97.0%) from 2009 to 2014. The prevalence of XDRAB increased in all the three surveillance regions over the years and was significantly higher in Intensive Care Unit (ICU) wards than non-ICU wards. This longitudinal multicenter surveillance program revealed the nationwide emergence of A. baumannii in China and showed a significant increase in prevalence from 2004 to 2014. High levels of bacterial resistance were detected among samples collected from clinical settings in China, with IRAB and XDRAB being especially prevalent. This study will help to guide empirical therapy and identify at-risk groups requiring more intense interventional infection control

  6. Detection of selected antibiotic resistance genes using multiplex PCR assay in mastitis pathogens in the Czech Republic

    Directory of Open Access Journals (Sweden)

    Vladimir Pyatov

    2017-01-01

    Full Text Available The aim of this research was to develop multiplex polymerase chain reaction assays for the detection of aminoglycoside (strA, strB, sulphonamide (sulI, sulII, tetracycline (tetA, tetB, tetK, tetM, tetO, macrolide and lincosamide (msrA, ermA, ermB, ermC, mefA/E genes of resistance in mastitis pathogens (Escherichia coli, Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae and Streptococcus dysgalactiae. Applying the established assays, we investigated the distribution of antibiotic resistance genes in the above mentioned species isolated from milk samples in the Czech Republic. Each assay consisted of seven pairs of primers. Six of them amplified fragments of antibiotic resistance genes and one pair a fragment of a species specific gene. Polymerase chain reaction conditions were optimized to amplify seven gene fragments simultaneously in one reaction. In total, 249 isolates were used, among which 111 were positive for E. coli, 52 for S. aureus and 86 for Streptococcus spp. The majority (60.2% of bacteria carried at least one antibiotic resistance gene and 44.6% were multidrug-resistant. The designed multiplex polymerase chain reaction assays may be applied as diagnostic method to replace or complement standard techniques of antibiotic susceptibility testing in the mentioned pathogens.

  7. Comparative Genotypes, Staphylococcal Cassette Chromosome mec (SCCmec) Genes and Antimicrobial Resistance amongst Staphylococcus epidermidis and Staphylococcus haemolyticus Isolates from Infections in Humans and Companion Animals.

    Science.gov (United States)

    McManus, Brenda A; Coleman, David C; Deasy, Emily C; Brennan, Gráinne I; O' Connell, Brian; Monecke, Stefan; Ehricht, Ralf; Leggett, Bernadette; Leonard, Nola; Shore, Anna C

    2015-01-01

    This study compares the characteristics of Staphylococcus epidermidis (SE) and Staphylococcus haemolyticus (SH) isolates from epidemiologically unrelated infections in humans (Hu) (28 SE-Hu; 8 SH-Hu) and companion animals (CpA) (12 SE-CpA; 13 SH-CpA). All isolates underwent antimicrobial susceptibility testing, multilocus sequence typing and DNA microarray profiling to detect antimicrobial resistance and SCCmec-associated genes. All methicillin-resistant (MR) isolates (33/40 SE, 20/21 SH) underwent dru and mecA allele typing. Isolates were predominantly assigned to sequence types (STs) within a single clonal complex (CC2, SE, 84.8%; CC1, SH, 95.2%). SCCmec IV predominated among MRSE with ST2-MRSE-IVc common to both Hu (40.9%) and CpA (54.5%). Identical mecA alleles and nontypeable dru types (dts) were identified in one ST2-MRSE-IVc Hu and CpA isolate, however, all mecA alleles and 2/4 dts detected among 18 ST2-MRSE-IVc isolates were closely related, sharing >96.5% DNA sequence homology. Although only one ST-SCCmec type combination (ST1 with a non-typeable [NT] SCCmec NT9 [class C mec and ccrB4]) was common to four MRSH-Hu and one MRSH-CpA, all MRSH isolates were closely related based on similar STs, SCCmec genes (V/VT or components thereof), mecA alleles and dts. Overall, 39.6% of MR isolates harbored NT SCCmec elements, and ACME was more common amongst MRSE and CpA isolates. Multidrug resistance (MDR) was detected among 96.7% of isolates but they differed in the prevalence of specific macrolide, aminoglycoside and trimethoprim resistance genes amongst SE and SH isolates. Ciprofloxacin, rifampicin, chloramphenicol [fexA, cat-pC221], tetracycline [tet(K)], aminoglycosides [aadD, aphA3] and fusidic acid [fusB] resistance was significantly more common amongst CpA isolates. SE and SH isolates causing infections in Hu and CpA hosts belong predominantly to STs within a single lineage, harboring similar but variable SCCmec genes, mecA alleles and dts. Host and

  8. Comparative Genotypes, Staphylococcal Cassette Chromosome mec (SCCmec Genes and Antimicrobial Resistance amongst Staphylococcus epidermidis and Staphylococcus haemolyticus Isolates from Infections in Humans and Companion Animals.

    Directory of Open Access Journals (Sweden)

    Brenda A McManus

    Full Text Available This study compares the characteristics of Staphylococcus epidermidis (SE and Staphylococcus haemolyticus (SH isolates from epidemiologically unrelated infections in humans (Hu (28 SE-Hu; 8 SH-Hu and companion animals (CpA (12 SE-CpA; 13 SH-CpA. All isolates underwent antimicrobial susceptibility testing, multilocus sequence typing and DNA microarray profiling to detect antimicrobial resistance and SCCmec-associated genes. All methicillin-resistant (MR isolates (33/40 SE, 20/21 SH underwent dru and mecA allele typing. Isolates were predominantly assigned to sequence types (STs within a single clonal complex (CC2, SE, 84.8%; CC1, SH, 95.2%. SCCmec IV predominated among MRSE with ST2-MRSE-IVc common to both Hu (40.9% and CpA (54.5%. Identical mecA alleles and nontypeable dru types (dts were identified in one ST2-MRSE-IVc Hu and CpA isolate, however, all mecA alleles and 2/4 dts detected among 18 ST2-MRSE-IVc isolates were closely related, sharing >96.5% DNA sequence homology. Although only one ST-SCCmec type combination (ST1 with a non-typeable [NT] SCCmec NT9 [class C mec and ccrB4] was common to four MRSH-Hu and one MRSH-CpA, all MRSH isolates were closely related based on similar STs, SCCmec genes (V/VT or components thereof, mecA alleles and dts. Overall, 39.6% of MR isolates harbored NT SCCmec elements, and ACME was more common amongst MRSE and CpA isolates. Multidrug resistance (MDR was detected among 96.7% of isolates but they differed in the prevalence of specific macrolide, aminoglycoside and trimethoprim resistance genes amongst SE and SH isolates. Ciprofloxacin, rifampicin, chloramphenicol [fexA, cat-pC221], tetracycline [tet(K], aminoglycosides [aadD, aphA3] and fusidic acid [fusB] resistance was significantly more common amongst CpA isolates. SE and SH isolates causing infections in Hu and CpA hosts belong predominantly to STs within a single lineage, harboring similar but variable SCCmec genes, mecA alleles and dts. Host and

  9. Flexible double-headed cytosine-linked 2'-deoxycytidine nucleotides. Synthesis, polymerase incorporation to DNA and interaction with DNA methyltransferases

    Czech Academy of Sciences Publication Activity Database

    Kielkowski, Pavel; Cahová, Hana; Pohl, Radek; Hocek, Michal

    2016-01-01

    Roč. 24, č. 6 (2016), s. 1268-1276 ISSN 0968-0896 R&D Projects: GA ČR GBP206/12/G151 Institutional support: RVO:61388963 Keywords : nucleosides * nucleotides * pyrimidines * DNA methyltransferases * DNA polymerases Subject RIV: CC - Organic Chemistry Impact factor: 2.930, year: 2016

  10. PCR Screening of Antibiotic Resistance Genes in Faecal Samples from Australian and Chinese Children.

    Science.gov (United States)

    Ravensdale, Joshua T; Xian, Darren Ten Wei; Wei, Chooi Ming; Lv, Quanjun; Wen, Xiajian; Guo, Jing; Coorey, Ranil; LeSouëf, Peter; Lu, Fengmin; Zhang, Brad; Dykes, Gary A

    2018-03-31

    Recent public awareness campaigns on the risk of antibiotic resistance in pathogenic microbes has placed pressure on governments to enforce stricter antimicrobial stewardship policies on the hospital and agricultural industry. This study aimed to screen faecal samples from Australian and Chinese children for the presence of antibiotic resistance genes to identify demographics at risk of carriage of these genes and examine antimicrobial stewardship policies from the two countries which may influence carriage. Faecal samples from 46 Australian and 53 Chinese children were screened for the presence of six clinically relevant antibiotic resistance genes using PCR. Clinical and demographic data was also collected from each patient. Over 90% of faecal samples from Chinese children tested positive for β-lactam, macrolide, tetracycline, and aminoglycoside resistance genes, which was substantially higher than Australian samples. Besides country of origin, no clear trend could be seen to predict carriage of resistance genes. The exception to this was Chinese born children who immigrated to Australia having higher rates of carriage for bla TEM and tetM genes than children born and still living in Australia. These data indicated that Chinese children were more likely to carry certain antibiotic resistance genes than Australian children. The Chinese government has recently implemented strict policies to control the overuse of antibiotics in hospitals. However, many of these policies do not extend to the agricultural industry which could explain the differences seen in this study. Copyright © 2018. Published by Elsevier Ltd.

  11. RlmCD-mediated U747 methylation promotes efficient G748 methylation by methyltransferase RlmAII in 23S rRNA in Streptococcus pneumoniae; interplay between two rRNA methylations responsible for telithromycin susceptibility.

    Science.gov (United States)

    Shoji, Tatsuma; Takaya, Akiko; Sato, Yoshiharu; Kimura, Satoshi; Suzuki, Tsutomu; Yamamoto, Tomoko

    2015-10-15

    Adenine at position 752 in a loop of helix 35 from positions 745 to 752 in domain II of 23S rRNA is involved in binding to the ribosome of telithromycin (TEL), a member of ketolides. Methylation of guanine at position 748 by the intrinsic methyltransferase RlmA(II) enhances binding of telithromycin (TEL) to A752 in Streptococcus pneumoniae. We have found that another intrinsic methylation of the adjacent uridine at position 747 enhances G748 methylation by RlmA(II), rendering TEL susceptibility. U747 and another nucleotide, U1939, were methylated by the dual-specific methyltransferase RlmCD encoded by SP_1029 in S. pneumoniae. Inactivation of RlmCD reduced N1-methylated level of G748 by RlmA(II) in vivo, leading to TEL resistance when the nucleotide A2058, located in domain V of 23S rRNA, was dimethylated by the dimethyltransferase Erm(B). In vitro methylation of rRNA showed that RlmA(II) activity was significantly enhanced by RlmCD-mediated pre-methylation of 23S rRNA. These results suggest that RlmCD-mediated U747 methylation promotes efficient G748 methylation by RlmA(II), thereby facilitating TEL binding to the ribosome. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Specialized (iso)eugenol-4-O-methyltransferases (s-IEMTs) and methods of making and using the same

    Science.gov (United States)

    Liu, Chang-Jun; Cai, Yuanheng

    2017-01-31

    Specialized (iso)eugenol 4-O-methyltransferase (s-IEMT) enzymes having increased capacity for methylation of monolignols are disclosed. The s-IEMTs have unique activity favoring methylation of coniferyl alcohol versus sinapyl alcohol. Various s-IEMTs methylate ferulic acid. Means for producing the various s-IEMTs are provided. The s-IEMTs are useful for modification of lignin content and production of aromatic compounds.

