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Sample records for antibioticresistance enzymes aminoglycoside

  1. Crystallographic Studies of Two Bacterial AntibioticResistance Enzymes: Aminoglycoside Phosphotransferase (2')-Ic and GES-1\\beta-lactamase

    Energy Technology Data Exchange (ETDEWEB)

    Brynes, Laura; /Rensselaer Poly.

    2007-10-31

    Guiana Extended-Spectrum-1 (GES-1) and Aminoglycoside phosphotransferase (2')-Ic (APH(2')-Ic) are two bacteria-produced enzymes that essentially perform the same task: they provide resistance to an array of antibiotics. Both enzymes are part of a growing resistance problem in the medical world. In order to overcome the ever-growing arsenal of antibiotic-resistance enzymes, it is necessary to understand the molecular basis of their action. Accurate structures of these proteins have become an invaluable tool to do this. Using protein crystallography techniques and X-ray diffraction, the protein structure of GES-1 bound to imipenem (an inhibitor) has been solved. Also, APH(2')-Ic has been successfully crystallized, but its structure was unable to be solved using molecular replacement using APH(2')-Ib as a search model. The structure of GES-1, with bound imipenem was solved to a resolution of 1.89A, and though the inhibitor is bound with only moderate occupancy, the structure shows crucial interactions inside the active site that render the enzyme unable to complete the hydrolysis of the {beta}-lactam ring. The APH(2')-Ic dataset could not be matched to the model, APH(2')-Ib, with which it shares 25% sequence identity. The structural information gained from GES-1, and future studies using isomorphous replacement to solve the APH(2')-Ic structure can aid directly to the creation of novel drugs to combat both of these classes of resistance enzymes.

  2. Microplate phosphocellulose binding assay for aminoglycoside-modifying enzymes.

    OpenAIRE

    Cooksey, R C; Metchock, B G; Thornsberry, C

    1986-01-01

    We modified the phosphocellulose binding assay for aminoglycoside-modifying enzymes (AMEs) by use of microdilution plates and a multichannel micropipette. Batteries of aminoglycoside substrates for screening organisms for the presence of AMEs as well as for subclassifying enzymes were prepared and stored in microdilution plates. When tested in parallel with the conventional tube reaction assay, the microplate assay yielded comparable radioactive counts and therefore equally correct identifica...

  3. Strategies to overcome the action of aminoglycoside-modifying enzymes for treating resistant bacterial infections

    OpenAIRE

    Labby, Kristin J.; Garneau-Tsodikova, Sylvie

    2013-01-01

    Shortly after the discovery of the first antibiotics, bacterial resistance began to emerge. Many mechanisms give rise to resistance; the most prevalent mechanism of resistance to the aminoglycoside (AG) family of antibiotics is the action of aminoglycoside-modifying enzymes (AMEs). Since the identification of these modifying enzymes, many efforts have been put forth to prevent their damaging alterations of AGs. These diverse strategies are discussed within this review, including: creating new...

  4. Serum Aminoglycoside Assay by Enzyme-Mediated Immunoassay (EMIT): Correlation with Radioimmunoassay, Fluoroimmunoassay, and Acetyltransferase and Microbiological Assays

    OpenAIRE

    White, L O; Scammell, L. M.; Reeves, D S

    1981-01-01

    Enzyme-mediated immunoassay (EMIT) serum aminoglycoside assay results were accurate and precise and correlated well with radioimmunoassay, fluoroimmunoassay, and acetyltransferase and microbiological assay determinations.

  5. Dissecting the cosubstrate structure requirements of the Staphylococcus aureus aminoglycoside resistance enzyme ANT(4').

    Science.gov (United States)

    Porter, Vanessa R; Green, Keith D; Zolova, Olga E; Houghton, Jacob L; Garneau-Tsodikova, Sylvie

    2010-12-01

    Aminoglycosides are important antibiotics used against a wide range of pathogens. As a mechanism of defense, bacteria have evolved enzymes able to inactivate these drugs by regio-selectively adding a variety of functionalities (acetyl, phospho, and nucelotidyl groups) to their scaffolds. The aminoglycoside nucleotidyltransferase ANT(4') is one of the most prevalent and unique modifying-enzymes. Here, by TLC, HRMS, and colorimetric assays, we demonstrate that the resistance enzyme ANT(4') from Staphylococcus aureus is highly substrate and cosubstrate promiscuous. We show that deoxy-ribonucleotide triphosphates (dNTPs) are better cosubstrates than NTPs. We demonstrate that the position of the triphosphate group (5' and not 3') on the ribose/deoxyribose ring is important for recognition by ANT(4'), and that NTPs with larger substituents at the 3'-position of the ribose ring are not cosubstrates for ANT(4'). We confirm that for all aminoglycosides tested, the respective nucleotidylated products are completely inactive. These results provide valuable insights into the development of strategies to combat the ever-growing bacterial resistance problem. PMID:21040710

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

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    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.

  7. Relationship between antimicrobial resistance and aminoglycoside-modifying enzyme gene expressions in Acinetobacter baumannii

    Institute of Scientific and Technical Information of China (English)

    SHI Wei-feng; JIANG Jian-ping; MI Zu-huang

    2005-01-01

    Background Acinetobacter baumannii is one of the main gram-negative bacilli in clinical practice. Nosocomial infections caused by multi-drug resistance Acinetobacter baumannii is very difficult to treat. This study was designed to investigate the antimicrobial resistance characteristics and four resistant gene expressions of aminoglycoside-modifying enzymes including N-acetyltransferases and O-phosphotransferases in Acinetobacter baumannii. Methods Bacterial identification and antimicrobial susceptibility test were performed by PhoenixTM system in 247 strains of Acinetobacter baumannii. Minimal inhibitory concentrations (MICs) of seven aminoglycosides including gentamicin, amikacin, kanamycin, tobramycin, netilmicin, neomycin and streptomycin in 15 strains of multi-drug resistant Acinetobacter baumannii were detected by agar dilution. Four aminoglycoside-modifying enzyme genes were amplified by polymerase chain reaction (PCR) and verified by DNA sequencer.Results The resistance rates of 247 strains of Acinetobacter baumannii against cefotaxime, levofloxacin, piperacillin, aztreonam, tetracycline, ciprofloxacin and chloramphenicol were more than 50%. Imipenem and meropenem showed high antibacterial activities with resistance rates of 3.2% and 4.1%. MIC50 and MIC90 of gentamicin, amikacin, streptomycin and kanamycin in 15 strains of multi-drug resistant Acinetobacter baumanii were all more than 1024 mg/L, and the resistance rates were 100%, 100%, 100% and 93.3%, respectively. But their resistance rates to tobramycin, netilmicin and neomycin were 86.7%, 93.3% and 46.7%, respectively. Three modifying enzyme genes, including aacC1, aacC2 and aacA4 genes, were found in 15 strains, but aphA6 had not been detected. Their positive rates were 93.3%, 20.0% and 20.0%, respectively. These three genes existed simultaneously in No.19 strain. Nucleotide sequences of aacC1, aacC2 and aacA4 genes shared 100%, 97.9% and 99.7% identities with GenBank genes (AY307113, S68058 and AY

  8. Identification of aminoglycoside-acetylating enzymes by high-pressure liquid chromatographic determination of their reaction products.

    OpenAIRE

    Lovering, A M; White, L. O.; Reeves, D S

    1984-01-01

    A method to identify the aminoglycoside-acetyltransferase (AAC) enzymes AAC(3), AAC(2') and AAC(6') by high-pressure liquid chromatographic characterization of their products of reaction with tobramycin or sisomicin is described. Conditions are given for the chromatography of kanamycin A, netilmicin, neomycin, and apramycin, and their products of reaction, if any, with the three AAC enzymes are listed.

  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. Distribution of genes encoding aminoglycoside-modifying enzymes among clinical isolates of methicillin-resistant staphylococci

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    N Perumal

    2016-01-01

    Full Text Available The objective of this study was to determine the distribution of genes encoding aminoglycoside-modifying enzymes (AMEs and staphylococcal cassette chromosome mec (SCCmec elements among clinical isolates of methicillin-resistant staphylococci (MRS. Antibiotic susceptibility test was done using Kirby-Bauer disk diffusion method. The presence of SCCmec types and AME genes, namely, aac (6′-Ie-aph (2′′, aph (3′-IIIa and ant (4′-Ia was determined using two different multiplex polymerase chain reaction. The most encountered AME genes were aac (6′-Ie-aph (2′′ (55.4% followed by aph (3′-IIIa (32.3% and ant (4′-Ia gene (9%. SCCmec type I (34% was predominant in this study. In conclusion, the aac (6′-Ie-aph (2′′ was the most common AME gene and SCCmec type I was most predominant among the MRS isolates.