  13. Nuclear modifier MTO2 modulates the aminoglycoside-sensitivity of mitochondrial 15S rRNA C1477G mutation in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Xiangyu He

    Full Text Available The phenotypic manifestations of mitochondrial DNA (mtDNA mutations are modulated by mitochondrial DNA haplotypes, nuclear modifier genes and environmental factors. The yeast mitochondrial 15S rRNA C1477G (P(R or P(R 454 mutation corresponds to the human 12S rRNA C1494T and A1555G mutations, which are well known as primary factors for aminoglycoside-induced nonsyndromic deafness. Here we report that the deletion of the nuclear modifier gene MTO2 suppressed the aminoglycoside-sensitivity of mitochondrial 15S rRNA C1477G mutation in Saccharomyces cerevisiae. First, the strain with a single mtDNA C1477G mutation exhibited hypersensitivity to neomycin. Functional assays indicated that the steady-state transcription level of mitochondrial DNA, the mitochondrial respiratory rate, and the membrane potential decreased significantly after neomycin treatment. The impaired mitochondria could not produce sufficient energy to maintain cell viability. Second, when the mto2 null and the mitochondrial C1477G mutations co-existed (mto2(P(R, the oxygen consumption rate in the double mutant decreased markedly compared to that of the control strains (MTO2(P(S, mto2(P(S and MTO2(P(R. The expression levels of the key glycolytic genes HXK2, PFK1 and PYK1 in the mto2(P(R strain were stimulated by neomycin and up-regulated by 89%, 112% and 55%, respectively. The enhanced glycolysis compensated for the respiratory energy deficits, and could be inhibited by the glycolytic enzyme inhibitor. Our findings in yeast will provide a new insight into the pathogenesis of human deafness.

  14. Rational design of a live attenuated dengue vaccine: 2'-o-methyltransferase mutants are highly attenuated and immunogenic in mice and macaques.

    Directory of Open Access Journals (Sweden)

    Roland Züst

    Full Text Available Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2'-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2'-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2'-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response.

  15. The impact of nosocomially-acquired resistant Pseudomonas aeruginosa infection in a burn unit.

    Science.gov (United States)

    Armour, Alexis D; Shankowsky, Heather A; Swanson, Todd; Lee, Jonathan; Tredget, Edward E

    2007-07-01

    Nosocomially-acquired Pseudomonas aeruginosa remains a serious cause of infection and septic mortality in burn patients. This study was conducted to quantify the impact of nosocomially-transmitted resistant P. aeruginosa in a burn population. Using a TRACS burn database, 48 patients with P. aeruginosa resistant to gentamicin were identified (Pseudomonas group). Thirty-nine were case-matched to controls without resistant P. aeruginosa cultures (control group) for age, total body surface area, admission year, and presence of inhalation injury. Mortality and various morbidity endpoints were examined, as well as antibiotic costs. There was a significantly higher mortality rate in the Pseudomonas group (33% vs. 8%, p products used (packed cells 51.1 +/- 8.0 vs. 21.1 +/- 3.4, p < 0.01; platelets 11.9 +/- 3.0 vs. 1.4 +/- 0.7, p < 0.01) were all significantly higher in the Pseudomonas group. Cost of antibiotics was also significantly higher ($2,658.52 +/- $647.93 vs. $829.22 +/- $152.82, p < 0.01). Nosocomial colonization or infection, or both, of burn patients with aminoglycoside-resistant P. aeruginosa is associated with significantly higher morbidity, mortality, and cost of care. Increased resource consumption did not prevent significantly higher mortality rates when compared with that of control patients. Thus, prevention, identification, and eradication of nosocomial Pseudomonas contamination are critical for cost-effective, successful burn care.

  16. Plasmid-mediated quinolone resistance; interactions between human, animal and environmental ecologies

    Directory of Open Access Journals (Sweden)

    Laurent ePOIREL

    2012-02-01

    Full Text Available Resistance to quinolones and fluoroquinolones is being increasingly reported among human but also veterinary isolates during the last two to three decades, very likely as a consequence of the large clinical usage of those antibiotics. Even if the principle mechanisms of resistance to quinolones are chromosome-encoded, due to modifications of molecular targets (DNA gyrase and topoisomerase IV, decreased outer-membrane permeability (porin defect and overexpression of naturally-occurring efflux, the emergence of plasmid-mediated quinolone resistance (PMQR has been reported since 1998. Although these PMQR determinants confer low-level resistance to quinolones and/or fluoroquinolones, they are a favorable background for selection of additional chromosome-encoded quinolone resistance mechanisms. Different transferable mechanisms have been identified, corresponding to the production of Qnr proteins, of the aminoglycoside acetyltransferase AAC(6’-Ib-cr, or of the QepA-type or OqxAB-type efflux pumps. Qnr proteins protect target enzymes (DNA gyrase and type IV topoisomerase from quinolone inhibition (mostly nalidixic acid. The AAC(6’-Ib-cr determinant acetylates several fluoroquinolones, such as norfloxacin and ciprofloxacin. Finally, the QepA and OqxAB efflux pumps extrude fluoroquinolones from the bacterial cell. A series of studies have identified the environment to be a reservoir of PMQR genes, with farm animals and aquatic habitats being significantly involved. In addition, the origin of the qnr genes has been identified, corresponding to the waterborne species Shewanella sp. Altogether, the recent observations suggest that the aquatic environment might constitute the original source of PMQR genes, that would secondly spread among animal or human isolates.

  17. Prevalence and antibiotic resistance of 15 minor staphylococcal species colonizing orthopedic implants.

    Science.gov (United States)

    Arciola, C R; Campoccia, D; An, Y H; Baldassarri, L; Pirini, V; Donati, M E; Pegreffi, F; Montanaro, L

    2006-04-01

    Several species belonging to Staphylococcus genus (non Sau/ non Sep species) exhibit increasing abilities as opportunistic pathogens in colonisation of periprosthesis tissues. Here we report on antibiotic resistance of 193 strains, belonging to non Sau/ non Sep species, consecutively collected from orthopedic implant infections in a period of about 40 months. The 193 strains (representing 17% of all staphylococci isolated) were analysed for their antibiotic resistance to 16 different drugs. Five species turned out more prevalent, ranging from 1 to 5%: S. hominis (4.2%), S. haemolyticus (3.7%), S. capitis (2.7%), S. warneri (2.6%), and S. cohnii (1.6%). Among these, the prevalence of antibiotic resistance to penicillins was similar, ranging from 51% to 66%. Conversely, significant differences were observed for all the remaining antibiotics. For S. haemolyticus the resistances to oxacillin and imipenem, the four aminoglycosides and erythromycin were at least twice that of the other three species which were compared. S. warneri was on the contrary the species with the lowest occurrence of resistant strains. Ten species appeared only rarely at the infection sites: S. lugdunensis, S. caprae, S. equorum, S. intermedius, S. xylosus, S. simulans, S. saprophyticus, S. pasteuri, S. sciuri, and S. schleiferi. The behaviours of these species, often resistant to penicillins, were individually analysed. Differences in both the frequencies and the panels of antibiotic resistances observed among the non Sau/ non Sep species: i) suggest that horizontal spreading of resistance factors, if acting, was not sufficient per se to level their bio-diversities; ii) highlight and confirm the worrisome appearance within the Staphylococcus genus of emerging "new pathogens", not homogeneous for their virulence and antibiotic resistance prevalence, which deserve to be recognised and treated individually.

  18. O6-methylguanine-DNA methyltransferase activity is associated with response to alkylating agent therapy and with MGMT promoter methylation in glioblastoma and anaplastic glioma

    Science.gov (United States)

    Bobola, Michael S.; Alnoor, Mohammad; Chen, John Y.-S.; Kolstoe, Douglas D.; Silbergeld, Daniel L.; Rostomily, Robert C.; Blank, A.; Chamberlain, Marc C.; Silber, John R.

    2014-01-01

    Background CpG methylation in the O6-methylguanine-DNA methyltransferase (MGMT) promoter is associated with better outcome following alkylating agent chemotherapy in glioblastoma (GBM) and anaplastic glioma (AG). To what extent improved response reflects low or absent MGMT activity in glioma tissue has not been unequivocally assessed. This information is central to developing anti-resistance therapies. Methods We examined the relationship of MGMT activity in 91 GBMs and 84 AGs with progression-free survival (PFS) following alkylator therapy and with promoter methylation status determined by methylation-specific PCR (MSP). Results Cox regression analysis revealed that GBMs with high activity had a significantly greater risk for progression in dichotomous (P ≤ 0.001) and continuous (P ≤ 0.003) models, an association observed for different alkylator regimens, including concurrent chemo-radiation with temozolomide. Analysis of MGMT promoter methylation status in 47 of the GBMs revealed that methylated tumors had significantly lower activity (P ≤ 0.005) and longer PFS (P ≤ 0.036) compared to unmethylated tumors, despite overlapping activities. PFS was also significantly greater in methylated vs. unmethylated GBMs with comparable activity (P ≤ 0.005), and among unmethylated tumors with less than median activity (P ≤ 0.026), suggesting that mechanisms in addition to MGMT promote alkylator resistance. Similar associations of MGMT activity with PFS and promoter methylation status were observed for AGs. Conclusions Our results provide strong support for the hypotheses that MGMT activity promotes alkylator resistance and reflects promoter methylation status in malignant gliomas. General significance MGMT activity is an attractive target for anti-resistance therapy regardless of methylation status. PMID:25558448

  19. A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine.

    Science.gov (United States)

    Li, L; Popko, J L; Zhang, X H; Osakabe, K; Tsai, C J; Joshi, C P; Chiang, V L

    1997-05-13

    S-adenosyl-L-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin. The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants. In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied. However, little is known about lignin pathway OMTs in gymnosperms. We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT). The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences. However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis. The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem. Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem.

  20. A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine

    Science.gov (United States)

    Li, Laigeng; Popko, Jacqueline L.; Zhang, Xing-Hai; Osakabe, Keishi; Tsai, Chung-Jui; Joshi, Chandrashekhar P.; Chiang, Vincent L.

    1997-01-01

    S-adenosyl-l-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin. The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants. In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied. However, little is known about lignin pathway OMTs in gymnosperms. We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT). The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences. However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis. The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem. Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem. PMID:9144260

  1. Genetic relatedness and molecular characterization of multidrug resistant Acinetobacter baumannii isolated in central Ohio, USA

    Directory of Open Access Journals (Sweden)

    Tadesse Daniel

    2009-06-01

    Full Text Available Abstract Background Over the last decade, nosocomial infections due to Acinetobacter baumannii have been described with an increasing trend towards multidrug resistance, mostly in intensive care units. The aim of the present study was to determine the clonal relatedness of clinical isolates and to elucidate the genetic basis of imipenem resistance. Methods A. baumannii isolates (n = 83 originated from two hospital settings in central Ohio were used in this study. Pulsed-field gel electrophoresis genotyping and antimicrobial susceptibility testing for clinically relevant antimicrobials were performed. Resistance determinants were characterized by using different phenotypic (accumulation assay for efflux and genotypic (PCR, DNA sequencing, plasmid analysis and electroporation approaches. Results The isolates were predominantly multidrug resistant (>79.5% and comprised of thirteen unique pulsotypes, with genotype VII circulating in both hospitals. The presence of blaOXA-23 in 13% (11/83 and ISAba1 linked blaOXA-66 in 79.5% (66/83 of clinical isolates was associated with high level imipenem resistance. In this set of OXA producing isolates, multidrug resistance was bestowed by blaADC-25, class 1 integron-borne aminoglycoside modifying enzymes, presence of sense mutations in gyrA/parC and involvement of active efflux (with evidence for the presence of adeB efflux gene. Conclusion This study underscores the major role of carbapenem-hydrolyzing class D β-lactamases, and in particular the acquired OXA-23, in the dissemination of imipenem-resistant A. baumannii. The co-occurrence of additional resistance determinant could also be a significant threat.

  2. Overexpressing both ATP sulfurylase and selenocysteine methyltransferase enhances selenium phytoremediation traits in Indian mustard

    International Nuclear Information System (INIS)

    LeDuc, Danika L.; AbdelSamie, Manal; Montes-Bayon, Maria; Wu, Carol P.; Reisinger, Sarah J.; Terry, Norman

    2006-01-01

    A major goal of our selenium (Se) phytoremediation research is to use genetic engineering to develop fast-growing plants with an increased ability to tolerate, accumulate, and volatilize Se. To this end we incorporated a gene (encoding selenocysteine methyltransferase, SMT) from the Se hyperaccumulator, Astragalus bisulcatus, into Indian mustard (LeDuc, D.L., Tarun, A.S., Montes-Bayon, M., Meija, J., Malit, M.F., Wu, C.P., AbdelSamie, M., Chiang, C.-Y., Tagmount, A., deSouza, M., Neuhierl, B., Boeck, A., Caruso, J., Terry, N., 2004. Overexpression of selenocysteine methyltransferase in Arabidopsis and Indian mustard increases selenium tolerance and accumulation Plant Physiol. 135, 377-383.). The resulting transgenic plants successfully enhanced Se phytoremediation in that the plants tolerated and accumulated Se from selenite significantly better than wild type. However, the advantage conferred by the SMT enzyme was much less when Se was supplied as selenate. In order to enhance the phytoremediation of selenate, we developed double transgenic plants that overexpressed the gene encoding ATP sulfurylase (APS) in addition to SMT, i.e., APS x SMT. The results showed that there was a substantial improvement in Se accumulation from selenate (4 to 9 times increase) in transgenic plants overexpressing both APS and SMT. - Simultaneous overexpression of APS and SMT genes in Indian mustard greatly increases ability to accumulate selenate

  3. Characterization of Multidrug Resistant E. faecalis Strains from Pigs of Local Origin by ADSRRS-Fingerprinting and MALDI -TOF MS; Evaluation of the Compatibility of Methods Employed for Multidrug Resistance Analysis.

    Directory of Open Access Journals (Sweden)

    Aneta Nowakiewicz

    Full Text Available The aim of this study was to characterize multidrug resistant E. faecalis strains from pigs of local origin and to analyse the relationship between resistance and genotypic and proteomic profiles by amplification of DNA fragments surrounding rare restriction sites (ADSRRS-fingerprinting and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI -TOF MS. From the total pool of Enterococcus spp. isolated from 90 pigs, we selected 36 multidrug resistant E. faecalis strains, which represented three different phenotypic resistance profiles. Phenotypic resistance to tetracycline, macrolides, phenicols, and lincomycin and high-level resistance to aminoglycosides were confirmed by the occurrence of at least one corresponding resistance gene in each strain. Based on the analysis of the genotypic and phenotypic resistance of the strains tested, five distinct resistance profiles were generated. As a complement of this analysis, profiles of virulence genes were determined and these profiles corresponded to the phenotypic resistance profiles. The demonstration of resistance to a wide panel of antimicrobials by the strains tested in this study indicates the need of typing to determine the spread of resistance also at the local level. It seems that in the case of E. faecalis, type and scope of resistance strongly determines the genotypic pattern obtained with the ADSRRS-fingerprinting method. The ADSRRS-fingerprinting analysis showed consistency of the genetic profiles with the resistance profiles, while analysis of data with the use of the MALDI- TOF MS method did not demonstrate direct reproduction of the clustering pattern obtained with this method. Our observations were confirmed by statistical analysis (Simpson's index of diversity, Rand and Wallace coefficients. Even though the MALDI -TOF MS method showed slightly higher discrimination power than ADSRRS-fingerprinting, only the latter method allowed reproduction of the

  4. The Genomic Basis of Intrinsic and Acquired Antibiotic Resistance in the Genus Serratia

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    Luisa Sandner-Miranda

    2018-05-01

    Full Text Available Serratia marcescens, a member of the Enterobacteriaceae family, was long thought to be a non-pathogenic bacterium prevalent in environmental habitats. Together with other members of this genus, it has emerged in recent years as an opportunistic nosocomial pathogen causing various types of infections. One important feature of pathogens belonging to this genus is their intrinsic and acquired resistance to a variety of antibiotic families, including β-lactam, aminoglycosides, quinolones and polypeptide antibiotics. The aim of this study was to elucidate which genes participate in the intrinsic and acquired antibiotic resistance of this genus in order to determine the Serratia genus resistome. We performed phylogenomic and comparative genomic analyses using 32 Serratia spp. genomes deposited in the NCBI GenBank from strains isolated from different ecological niches and different lifestyles. S. marcescens strain SmUNAM836, which was previously isolated from a Mexican adult with obstructive pulmonary disease, was included in this study. The results show that most of the antibiotic resistance genes (ARGs were found on the chromosome, and to a lesser degree, on plasmids and transposons acquired through horizontal gene transfer. Four strains contained the gyrA point mutation in codon Ser83 that confers quinolone resistance. Pathogenic and environmental isolates presented a high number of ARGs, especially genes associated with efflux systems. Pathogenic strains, specifically nosocomial strains, presented more acquired resistance genes than environmental isolates. We may conclude that the environment provides a natural reservoir for antibiotic resistance, which has been underestimated in the medical field.

  5. The Genomic Basis of Intrinsic and Acquired Antibiotic Resistance in the Genus Serratia

    Science.gov (United States)

    Sandner-Miranda, Luisa; Vinuesa, Pablo; Cravioto, Alejandro; Morales-Espinosa, Rosario

    2018-01-01

    Serratia marcescens, a member of the Enterobacteriaceae family, was long thought to be a non-pathogenic bacterium prevalent in environmental habitats. Together with other members of this genus, it has emerged in recent years as an opportunistic nosocomial pathogen causing various types of infections. One important feature of pathogens belonging to this genus is their intrinsic and acquired resistance to a variety of antibiotic families, including β-lactam, aminoglycosides, quinolones and polypeptide antibiotics. The aim of this study was to elucidate which genes participate in the intrinsic and acquired antibiotic resistance of this genus in order to determine the Serratia genus resistome. We performed phylogenomic and comparative genomic analyses using 32 Serratia spp. genomes deposited in the NCBI GenBank from strains isolated from different ecological niches and different lifestyles. S. marcescens strain SmUNAM836, which was previously isolated from a Mexican adult with obstructive pulmonary disease, was included in this study. The results show that most of the antibiotic resistance genes (ARGs) were found on the chromosome, and to a lesser degree, on plasmids and transposons acquired through horizontal gene transfer. Four strains contained the gyrA point mutation in codon Ser83 that confers quinolone resistance. Pathogenic and environmental isolates presented a high number of ARGs, especially genes associated with efflux systems. Pathogenic strains, specifically nosocomial strains, presented more acquired resistance genes than environmental isolates. We may conclude that the environment provides a natural reservoir for antibiotic resistance, which has been underestimated in the medical field.

  6. High diversity of genes and plasmids encoding resistance to third-generation cephalosporins and quinolones in clinical Escherichia coli from commercial poultry flocks in Italy

    DEFF Research Database (Denmark)

    Niero, Giulia; Bortolaia, Valeria; Vanni, Michele

    2018-01-01

    = 98) and layers (n = 22) between 2008 and 2012. 3GC-resistant isolates were screened for extended-spectrum and AmpC β-lactamase (ESBL/AmpC), while all isolates were tested for plasmid-mediated quinolone resistance (PMQR) genes. ESBL/AmpC- and PMQR-positive isolates were typed by pulsed-field gel......% of isolates from turkeys, broilers and layers, respectively. We identified seven ESBL/AmpC-encoding plasmid types, usually conjugative (78%), with a marked prevalence of IncI1/pST3 plasmids carrying blaCTX-M-1. PMQR occurred less frequently among isolates from turkeys (0.9%) compared to those from broilers (5......%) and layers (4%). The PMQR genes qnrS, qnrB19 and oqxA/B were located on three plasmid types and two non-typeable plasmids, mostly (85%) conjugative. ESBL/AmpC- and PMQR-positive isolates were genetically unrelated and 64% of them were additionally resistant to aminoglycosides, sulfonamides and tetracyclines...