  11. The prevalence of aminoglycoside-modifying enzyme genes (aac (6'-I, aac (6'-II, ant (2"-I, aph (3'-VI in Pseudomonas aeruginosa

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    Farzam Vaziri

    2011-01-01

    Full Text Available INTRODUCTION: Pseudomonas aeruginosa (P. aeruginosa is one of the primary opportunistic pathogens responsible for nosocomial infections. Aminoglycosides are an import ant component of antipseudomonal chemotherapy. The inactivation of drugs by modifying enzymes is the most common mechanism of aminoglycoside resistance. OBJECTIVES: The inactivation of aminoglycosides by modifying enzymes is the primary resistance mechanism employed by P. aeruginosa. The aim of the present study was to investigate the occurrence of aminoglycoside resistance and the prevalence of four import ant modifying enzyme genes (aac (6'-I, aac (6'-II, ant (2"-I, aph (3'-VI in P. aeruginosa in Iran. METHODS: A total of 250 clinical isolates of P. aeruginosa were collected from several hospitals in seven cities in Iran. Antimicrobial susceptibility tests (using the disk diffusion method and E-tests were performed for all 250 isolates. In addition, all isolates were screened for the presence of modifying enzyme genes by polymerase chain reaction. RESULTS: The resistance rates, as determined by the disk diffusion method, were as follows: gentamicin 43%, tobramycin 38%, and amikacin 24%. Of the genes examined, aac (6'-II (36% was the most frequently identified gene in phenotypic resist ant isolates, followed by ant (2"-I, aph (3'-VI, and aac (6'-I. CONCLUSIONS: Aminoglycoside resistance in P. aeruginosa remains a signific ant problem in Iran. Therefore, there is considerable local surveillance of aminoglycoside resistance.

  12. The prevalence of aminoglycoside-modifying enzyme genes (aac (6′)-I, aac (6′)-II, ant (2″)-I, aph (3′)-VI) in Pseudomonas aeruginosa

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    Farzam Vaziri; Shahin Najar Peerayeh; Qorban Behzadian Nejad; Abbas Farhadian

    2011-01-01

    INTRODUCTION: Pseudomonas aeruginosa (P. aeruginosa) is one of the primary opportunistic pathogens responsible for nosocomial infections. Aminoglycosides are an import ant component of antipseudomonal chemotherapy. The inactivation of drugs by modifying enzymes is the most common mechanism of aminoglycoside resistance. OBJECTIVES: The inactivation of aminoglycosides by modifying enzymes is the primary resistance mechanism employed by P. aeruginosa. The aim of the present study was to investig...

  13. Molecular identification of aminoglycoside-modifying enzymes in clinical isolates of Escherichia coli resistant to amoxicillin/clavulanic acid isolated in Spain.

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    Fernández-Martínez, Marta; Miró, Elisenda; Ortega, Adriana; Bou, Germán; González-López, Juan José; Oliver, Antonio; Pascual, Alvaro; Cercenado, Emilia; Oteo, Jesús; Martínez-Martínez, Luis; Navarro, Ferran

    2015-08-01

    The activity of eight aminoglycosides (amikacin, apramycin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin and tobramycin) against a collection of 257 amoxicillin/clavulanic acid (AMC)-resistant Escherichia coli isolates was determined by microdilution. Aminoglycoside resistance rates, the prevalence of aminoglycoside-modifying enzyme (AME) genes, the relationship between AME gene detection and resistance phenotype to aminoglycosides, and the association of AME genes with mechanisms of AMC resistance in E. coli isolates in Spain were investigated. Aminoglycoside-resistant isolates were screened for the presence of genes encoding common AMEs [aac(3)-Ia, aac(3)-IIa, aac(3)-IVa, aac(6')-Ib, ant(2″)-Ia, ant(4')-IIa and aph(3')-Ia] or 16S rRNA methylases (armA, rmtB, rmtC and npmA). In total, 105 isolates (40.9%) were resistant to at least one of the aminoglycosides tested. Amikacin, apramycin and arbekacin showed better activity, with MIC90 values of 2mg/L (arbekacin) and 8mg/L (amikacin and apramycin). Kanamycin presented the highest MIC90 (128mg/L). The most common AME gene was aac(6')-Ib (36 strains; 34.3%), followed by aph(3')-Ia (31 strains; 29.5%), ant(2″)-Ia (29 strains; 27.6%) and aac(3)-IIa (23 strains; 21.9%). aac(3)-Ia, aac(3)-IVa, ant(4')-IIa and the four methylases were not detected. The ant(2″)-Ia gene was usually associated with OXA-1 [21/30; 70%], whilst 23/25 (92%) strains producing CTX-M-15 had the aac(6')-Ib gene. The most prevalent AME gene was aac(6')-Ib (18/41; 44%) in nosocomial isolates, whilst ant(2″)-Ia and aph(3')-Ia genes (20/64; 31%) were more frequent in strains of community origin. In 64.6% isolates the phenotypic profile correlated with the presence of commonly encountered AMEs.