  7. Hearing loss in children treated for multidrug-resistant tuberculosis.

    Science.gov (United States)

    Seddon, James A; Thee, Stephanie; Jacobs, Kayleen; Ebrahim, Adam; Hesseling, Anneke C; Schaaf, H Simon

    2013-04-01

    The aminoglycosides and polypeptides are vital drugs for the management of multidrug-resistant (MDR) tuberculosis (TB). Both classes of drug cause hearing loss. We aimed to determine the extent of hearing loss in children treated for MDR-TB. In this retrospective study, children (Hearing was assessed and classified using audiometry and otoacoustic emissions. Ninety-four children were included (median age: 43 months). Of 93 tested, 28 (30%) were HIV-infected. Twenty-three (24%) children had hearing loss. Culture-confirmed, as opposed to presumed, diagnosis of TB was a risk factor for hearing loss (OR: 4.12; 95% CI: 1.13-15.0; p = 0.02). Seven of 11 (64%) children classified as having hearing loss using audiometry had progression of hearing loss after finishing the injectable drug. Hearing loss is common in children treated for MDR-TB. Alternative drugs are required for the treatment of paediatric MDR-TB. Copyright © 2012 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

  8. Epigenetic changes of Arabidopsis genome associated with altered DNA methyltransferase and demethylase expressions after gamma irradiation

    International Nuclear Information System (INIS)

    Kim, Ji Eun; Cho, Eun Ju; Kim, Ji Hong; Chung, Byung Yeoup; Kim, Jin Hong

    2012-01-01

    DNA methylation at carbon 5 of cytosines is a hall mark of epigenetic inactivation and heterochromatin in both plants and mammals. In Arabidopsis, DNA methylation has two roles that protect the genome from selfish DNA elements and regulate gene expression. Plant genome has three types of DNA methyltransferase, METHYLTRANSFERASE 1 (MET1), DOMAINREARRANGED METHYLASE (DRM) and CHROMOMETHYLASE 3 (CMT3) that are capable of methylating CG, CHG (where H is A, T, or C) and CHH sites, respectively. MET1 is a maintenance DNA methyltransferase that controls CG methylation. Two members of the DRM family, DRM1 and DRM2, are responsible for de novo methylation of CG, CHG, and CHH sites but show a preference for CHH sites. Finally, CMT3 principally carries out CHG methylation and is involved in both de novo methylation and maintenance. Alternatively, active DNA demethylation may occur through the glycosylase activity by removing the methylcytosines from DNA. It may have essential roles in preventing transcriptional silencing of transgenes and endogenous genes and in activating the expression of imprinted genes. DNA demetylation in Arabidopsis is mediated by the DEMETER (DME) family of bifunctional DNA glycosylase. Three targets of DME are MEA (MEDEA), FWA (FLOWERING WAGENINGEN), and FIS2 (FERTILIZATION INDEPENDENT SEED 2). The DME family contains DEMETER-LIKE 2 (DML2), DML3, and REPRESSOR OF SILENING 1 (ROS1). DNA demetylation by ROS1, DML2, and DML3 protect the hypermethylation of specific genome loci. ROS1 is necessary to suppress the promoter methylation and the silencing of endogenous genes. In contrast, the function of DML2 and DML3 has not been reported. Several recent studies have suggested that epigenetic alterations such as change in DNA methylation and histone modification should be caused in plant genomes upon exposure to ionizing radiation. However, there is a lack of data exploring the underlying mechanisms. Therefore, the present study aims to characterize and

  9. Epigenetic changes of Arabidopsis genome associated with altered DNA methyltransferase and demethylase expressions after gamma irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ji Eun; Cho, Eun Ju; Kim, Ji Hong; Chung, Byung Yeoup; Kim, Jin Hong [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2012-05-15

    DNA methylation at carbon 5 of cytosines is a hall mark of epigenetic inactivation and heterochromatin in both plants and mammals. In Arabidopsis, DNA methylation has two roles that protect the genome from selfish DNA elements and regulate gene expression. Plant genome has three types of DNA methyltransferase, METHYLTRANSFERASE 1 (MET1), DOMAINREARRANGED METHYLASE (DRM) and CHROMOMETHYLASE 3 (CMT3) that are capable of methylating CG, CHG (where H is A, T, or C) and CHH sites, respectively. MET1 is a maintenance DNA methyltransferase that controls CG methylation. Two members of the DRM family, DRM1 and DRM2, are responsible for de novo methylation of CG, CHG, and CHH sites but show a preference for CHH sites. Finally, CMT3 principally carries out CHG methylation and is involved in both de novo methylation and maintenance. Alternatively, active DNA demethylation may occur through the glycosylase activity by removing the methylcytosines from DNA. It may have essential roles in preventing transcriptional silencing of transgenes and endogenous genes and in activating the expression of imprinted genes. DNA demetylation in Arabidopsis is mediated by the DEMETER (DME) family of bifunctional DNA glycosylase. Three targets of DME are MEA (MEDEA), FWA (FLOWERING WAGENINGEN), and FIS2 (FERTILIZATION INDEPENDENT SEED 2). The DME family contains DEMETER-LIKE 2 (DML2), DML3, and REPRESSOR OF SILENING 1 (ROS1). DNA demetylation by ROS1, DML2, and DML3 protect the hypermethylation of specific genome loci. ROS1 is necessary to suppress the promoter methylation and the silencing of endogenous genes. In contrast, the function of DML2 and DML3 has not been reported. Several recent studies have suggested that epigenetic alterations such as change in DNA methylation and histone modification should be caused in plant genomes upon exposure to ionizing radiation. However, there is a lack of data exploring the underlying mechanisms. Therefore, the present study aims to characterize and

  10. Isolation and identification of antibiotic resistance genes in Staphylococcus aureus isolates from respiratory system infections in shahrekord, Iran

    Directory of Open Access Journals (Sweden)

    Maryam Reisi

    2014-07-01

    Full Text Available   Introduction : Staphylococcus aureus is considered as one of pathogenic agents in humans, that engages different body parts including respiratory system and causes to spend lots of costs and extending patient’s treatment period. This study which is performed to separate and investigate the pattern of antibiotic resistance in Staphylococcus aureus isolates from upper respiratory system infections in Shahrekord.   Materials and methods: This study was done by sectional-descriptive method On 200 suspicious persons to the upper respiratory system infections who were referred to the Imam Ali clinic in Shahrekord in 2012. After isolation of Staphylococcus aureus from cultured nose discharges, antibiotic resistance genes were identified by polymerase chain reaction (PCR by using defined primer pairs .   Results : Among 200 investigated samples in 60 cases (30% Staphylococcus aureus infection (by culturing and PCR method was determined. Isolates showed the lowest amount of antibiotic resistance to vancomycin (0.5% and the highest amount of resistance to the penicillin G and cefotaxime (100%. mecA gene (encoding methicillin resistance with frequency of 85.18% and aacA-D gene (encoding resistance to aminoglycosides with frequency of 28.33% showed the highest and lowest frequency of antibiotic resistance genes coding in Staphylococcus aureus isolates respectively .   Discussion and conclusion : Notable prevalence of resistant Staphylococcus aureus isolates in community acquired respiratory infections, recommend continuous control necessity to impede the spreading of these bacteria and their infections.  

  11. Determination of catechol O-methyltransferase activity in relation to melanin metabolism using high-performance liquid chromatography with fluorimetric detection

    NARCIS (Netherlands)

    Smit, N. P.; Pavel, S.; Kammeyer, A.; Westerhof, W.

    1990-01-01

    A new sensitive method for the determination of catechol O-methyltransferase activity has been developed. The method is based on the O-methylation of the indolic intermediates of melanin metabolism. The substrate, 5,6-dihydroxyindole-2-carboxylic acid, is converted by the enzyme to two O-methylated

  12. Preliminary individualized chemotherapy for malignant astrocytomas based on O6-methylguanine-deoxyribonucleic acid methyltransferase methylation analysis.

    Science.gov (United States)

    Watanabe, Takao; Katayama, Yoichi; Ogino, Akiyoshi; Ohta, Takashi; Yoshino, Atsuo; Fukushima, Takao

    2006-08-01

    O(6)-methylguanine-deoxyribonucleic acid methyltransferase gene (MGMT) methylation is apparently correlated with responsiveness to nitrosourea chemotherapy, suggesting this alkylating agent should be effective against MGMT-methylated tumors. MGMT appears not to be linked to platinum resistance, so platinum chemotherapy should be used for MGMT-unmethylated tumors. This study was a preliminary trial of individualized chemotherapy based on MGMT methylation status in a total of 20 patients with newly diagnosed malignant astrocytomas (9 anaplastic astrocytomas and 11 glioblastomas multiforme). The procarbazine, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-2(2-chloroethyl)-3-nitrosourea, and vincristine (PAV) regimen was administered to seven patients with MGMT-methylated tumors, and the carboplatin and etoposide (CE) regimen was administered to 13 patients with MGMT-unmethylated tumors. Objective response to the PAV therapy was noted in all three patients with measurable residual tumor (2 complete responses and 1 partial response). Five of the seven patients continued to be disease-free after initiation of the PAV therapy. Objective response to the CE therapy was seen in only one of seven patients with measurable residual tumor (1 partial response). Three of the 13 patients were free from progression, whereas the remaining 10 patients showed early progression. The PAV regimen is effective against MGMT-methylated malignant astrocytomas, but the CE regimen is not useful at the given dose and schedule in MGMT-unmethylated tumors.

  13. Phytochemical Prospection and Modulation of Antibiotic Activity In Vitro by Lippia origanoides H.B.K. in Methicillin Resistant Staphylococcus aureus

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    Humberto Medeiros Barreto

    2014-01-01

    Full Text Available The Lippia origanoides H.B.K. ethanol extract (LOEE and hexane (LOHEX, dichloromethane (LODCM, and ethyl acetate (LOEA fractions were tested for their antimicrobial activity alone or in combination with antibiotics against a methicillin resistant Staphylococcus aureus (MRSA strain. The natural products did not show antimicrobial activity against multidrug resistant strain at the clinically significant concentrations tested. However, a modulatory effect in the antibacterial activity of the neomycin and amikacin was verified when LOEE, LOHEX and LODCM were added to the growth medium at subinhibitory concentrations. A similar modulation was found when the natural products were changed for chlorpromazine, an inhibitor of bacterial efflux pumps, suggesting the involvement of resistance mediated by efflux system in the MRSA tested. The fractions LOHEX and LODCM showed a modulatory activity bigger than their majority compounds (carvacrol, thymol, and naringenin, indicating that this activity is not due to their majority compounds only, but it is probably due to a synergism between their chemical components. These results indicate that L. origanoides H.B.K. can be a source of phytochemicals able to modify the phenotype of resistance to aminoglycosides in MRSA.

  14. Phenotypic Detection of Metallo-Beta-Lactamases in Carbapenem Resistant Acinetobacter baumannii Isolated from Pediatric Patients in Pakistan

    Directory of Open Access Journals (Sweden)

    Muneeza Anwar

    2016-01-01

    Full Text Available Multidrug resistant A. baumannii has emerged as an important and problematic human pathogen as it is the causative agent of several types of infections especially in neonates and immunocompromised patients because they have least capacity to fight against infections. Carbapenems are used as last resort antibiotics for treating these infections but currently resistance against carbapenems due to MBL production is on the rise. The objective of this study was to determine the frequency of antibiotic resistance in A. baumannii and also to compare the efficacy of combined disk test and double disk synergy test for detection of metallo-beta-lactamases. A total of 112 A. baumannii were identified from various clinical samples and antibiotic susceptibility profile was determined by Kirby-Bauer Disk Diffusion method. Out of 112, 66 (58.9% isolates were resistant to both imipenem and meropenem (OXOID. These resistant isolates were tested for carbapenemase production, and 55 (83.3% were carbapenemase producers by Modified Hodge Test. These isolates were further tested for MBL production by combined disk test and double disk synergy test. Out of 66, 49 isolates were positive by both methods, CDT and DDST, and only one isolate was detected as negative (with kappa value = 0.038. All MBL producing strains showed remarkable resistance to cephalosporins, fluoroquinolones, aminoglycosides, and piperacillin/tazobactam (OXOID. The antibiotic resistance was very high in A. baumannii which were isolated from children in Pakistan specially attending a nephrology unit.

  15. Mounting resistance of uropathogens to antimicrobial agents: A retrospective study in patients with chronic bacterial prostatitis relapse.

    Science.gov (United States)

    Stamatiou, Konstantinos; Pierris, Nikolaos

    2017-07-01

    Despite recent progress in the management of chronic bacterial prostatitis (CBP), many cases relapse. Increased drug resistance patterns of responsible bacteria have been proposed as the most probable causative factor. Driven by the limited number of previous studies addressing this topic, we aimed to study whether antibiotic resistance increases in patients with CBP when relapse occurs. A secondary aim of this study was to determine the resistance patterns of responsible bacteria from patients with CBP. The study material consisted of bacterial isolates from urine and/or prostatic secretions obtained from patients with CBP. Bacterial identification was performed by using the Vitek 2 Compact system and susceptibility testing was performed by disc diffusion and/or the Vitek 2 system. Interpretation of susceptibility results was based on Clinical and Laboratory Standards Institute guidelines. A total of 253 samples from patients diagnosed with CBP for the first time (group A) and 137 samples from relapsing patients with a history of CBP and previous antibiotic treatment (group B) were analyzed. A significant reduction in bacterial resistance to the less used antibiotics (TMP-SMX, tetracyclines, aminoglycosides, penicillins, and macrolides) was noted. An increase in resistance to quinolones of many bacteria that cause CBP was also noted with the increase in resistance of enterococcus strains being alarming. Comparison of the resistance profile of CBP-responsible bacteria between samples from first-time-diagnosed patients and samples from relapsing patients revealed notable differences that could be attributed to previous antibiotic treatment.

  16. Risk factors associated with fluoroquinolone-resistant enterococcal urinary tract infections in a tertiary care university hospital in north India.

    Science.gov (United States)

    Banerjee, Tuhina; Anupurba, Shampa

    2016-10-01

    Fluoroquinolone resistance in both Gram-positive and Gram-negative bacteria has increased with the widespread use of fluoroquinolones. Fluoroquinolone resistance in Gram-negative bacilli has been widely studied, though staphylococci and enterococci are also notably resistant. Enterococci being the second most common cause of healthcare-associated urinary tract infections (UTIs) fluoroquinolones are often the drug of choice. This study was undertaken to assess the risk factors associated with fluoroquinolone-resistant enterococcal UTI in a tertiary level health facility in north India. A total of 365 patients with UTI caused by enterococci were studied over a period of two years. Patients with ciprofloxacin-resistant and susceptible UTI were considered as cases and controls, respectively. Resistance profile of the isolates against common antibiotics was studied by minimum inhibitory concentration (MIC) determination. Mechanisms for fluoroquinolone resistance was studied by efflux pump inhibitor activity and multiplex PCR targeting the qnr genes. A total of 204 (55.89%) cases and 161 (44.1%) controls were identified. The fluoroquinolone-resistant isolates were significantly resistant to ampicillin, high strength aminoglycosides and vancomycin. The majority (78%) of the resistant isolates showed efflux pump activity. Treatment in indoor locations, presence of urinary catheters and pregnancy along with recent exposure to antibiotics especially fluoroquinolones, third generation cephalosporins and piperacillin-tazobactam were identified as independent risk factors. Our results showed that fluoroquinolone resistance in enterococcal UTI was largely associated with indoor usage of antibiotics and use of indwelling devices. Knowledge of risk factors is important to curb this emergence of resistance.

  17. Mutations in conserved helix 69 of 23S rRNA of Thermus thermophilus that affect capreomycin resistance but not posttranscriptional modifications

    DEFF Research Database (Denmark)

    Monshupanee, Tanakarn; Gregory, Steven T; Douthwaite, Stephen

    2008-01-01

    of previously reported capreomycin resistance base substitutions. Capreomycin resistance in other bacteria has been shown to result from inactivation of the TlyA methyltransferase which 2'-O methylates C1920 of 23S rRNA. Inactivation of the tlyA gene in T. thermophilus does not affect its sensitivity...... for resistance to the tuberactinomycin antibiotic capreomycin. Two base substitutions, A1913U and mU1915G, and a single base deletion, DeltamU1915, were identified in helix 69 of 23S rRNA, a structural element that forms part of an interribosomal subunit bridge with the decoding center of 16S rRNA, the site...... to capreomycin. Finally, none of the mutations in helix 69 interferes with methylation at C1920 or with pseudouridylation at positions 1911 and 1917. We conclude that the resistance phenotype is a consequence of structural changes introduced by the mutations....

  18. [Analysis of resistant genes of beta-lactam antibiotics from Pseudomonas aeruginosa in pediatric patients].

    Science.gov (United States)

    Dong, Fang; Xu, Xi-wei; Song, Wen-qi; Lü, Ping; Yang, Yong-hong; Shen, Xu-zhuang

    2008-11-18

    To analyze the antibiotic resistance of the Pseudomonas aeruginosa (PA) isolated from pediatric patients and the resistant genes of beta-lactam antibiotics thereof. 146 PA strains were isolated from pediatric patients. Agar dilution method recommended by the Clinical and Laboratory Standards Institute was used to examine the minimum inhibitory concentrations (MICs) of 12 antimicrobial agents, including the penicillins, third and fourth genet ration cephalosporins, carbapenemase, Aztreonam, beta-lactamase inhibitors, quinolones, and aminoglycosides. PCR was used to detect the expression of the genes TEM, SHV, OXA, PER, GES, CTX-M, IMP, VIM, DHA, MIR, FOX, and oprD2. The multi-drug resistance rates against different antibiotic were high among the 146 PA strains. The rates of imipenem and meropenem resistance were 41.1% and 35.6% respectively. Among the 146 PA strains, 46 (31.5%) were positive for the MBL genotype; 38 (82.6%) carried the blaIMP gene, 8 (17.4%) carried the blaVIM gene, and 114 (78.1%) were oprD2 negative. The genes TEM, SHV, OXA, CTX-M, PER, VEB, GES, FOX, MIR, and DHA were not found in all strains. Many PA isolated from pediatric patients carry the genes IMP or VIM and losses oprD2 gene related to the expression of the outer membrane porin OprD2. The loss of the gene oprD2 is essential mechanism of beta-lactam antibiotics resistance in PA.

  19. Identification, antimicrobial resistance and genotypic characterization of Enterococcus spp. isolated in Porto Alegre, Brazil

    Science.gov (United States)

    Bender, Eduardo André; de Freitas, Ana Lúcia Peixoto; Reiter, Keli Cristine; Lutz, Larissa; Barth, Afonso Luís

    2009-01-01

    In the past two decades the members of the genus Enterococcus have emerged as important nosocomial pathogens worldwide. In the present study, we evaluated the antimicrobial resistance and genotypic characteristics of 203 Enterococcus spp. recovered from different clinical sources from two hospitals in Porto Alegre, Rio Grande do Sul, Brazil. The species were identified by conventional biochemical tests and by an automated system. The genetic diversity of E. faecalis presenting high-level aminoglycoside resistance (HLAR) was assessed by pulsed-field gel electrophoresis of chromosomal DNA after SmaI digestion. The E. faecalis was the most frequent specie (93.6%), followed by E. faecium (4.4%). The antimicrobial resistance profile was: 2.5% to ampicillin, 0.5% to vancomycin, 0.5% teicoplanin, 33% to chloramphenicol, 2% to nitrofurantoin, 66.1% to erythromycin, 66.5% to tetracycline, 24.6% to rifampicin, 30% to ciprofloxacin and 87.2% to quinupristin-dalfopristin. A total of 10.3% of the isolates proved to be HLAR to both gentamicin and streptomycin (HLR-ST/GE), with 23.6% resistant only to gentamicin (HLR-GE) and 37.4% only to streptomycin (HLR-ST). One predominant clonal group was found among E. faecalis HLR-GE/ST. The prevalence of resistance among beta-lactam antibiotics and glycopeptides was very low. However, in this study there was an increased number of HLR Enterococcus which may be spreading intra and inter-hospital. PMID:24031416

  20. Effects of salicylates and aminoglycosides on spontaneous otoacoustic emissions in the Tokay gecko.

    Science.gov (United States)

    Stewart, C E; Hudspeth, A J

    2000-01-04

    The high sensitivity and sharp frequency discrimination of hearing depend on mechanical amplification in the cochlea. To explore the basis of this active process, we examined the pharmacological sensitivity of spontaneous otoacoustic emissions (SOAEs) in a lizard, the Tokay gecko. In a quiet environment, each ear produced a complex but stable pattern of emissions. These SOAEs were reversibly modulated by drugs that affect mammalian otoacoustic emissions, the salicylates and the aminoglycoside antibiotics. The effect of a single i.p. injection of sodium salicylate depended on the initial power of the emissions: ears with strong control SOAEs displayed suppression at all frequencies, whereas those with weak control emissions showed enhancement. Repeated oral administration of acetylsalicylic acid reduced all emissions. Single i.p. doses of gentamicin or kanamycin suppressed SOAEs below 2.6 kHz, while modulating those above 2.6 kHz in either of two ways. For ears whose emission power at 2.6-5.2 kHz encompassed more than half of the total, individual emissions displayed facilitation as great as 35-fold. For the remaining ears, emissions dropped to as little as one-sixth of their initial values. The similarity of the responses of reptilian and mammalian cochleas to pharmacological intervention provides further evidence for a common mechanism of cochlear amplification.

  1. Pure tone audiograms and possible aminoglycoside-induced hearing loss in belugas (Delphinapterus leucas)

    Science.gov (United States)

    Finneran, James J.; Carder, Donald A.; Dear, Randall; Belting, Traci; McBain, Jim; Dalton, Les; Ridgway, Sam H.