  14. An aminoglycoside sensing riboswitch controls the expression of aminoglycoside resistance acetyltransferase and adenyltransferases.

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    Chen, Dongrong; Murchie, Alastair I H

    2014-10-01

    The emergence of antibiotic resistance in human pathogens is an increasing threat to public health. The fundamental mechanisms that control the high levels of expression of antibiotic resistance genes are not yet completely understood. The aminoglycosides are one of the earliest classes of antibiotics that were introduced in the 1940s. In the clinic aminoglycoside resistance is conferred most commonly through enzymatic modification of the drug although resistance through enzymatic modification of the target rRNA through methylation or the overexpression of efflux pumps is also appearing. An aminoglycoside sensing riboswitch has been identified that controls expression of the aminoglycoside resistance genes that encode the aminoglycoside acetyltransferase (AAC) and aminoglycoside nucleotidyltransferase (ANT) (adenyltransferase (AAD)) enzymes. AAC and ANT cause resistance to aminoglycoside antibiotics through modification of the drugs. Expression of the AAC and ANT resistance genes is regulated by aminoglycoside binding to the 5' leader RNA of the aac/aad genes. The aminoglycoside sensing RNA is also associated with the integron cassette system that captures antibiotic resistance genes. Specific aminoglycoside binding to the leader RNA induces a structural transition in the leader RNA, and consequently induction of resistance protein expression. Reporter gene expression, direct measurements of drug RNA binding, chemical probing and UV cross-linking combined with mutational analysis demonstrated that the leader RNA functioned as an aminoglycoside sensing riboswitch in which drug binding to the leader RNA leads to the induction of aminoglycoside antibiotic resistance. This article is part of a Special Issue entitled: Riboswitches. PMID:24631585

  15. DNA-Aptamers Binding Aminoglycoside Antibiotics

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    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. Pharmacokinetics of Aminoglycosides

    Institute of Scientific and Technical Information of China (English)

    Lokangu Lombo(Congo); HE Hua

    2004-01-01

    The Pharmacokinetics informations of aminoglycosides, their monograph and clinical Pharmacokinetics parameters are reported in this review. The Aminoglycosides are highly polarity and in reserve for serious infections caused by aerobic gram-negative bacteria and some gram-positive bacteria but their toxicity are major limitations in clinical use.

  17. Endotoxemia-mediated inflammation potentiates aminoglycoside-induced ototoxicity

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    Koo, J.-W.; Quintanilla-Dieck, L.; Jiang, M.; Liu, J.; Urdang, Z. D.; Allensworth, J. J.; Cross, C. P.; Li, H.; Steyger, P. S.

    2015-01-01

    The ototoxic aminoglycoside antibiotics are essential to treat severe bacterial infections, particularly in neonatal intensive care units. Using a bacterial lipopolysaccharide (LPS) experimental model of sepsis, we tested whether LPS-mediated inflammation potentiates cochlear uptake of aminoglycosides and permanent hearing loss in mice. Using confocal microscopy and enzyme-linked immunosorbent assays, we found that low-dose LPS (endotoxemia) greatly increased cochlear concentrations of aminoglycosides and resulted in vasodilation of cochlear capillaries without inducing paracellular flux across the blood-labyrinth barrier (BLB), or elevating serum concentrations of the drug. Additionally, endotoxemia increased expression of both serum and cochlear inflammatory markers. These LPS-induced changes, classically mediated by Toll-like Receptor 4 (TLR4), were attenuated in TLR4-hyporesponsive mice. Multiday dosing with aminoglycosides during chronic endotoxemia induced greater hearing threshold shifts and sensory cell loss compared to mice without endotoxemia. Thus, endotoxemia-mediated inflammation enhanced aminoglycoside trafficking across the BLB, and potentiated aminoglycoside-induced ototoxicity. These data indicate that patients with severe infections are at greater risk of aminoglycoside-induced hearing loss than previously recognized. PMID:26223301

  18. Genotyping of aminoglycoside-modifying enzymes in multidrug-resistant Pseudomonas aeruginosa%铜绿假单胞菌中氨基糖苷类基因的分布与耐药性研究

    Institute of Scientific and Technical Information of China (English)