    2005-06-01

    A behavioral response paradigm was used to measure pure-tone hearing sensitivities in two belugas (Delphinapterus leucas). Tests were conducted over a 20-month period at the Point Defiance Zoo and Aquarium, in Tacoma, WA. Subjects were two males, aged 8-10 and 9-11 during the course of the study. Subjects were born in an oceanarium and had been housed together for all of their lives. Hearing thresholds were measured using a modified up/down staircase procedure and acoustic response paradigm where subjects were trained to produce audible responses to test tones and to remain quiet otherwise. Test frequencies ranged from approximately 2 to 130 kHz. Best sensitivities ranged from approximately 40 to 50 dB re 1 μPa at 50-80 kHz and 30-35 kHz for the two subjects. Although both subjects possessed traditional ``U-shaped'' mammalian audiograms, one subject exhibited significant high-frequency hearing loss above 37 kHz compared to previously published data for belugas. Hearing loss in this subject was estimated to approach 90 dB for frequencies above 50 kHz. Similar ages, ancestry, and environmental conditions between subjects, but a history of ototoxic drug administration in only one subject, suggest that the observed hearing loss was a result of the aminoglycoside antibiotic amikacin. .

  2. Cationic Amphiphiles Increase Activity of Aminoglycoside Antibiotic Tobramycin in the Presence of Airway Polyelectrolytes

    International Nuclear Information System (INIS)

    Purdy Drew, Kirstin R.; Sanders, Lori K.; Culumber, Zachary W.; Zribi, Olena; Wong, Gerard C.L.

    2009-01-01

    It is empirically known that anionic polyelectrolytes present in cystic fibrosis (CF) airways due to bacterial infection significantly decrease the activity of cationic antimicrobials via electrostatic binding. In this work, we use synchrotron small-angle X-ray scattering to investigate the interaction between tobramycin, an aminoglycoside antibiotic commonly administered to CF patients via inhalation, with DNA, which is found in high concentrations in the CF airway. We find that interactions between DNA and tobramycin are significantly modified by the presence of mixtures of amphiphilic molecules. We measure a hierarchy of self-assembled structures formed between tobramycin, DNA, and the amphiphile mixtures and show how interactions between these components can be controlled. Results indicate that mixtures of cationic and negative curvature amphiphiles optimized for DNA binding via charge matching and curvature matching can competitively displace bound tobramycin from DNA and thereby drastically suppress tobramycin-DNA binding and resultant antimicrobial inactivation. Growth inhibition assays confirm the increased activity of tobramycin in the presence of DNA with the addition of the amphiphiles. These results suggest that optimized cationic amphiphile solutions have the potential to enhance antimicrobial function in highly infected environments that contain increased concentrations of anionic inflammatory polymers.

  3. Cationic Amphiphiles Increase Activity of Aminoglycoside Antibiotic Tobramycin in the Presence of Airway Polyelectrolytes

    International Nuclear Information System (INIS)

    Drew, K.R.Purdy; Sanders, L.K.; Culumber, Z.W.; Zribi, O.; Wong, G.C.L.

    2009-01-01

    It is empirically known that anionic polyelectrolytes present in cystic fibrosis (CF) airways due to bacterial infection significantly decrease the activity of cationic antimicrobials via electrostatic binding. In this work, we use synchrotron small-angle X-ray scattering to investigate the interaction between tobramycin, an aminoglycoside antibiotic commonly administered to CF patients via inhalation, with DNA, which is found in high concentrations in the CF airway. We find that interactions between DNA and tobramycin are significantly modified by the presence of mixtures of amphiphilic molecules. We measure a hierarchy of self-assembled structures formed between tobramycin, DNA, and the amphiphile mixtures and show how interactions between these components can be controlled. Results indicate that mixtures of cationic and negative curvature amphiphiles optimized for DNA binding via charge matching and curvature matching can competitively displace bound tobramycin from DNA and thereby drastically suppress tobramycin-DNA binding and resultant antimicrobial inactivation. Growth inhibition assays confirm the increased activity of tobramycin in the presence of DNA with the addition of the amphiphiles. These results suggest that optimized cationic amphiphile solutions have the potential to enhance antimicrobial function in highly infected environments that contain increased concentrations of anionic inflammatory polymers

  4. Predominance of carbapenem-resistant Pseudomonas aeruginosa isolates carrying blaIMP and blaVIM metallo-β-lactamases in a major hospital in Costa Rica.

    Science.gov (United States)

    Toval, Francisco; Guzmán-Marte, Anel; Madriz, Vivian; Somogyi, Teresita; Rodríguez, César; García, Fernando

    2015-01-01

    This study aimed to assess the molecular basis of the resistance to carbapenems in clinical isolates of Pseudomonas aeruginosa recovered from a tertiary-level health facility in San José, Costa Rica. A total of 198 non-duplicated isolates were evaluated for their susceptibility to β-lactams, aminoglycosides and fluoroquinolones. The production of metallo-β-lactamases (MBLs), the presence of MBL encoding genes (blaIMP, blaVIM and blaGIM-1) and the occurrence of these genes within class 1 integrons were investigated. In addition, an ERIC2 PCR fingerprinting method was used to elucidate the distribution of the detected MBL genes within the strain collection. Of the 198 isolates tested, 125 (63.1 %) were categorized as carbapenem-resistant. The majority (88.8 %) of the carbapemen-resistant isolates also showed resistance to ceftazidime, cefepime, aztreonam, ticarcillin/clavulanic acid, amikacin, gentamicin, tobramycin, ciprofloxacin and gatifloxacin. Among the carbapenem-resistant isolates, 102 (81.6 %) showed MBL activity. Strikingly, both blaIMP and blaVIM genes were simultaneously detected in most (94.1 %) of the 102 MBL producers. Five carbapenem-resistant MBL producers were positive only for blaIMP genes. Almost 70 % of the isolates examined harboured the intI1 gene, accompanied by the sul1 and qacEΔ1 genes in 136 (99 %) and 122 (89 %) isolates, respectively. The majority (94.4 %) of the carbapenem-resistant isolates carried the intI1 gene, in contrast to 26 % of the carbapenem-susceptible isolates. Ninety-three out of 96 (96.9 %) isolates carrying both blaIMP and blaVIM genes also harboured the intI1, sul1 and qacEΔ1 genes. Gene cassettes from carbapenem-susceptible and MBL-negative carbapenem-resistant isolates encoded aminoglycoside-resistance enzymes (aadA2, aadA4 and aadA6) as well as orfD and qacF genes. RAPD analysis distributed 126 of the isolates in 29 clusters. Eighty of the 90 blaIMP (+) blaVIM (+) isolates were sorted into 16

  5. Expression, purification, crystallization and preliminary crystallographic study of isolated modules of the mouse coactivator-associated arginine methyltransferase 1

    Energy Technology Data Exchange (ETDEWEB)

    Troffer-Charlier, Nathalie; Cura, Vincent; Hassenboehler, Pierre; Moras, Dino; Cavarelli, Jean, E-mail: cava@igbmc.u-strasbg.fr [IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Département de Biologie et Génomique Structurales, 1 Rue Laurent Fries, Illkirch, F-67404 (France); INSERM, U596, Illkirch, F-67400 (France); CNRS, UMR7104, Illkirch, F-67400 (France); Université Louis Pasteur, Faculté des Sciences de la Vie, Strasbourg, F-67000 (France)

    2007-04-01

    Isolated modules of mouse coactivator-associated arginine methyltransferase 1 encompassing the protein arginine N-methyltransferase catalytic domain have been overexpressed, purified and crystallized. X-ray diffraction data have been collected and have enabled determination of the structures by multiple isomorphous replacement using anomalous scattering. Coactivator-associated arginine methyltransferase 1 (CARM1) plays a crucial role in gene expression as a coactivator of several nuclear hormone receptors and also of non-nuclear receptor systems. Its recruitment by the transcriptional machinery induces protein methylation, leading to chromatin remodelling and gene activation. CARM1{sub 28–507} and two structural states of CARM1{sub 140–480} were expressed, purified and crystallized. Crystals of CARM1{sub 28–507} belong to space group P6{sub 2}22, with unit-cell parameters a = b = 136.0, c = 125.3 Å; they diffract to beyond 2.5 Å resolution using synchrotron radiation and contain one monomer in the asymmetric unit. The structure of CARM1{sub 28–507} was solved by multiple isomorphous replacement and anomalous scattering methods. Crystals of apo CARM1{sub 140–480} belong to space group I222, with unit-cell parameters a = 74.6, b = 99.0, c = 207.4 Å; they diffract to beyond 2.7 Å resolution and contain two monomers in the asymmetric unit. Crystals of CARM1{sub 140–480} in complex with S-adenosyl-l-homocysteine belong to space P2{sub 1}2{sub 1}2, with unit-cell parameters a = 74.6, b = 98.65, c = 206.08 Å; they diffract to beyond 2.6 Å resolution and contain four monomers in the asymmetric unit. The structures of apo and holo CARM1{sub 140–480} were solved by molecular-replacement techniques from the structure of CARM1{sub 28–507}.

  6. Culture and Next-generation sequencing-based drug susceptibility testing unveil high levels of drug-resistant-TB in Djibouti: results from the first national survey.

    Science.gov (United States)

    Tagliani, Elisa; Hassan, Mohamed Osman; Waberi, Yacine; De Filippo, Maria Rosaria; Falzon, Dennis; Dean, Anna; Zignol, Matteo; Supply, Philip; Abdoulkader, Mohamed Ali; Hassangue, Hawa; Cirillo, Daniela Maria

    2017-12-15

    Djibouti is a small country in the Horn of Africa with a high TB incidence (378/100,000 in 2015). Multidrug-resistant TB (MDR-TB) and resistance to second-line agents have been previously identified in the country but the extent of the problem has yet to be quantified. A national survey was conducted to estimate the proportion of MDR-TB among a representative sample of TB patients. Sputum was tested using XpertMTB/RIF and samples positive for MTB and resistant to rifampicin underwent first line phenotypic susceptibility testing. The TB supranational reference laboratory in Milan, Italy, undertook external quality assurance, genotypic testing based on whole genome and targeted-deep sequencing and phylogenetic studies. 301 new and 66 previously treated TB cases were enrolled. MDR-TB was detected in 34 patients: 4.7% of new and 31% of previously treated cases. Resistance to pyrazinamide, aminoglycosides and capreomycin was detected in 68%, 18% and 29% of MDR-TB strains respectively, while resistance to fluoroquinolones was not detected. Cluster analysis identified transmission of MDR-TB as a critical factor fostering drug resistance in the country. Levels of MDR-TB in Djibouti are among the highest on the African continent. High prevalence of resistance to pyrazinamide and second-line injectable agents have important implications for treatment regimens.

  7. Mutations in the histamine N-methyltransferase gene, HNMT, are associated with nonsyndromic autosomal recessive intellectual disability

    OpenAIRE

    Heidari, Abolfazl; Tongsook, Chanakan; Najafipour, Reza; Musante, Luciana; Vasli, Nasim; Garshasbi, Masoud; Hu, Hao; Mittal, Kirti; McNaughton, Amy J. M.; Sritharan, Kumudesh; Hudson, Melissa; Stehr, Henning; Talebi, Saeid; Moradi, Mohammad; Darvish, Hossein

    2015-01-01

    Histamine (HA) acts as a neurotransmitter in the brain, which participates in the regulation of many biological processes including inflammation, gastric acid secretion and neuromodulation. The enzyme histamine N-methyltransferase (HNMT) inactivates HA by transferring a methyl group from S-adenosyl-l-methionine to HA, and is the only well-known pathway for termination of neurotransmission actions of HA in mammalian central nervous system. We performed autozygosity mapping followed by targeted...

  8. Feed additives shift gut microbiota and enrich antibiotic resistance in swine gut.

    Science.gov (United States)

    Zhao, Yi; Su, Jian-Qiang; An, Xin-Li; Huang, Fu-Yi; Rensing, Christopher; Brandt, Kristian Koefoed; Zhu, Yong-Guan

    2018-04-15

    Antibiotic resistance genes (ARGs) are emerging environmental contaminants posing a threat to public health. Antibiotics and metals are widely used as feed additives and could consequently affect ARGs in swine gut. In this study, high-throughput quantitative polymerase chain reaction (HT-qPCR) based ARG chip and next-generation 16S rRNA gene amplicon sequencing data were analyzed using multiple statistical approaches to profile the antibiotic resistome and investigate its linkages to antibiotics and metals used as feed additives and to the microbial community composition in freshly collected swine manure samples from three large-scale Chinese pig farms. A total of 146 ARGs and up to 1.3×10 10 total ARG copies per gram of swine feces were detected. ARGs conferring resistance to aminoglycoside, macrolide-lincosamide-streptogramin B (MLSB) and tetracycline were dominant in pig gut. Total abundance of ARGs was positively correlated with in-feed antibiotics, microbial biomass and abundance of mobile genetic elements (MGEs) (Padditives and community composition (16.5%). These results suggest that increased levels of in-feed additives could aggravate the enrichment of ARGs and MGEs in swine gut. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Multivariable Analysis of the Association Between Antimicrobial Use and Antimicrobial Resistance in Escherichia coli Isolated from Apparently Healthy Pigs in Japan.

    Science.gov (United States)

    Makita, Kohei; Goto, Masaki; Ozawa, Manao; Kawanishi, Michiko; Koike, Ryoji; Asai, Tetsuo; Tamura, Yutaka

    2016-01-01

    The objective of this study was to investigate the association between antimicrobial agent use and antimicrobial resistance in Escherichia coli isolated from healthy pigs using data from 2004 to 2007 in the Japanese Veterinary Antimicrobial Resistance Monitoring System (JVARM). Fecal E. coli isolates from 250 pigs (one isolate each from a pig per farm) were examined for antimicrobial resistance. Information on the use of antimicrobials within preceding 6 months and types of farms recorded in JVARM was collected and statistically analyzed against the resistance patterns. In the univariate analysis, associations between both therapeutic and feed additive use of antimicrobials, and resistance to dihydrostreptomycin, gentamicin, kanamycin, ampicillin, cefazolin, ceftiofur, oxytetracycline, chloramphenicol, trimethoprim, nalidixic acid, enrofloxacin, colistin, and bicozamycin, and husbandry factors were investigated. In multivariable analysis, generalized estimating equations were used to control geographical intraclass correlation. Confounding for structurally unrelated associations was tested using generalized linear models. The results suggested direct and cross selections in the associations between use of aminoglycosides in reproduction farms and resistance to kanamycin, use of tetracyclines in larger farms and resistance to oxytetracycline, use of beta-lactams and resistance to ampicillin, use of phenicols and resistance to chloramphenicol, and use of fluoroquinolones and resistance to nalidixic acid and enrofloxacin. Coselection was suggested in the use of tetracyclines and chloramphenicol resistance. The associations between use of beta-lactams and dihydrostreptomycin resistance, use of macrolides and ampicillin and oxytetracycline resistance, and use of colistin and kanamycin resistance were significant, but were confounded by the simultaneous use of homologous antimicrobials.

  10. Genetic complexity of fusidic acid-resistant small colony variants (SCV in Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Jonas Lannergård

    Full Text Available FusE mutants are fusidic acid-resistant small colony variants (SCVs of Staphylococcus aureus that can be selected with aminoglycosides. All FusE SCVs have mutations in rplF, encoding ribosomal protein L6. However, individual FusE mutants including some with the same mutation in rplF display auxotrophy for either hemin or menadione, suggesting that additional mutations are involved. Here we show that FusE SCVs can be divided into three genetic sub-groups and that some carry an additional mutation, in one of the genes required for hemin biosynthesis, or in one of the genes required for menadione biosynthesis. Reversion analysis and genome sequencing support the hypothesis that these combinations of mutations in the rplF, hem, and/or men genes can account for the SCV and auxotrophic phenotypes of FusE mutants.

  11. A histone H3K9M mutation traps histone methyltransferase Clr4 to prevent heterochromatin spreading

    Energy Technology Data Exchange (ETDEWEB)

    Shan, Chun-Min; Wang, Jiyong; Xu, Ke; Chen, Huijie; Yue, Jia-Xing; Andrews, Stuart; Moresco, James J.; Yates, John R.; Nagy, Peter L.; Tong, Liang; Jia, Songtao

    2016-09-20

    Histone lysine-to-methionine (K-to-M) mutations are associated with multiple cancers, and they function in a dominant fashion to block the methylation of corresponding lysines on wild type histones. However, their mechanisms of function are controversial. Here we show that in fission yeast, introducing the K9M mutation into one of the three histone H3 genes dominantly blocks H3K9 methylation on wild type H3 across the genome. In addition, H3K9M enhances the interaction of histone H3 tail with the H3K9 methyltransferase Clr4 in a SAM (S-adenosyl-methionine)-dependent manner, and Clr4 is trapped at nucleation sites to prevent its spreading and the formation of large heterochromatin domains. We further determined the crystal structure of an H3K9M peptide in complex with human H3K9 methyltransferase G9a and SAM, which reveales that the methionine side chain had enhanced van der Waals interactions with G9a. Therefore, our results provide a detailed mechanism by which H3K9M regulates H3K9 methylation.

  12. Resistência a β-lactâmicos em Acinetobacter spp isolados de efluente hospitalar no sul do Brasil Resistance to β-lactams among Acinetobacter spp isolated from hospital sewage in southern Brazil

    Directory of Open Access Journals (Sweden)

    Carolina de Souza Gusatti

    2009-04-01

    Full Text Available Acinetobacter spp é um importante patógeno causador de infecções nosocomiais que acomete pacientes imunocomprometidos e capaz de adquirir resistência a antimicrobianos com facilidade. Os esgotos hospitalares são importantes disseminadores de genes de resistência a antimicrobianos para a microbiota ambiental. Neste contexto, 30 cepas de Acinetobacter spp provenientes de efluente de um hospital em Porto Alegre, RS, foram analisados quanto ao perfil de susceptibilidade a β-lactamases, quinolonas e aminoglicosídeos através de antibiograma e testes de triagem para metalo beta-lactamases e β-lactamases de espectro estendido. O perfil encontrado revela cepas multi-resistentes e que mecanismos de resistência como a produção de β-lactamases de espectro estendido e bombas de efluxo podem estar presentes nesses isolados.Acinetobacter spp is an important pathogen that is responsible for nosocomial infections affecting immunocompromised patients, and it can easily acquire resistance to antimicrobial agents. Hospital sewage is an important means for disseminating genes for resistance to antimicrobial agents, to the microbiota of the environment. Within this context, 30 strains of Acinetobacter spp from the sewage of a hospital in Porto Alegre, Rio Grande do Sul, were analyzed regarding their profile of susceptibility to β-lactams, quinolones and aminoglycosides, by means of an antibiogram and tests to screen for metallo-β-lactamases and extended-spectrum β-lactamases. The profile obtained revealed the presence of multidrug-resistant strains and showed that resistance mechanisms such as the production of extended-spectrum β-lactamases and efflux pumps may be present in these strains.

  13. Bacteremia and candidemia in hematological malignancies

    DEFF Research Database (Denmark)

    Bruun, B; Bangsborg, Jette Marie; Hovgaard, D

    1988-01-01

    of coagulase-negative staphylococci was higher in the latter period while that of Staphylococcus aureus was lower. Of 67 strains of Enterobacteriaceae tested for an aminoglycoside, 6% were found to be resistant, whereas 8% of 48 Enterobacteriaceae strains were found to be cefotaxime resistant. Methicillin...

  14. Bacterial Aetiology and Antibiotic Resistance Pattern of Community-Acquired Urinary Tract Infections in Children in a Tertiary Care Hospital in Bangladesh

    Directory of Open Access Journals (Sweden)

    Lazina Sharmin

    2017-09-01

    Full Text Available Background: Urinary tract infections (UTIs in children are among the most common bacterial infections. Community-acquired urinary tract infections (CAUTI are often treated empirically with broad-spectrum antibiotics. Pattern of aetiologic agents and their antibiotic sensitivity may vary according to geographical and regional location. So, knowledge of antibiotic resistance trends is important for improving evidence-based recommendations for empirical treatment of UTIs. Objectives: To determine the common bacterial aetiologies of CAUTIs and their antibiotic resistance patterns in a tertiary care hospital, Savar. Materials and Methods: This cross-sectional descriptive study was conducted at Enam Medical College Hospital, Savar from May 2016 to April 2017. We collected clean-catch mid-stream urine samples from 257 patients having clinical diagnosis of UTI and submitted to the clinical microbiology laboratory for culture and sensitivity. Results: A total of 120 (46.7% samples were positive for bacterial growth. Escherichia coli (79% was the most common pathogen, followed by Klebsiella spp. (14%. Bacterial isolates showed high prevalence of resistance to multiple antibiotics. Resistance against amoxicillin/clavulanic acid, co-trimoxazole and ciprofloxacin was higher compared to newer quinolones and aminoglycosides. Conclusion: Esch. coli and Klebsiella spp. were the predominant bacterial pathogens. The resistance pattern to commonly prescribed antibiotics was quite high and alarming.

  15. Microflora of conjunctiva in children and its sensitivity and resistance to antibacterial drugs

    Directory of Open Access Journals (Sweden)

    T. N. Vorontsova

    2014-07-01

    Full Text Available Purpose: Investigation of microflora of conjunctiva and its resistance to antibacterial drugs in healthy children and patients with various inflammatory eye diseases.Methods: We examined 402 children (421 eyes in the age from 1 month till 17 years: 62 healthy children (70 eyes and 340 pa- tients with different inflammatory diseases of anterior segment of eye (351 eyes. the smear was done in all children for plating and definition of sensitivity of microflora to antibacterial drugs by method of diffusion to agar.Results: the plating was positive even in 72.9% of healthy children who entered the hospital for the planned surgery. Most often we revealed Staphylococcus epidermidis (44.3%, Staphylococcus aureus (12.8%, Streptococcus faecalis (5.7% and Enterobacter (2.9%. In children with inflammatory diseases Staphylococcus epidermidis and Staphylococcus aureus (62.6% were found fre- quently. the analysis of data showed high level of resistance of all microflora to aminoglycosides (neomycin 37.8% and tobramycin 32.7% and chloramphenicol — 37.1%. the lowest resistance of all microflora was registered to levofloxacin (11.1% and ciprofloxacin (10.5%. In gram-negative microflora we revealed the maximal sensitivity to ciprofloxacin, in gram-positive — to levofloxacin.We detected the maximal resistance of microflora to ampicillin (66.1%, and minimal — to cephalosporines (4.5% among the antibiotics of systemic application.Conclusion: the findings allow us to recommend drops containing levofloxacin (Signicef for clinical practice in pediatric ophthalmology. 

  16. Microflora of conjunctiva in children and its sensitivity and resistance to antibacterial drugs

    Directory of Open Access Journals (Sweden)

    T. N. Vorontsova

    2012-01-01

    Full Text Available Purpose: Investigation of microflora of conjunctiva and its resistance to antibacterial drugs in healthy children and patients with various inflammatory eye diseases.Methods: We examined 402 children (421 eyes in the age from 1 month till 17 years: 62 healthy children (70 eyes and 340 pa- tients with different inflammatory diseases of anterior segment of eye (351 eyes. the smear was done in all children for plating and definition of sensitivity of microflora to antibacterial drugs by method of diffusion to agar.Results: the plating was positive even in 72.9% of healthy children who entered the hospital for the planned surgery. Most often we revealed Staphylococcus epidermidis (44.3%, Staphylococcus aureus (12.8%, Streptococcus faecalis (5.7% and Enterobacter (2.9%. In children with inflammatory diseases Staphylococcus epidermidis and Staphylococcus aureus (62.6% were found fre- quently. the analysis of data showed high level of resistance of all microflora to aminoglycosides (neomycin 37.8% and tobramycin 32.7% and chloramphenicol — 37.1%. the lowest resistance of all microflora was registered to levofloxacin (11.1% and ciprofloxacin (10.5%. In gram-negative microflora we revealed the maximal sensitivity to ciprofloxacin, in gram-positive — to levofloxacin.We detected the maximal resistance of microflora to ampicillin (66.1%, and minimal — to cephalosporines (4.5% among the antibiotics of systemic application.Conclusion: the findings allow us to recommend drops containing levofloxacin (Signicef for clinical practice in pediatric ophthalmology. 

  17. Antimicrobial resistance and resistance genes in Salmonella strains isolated from broiler chickens along the slaughtering process in China.

    Science.gov (United States)

    Zhu, Yuanting; Lai, Haimei; Zou, Likou; Yin, Sheng; Wang, Chengtao; Han, Xinfeng; Xia, Xiaolong; Hu, Kaidi; He, Li; Zhou, Kang; Chen, Shujuan; Ao, Xiaolin; Liu, Shuliang

    2017-10-16

    A total of 189 Salmonella isolates were recovered from 627 samples which were collected from cecal contents of broilers, chicken carcasses, chicken meat after cutting step and frozen broiler chicken products along the slaughtering process at a slaughterhouse in Sichuan province of China. The Salmonella isolates were subjected to antimicrobial susceptibility testing to 10 categories of antimicrobial agents using the Kirby-Bauer disk diffusion method. Those antibiotics-resistant isolates were further investigated for the occurrence of resistance genes, the presence of class 1 integron as well as the associated gene cassettes, and the mutations within the gyrA and parC genes. Consequently, the prevalence of Salmonella was 30.14% (47.96% for cecal content, 18.78% for chicken carcasses, 31.33% for cutting meat and 14.00% for frozen meat, respectively). The predominant serotypes were S. Typhimurium (15.34%) and S. Enteritidis (69.84%). High resistance rates to the following drugs were observed: nalidixic acid (99.5%), ampicillin (87.8%), tetracycline (51.9%), ciprofloxacin (48.7%), trimethoprim/sulfamethoxazole (48.1%), and spectinomycin (34.4%). Antimicrobial resistance profiling showed that 60.8% of isolates were multidrug resistant (MDR), and MDR strains increased from 44.7% to 78.6% along the slaughtering line. 94.6% (n=157) of beta-lactam-resistant isolates harbored at least one resistance gene of bla TEM or bla CTX-M . The relatively low prevalence of aminoglycoside resistance genes (aac(3)-II, aac(3)-IV, and ant(2″)-I) was found in 49 (66.2%) of antibiotic-resistant isolates. The tetracycline resistance genes (tet(A), tet(B), tet(C), and tet(G) and sulfonamide resistance genes (sul1, sul2, and sul3) were identified in 84 (85.7%) and 89 (97.8%) antibiotic-resistant isolates respectively. floR was identified in 44 (97.8%) florfenicol-resistant isolates. Class 1 integron was detected in 37.4% (n=43) of the MDR isolates. Two different gene cassettes, bla OXA-30 -aad

  18. Antibiotic Resistance Genetic Markers and Integrons in White Soft Cheese: Aspects of Clinical Resistome and Potentiality of Horizontal Gene Transfer.

    Science.gov (United States)

    de Paula, Ana Caroline L; Medeiros, Julliane D; de Azevedo, Analice C; de Assis Chagas, Jéssica M; da Silva, Vânia L; Diniz, Cláudio G

    2018-02-19

    Antibiotic resistance poses an important threat to global public health and has become a challenge to modern medicine. The occurrence of antibiotic-resistant bacteria in a broad range of foods has led to a growing concern about the impact that food may have as a reservoir of antibiotic resistance genes. Considering Minas Frescal Cheese (MFC)-a typical Brazilian white soft cheese-and its economic and cultural values, in this study, medically relevant antimicrobial-resistance genetic markers (AR genes) were screened, and the occurrence of integrons were evaluated in manufactured MFC using culture-independent approaches. Through a fingerprinting analysis, the tested MFCs were brand-clustered, indicating reproducibility along the production chain. A common core of resistance markers in all brands evaluated and related antimicrobials such as β-lactams, tetracyclines, quinolones, and sulfonamide was detected. Several other markers, including efflux pumps and aminoglycosides-resistance were distributed among brands. Class 1 and 2 integrons were observed, respectively, in 77% and 97% of the samples. The presence of AR genes is of special interest due to their clinical relevance. Taken together, the data may suggest that the production chain of MFC might contribute to the spread of putative drug-resistant bacteria, which could greatly impact human health. Furthermore, detection of class 1 and class 2 integrons in MFC has led to discussions about resistance gene spread in this traditional cheese, providing evidence of potential horizontal transfer of AR genes to human gut microbiota.

  19. Antibiotic Resistance Genetic Markers and Integrons in White Soft Cheese: Aspects of Clinical Resistome and Potentiality of Horizontal Gene Transfer

    Directory of Open Access Journals (Sweden)

    Ana Caroline L. de Paula

    2018-02-01

    Full Text Available Antibiotic resistance poses an important threat to global public health and has become a challenge to modern medicine. The occurrence of antibiotic-resistant bacteria in a broad range of foods has led to a growing concern about the impact that food may have as a reservoir of antibiotic resistance genes. Considering Minas Frescal Cheese (MFC—a typical Brazilian white soft cheese—and its economic and cultural values, in this study, medically relevant antimicrobial-resistance genetic markers (AR genes were screened, and the occurrence of integrons were evaluated in manufactured MFC using culture-independent approaches. Through a fingerprinting analysis, the tested MFCs were brand-clustered, indicating reproducibility along the production chain. A common core of resistance markers in all brands evaluated and related antimicrobials such as β-lactams, tetracyclines, quinolones, and sulfonamide was detected. Several other markers, including efflux pumps and aminoglycosides-resistance were distributed among brands. Class 1 and 2 integrons were observed, respectively, in 77% and 97% of the samples. The presence of AR genes is of special interest due to their clinical relevance. Taken together, the data may suggest that the production chain of MFC might contribute to the spread of putative drug-resistant bacteria, which could greatly impact human health. Furthermore, detection of class 1 and class 2 integrons in MFC has led to discussions about resistance gene spread in this traditional cheese, providing evidence of potential horizontal transfer of AR genes to human gut microbiota.

  20. A decade-long commitment to antimicrobial resistance surveillance in Portugal

    Directory of Open Access Journals (Sweden)

    Catarina Moreira Marinho

    2016-10-01

    Full Text Available Antimicrobial resistance (AMR is a worldwide problem with serious health and economic repercussions. Since the 1940s, underuse, overuse, and misuse of antibiotics have had a significant environmental downside. Large amounts of antibiotics not fully metabolized after use in human and veterinary medicine, and other applications, are annually released into the environment. The result has been the development and dissemination of antibiotic-resistant bacteria due to many years of selective pressure. Surveillance of AMR provides important information that helps in monitoring and understanding how resistance mechanisms develop and disseminate within different environments. Surveillance data is needed to inform clinical therapy decisions, to guide policy proposals, and to assess the impact of action plans to fight AMR. The Functional Genomics and Proteomics Unit, based at the University of Trás-os-Montes and Alto Douro (UTAD in Vila Real, Portugal, has recently completed 10 years of research surveying AMR in bacteria, mainly commensal indicator bacteria such as enterococci and Escherichia coli from the microbiota of different animals. Samples from more than 75 different sources have been accessed, from humans to food-producing animals, pets, and wild animals. The typical microbiological workflow involved phenotypic studies followed by molecular approaches. Throughout the decade, 4,017 samples were collected and over 5,000 bacterial isolates obtained. High levels of AMR to several antimicrobial classes have been reported, including to β-lactams, glycopeptides, tetracyclines, aminoglycosides, sulphonamides and quinolones. Multi-resistant strains, some relevant to human and veterinary medicine like extended-spectrum β-lactamase-producing E. coli and vancomycin-resistant enterococci, have been repeatedly isolated even in non-synanthropic animal species. Of particular relevance are reports of AMR bacteria in wildlife from natural reserves and endangered