    郑敏巧; 王焰兵; 陈剑锋; 丁玎; 黄恩佩; 曾爱平

    2012-01-01

    OBJECTIVE To investigate genotyping of aminoglycoside-modifying enzymes (AMEs) in clinical isolates of multidrug-resistant Pseudomonas aeruginosa (MDRPA). METHODS A total of 40 strains of MRPA were isolated from hospitalized patients from Sep 2010 to Nov 2010. The genes of aminoglycoside-modifying enzymes were detected by polymerase chain reaction(PCR)i the statistical analysis of drug susceptibility testing results was performed , and the relationship between the genes of AMEs and drug resistance was analyzed. RESULTS Among 40 strains of MPDRPA, the resistance genes were detected in 36 strains with the detection rate of 90. 0%,8 genotypes of aminoglycoside passivation genes were amplified with PCR, most of the strains produced aminoglycoside passivation gene, followed by ACC and ANT, the positive rates of acc3 Ⅱ , ant6Ⅰ , acc3 Ⅳ , acc3 Ⅰ , and ant(3") Ⅰ were 80. 0% , 55.0% , 25. 0%, 15.0%, and 7. 5% , respectively. CONCLUSION The positive rates of AME genes are very high in clinical isolates of MDRPA, there were at least 5 genotypes including aac(3) Ⅱ . ant(6)Ⅰ , acc(6') Ⅰ , acc(3) Ⅰ , and ant(3") Ⅰ in AMEs.%目的 了解医院临床分离的多药耐药铜绿假单胞菌(MDRPA)中氨基糖苷类修饰酶(AMEs)基因存在状况.方法 收集临床2010年9-11月分离的铜绿假单胞菌40株进行氨基糖苷类基因检测,采用PCR法检测;统计铜绿假单胞菌的药敏结果,并分析氨基糖苷类基因和耐药性之间的关系.结果 40株铜绿假单胞菌中36株检出耐药基因,检出率为90.0%,PCR扩增出8种氨基糖苷钝化酶基因,大部分菌株均产生氨基糖苷钝化酶,其次为乙酰转移酶(ACC)和核苷转移酶(ANT),acc3Ⅱ、ant6Ⅰ、acc3Ⅳ、acc3Ⅰ、ant(3")Ⅰ基因的阳性率分别为80.0%、55.0%、25.0%、15.0%及7.5%.结论 医院临床分离的多药耐药铜绿假单胞菌中AMEs基因携带率很高,至少存在5种AMEs基因,分别为aac(3)Ⅱ、ant(6)Ⅰ、acc(6′)Ⅰ、acc(3

  19. Study of aminoglycoside modifying enzyme associated resistance genes in Enterococcus%产氨基糖苷类修饰酶肠球菌临床分离株相关耐药基因的研究

    Institute of Scientific and Technical Information of China (English)

    姚杰; 徐元宏; 王友梅; 刘灿

    2011-01-01

    目的 了解临床分离的肠球菌对高水平氨基糖苷类抗菌药物的耐药情况并对其氨基糖苷类修饰酶基因进行检测.方法 用琼脂稀释法检测112株粪肠球菌和118株屎肠球菌对高浓度庆大霉素(500 μg/ml)和高浓度链霉素(2 000 μg/ml)的最低抑菌浓度,随机选取氨基糖苷类高水平耐药的粪肠球菌和屎肠球菌各48株进行氨基糖苷类修饰酶基因检测,PCR扩增aac(6')/aph(2″)、aph(3')-Ⅲ和ant(6)-Ⅰ基因,并对其进行测序分析.结果 112株粪肠球菌和118株屎肠球菌对高浓度庆大霉素的耐药率分别为53.6%和78.8%,对高浓度链霉素的耐药率分别为39.3%和64.4%,且粪肠球菌与屎肠球菌的耐药率差异有统计学意义(P<0.01).aac(6')/aph(2″)基因阳性分别为38株和45株,占80.2%和93.8%;aph(3')-Ⅲ基因阳性分别为14株和19株,占29.2%和39.6%;ant(6)-Ⅰ基因阳性分别为15株和21株,占31.3%和43.8%.其中同时检测出两种和两种以上氨基糖苷类修饰酶基因的菌株有40株,高达41.7%.结论 氨基糖苷类高水平耐药肠球菌已成为医院感染的重要耐药菌,AAC(6')/APH(2″)酶的产生是肠球菌最为常见的氨基糖苷类耐药机制.%Objective To investigate enterococci isolated from clinical specimens on the high level of aminoglycoside antibiotic resistance and the detection of aminoglycoside modifying enzyme gene. Methods 112 strains of E.faecalis and 118 strains of E. faecium to the high-level gentamicin(500 μg/ml)and high-level of streptomycin (2 000 μg/ml)minimum inhibitory concentration were detected by agar ditution method. The aac (6')/aph (2"),aph(3')-Ⅲ and ant(6)- Ⅰ genes were amplified by PCR. Finally,their genotype were determined by DNA sequencing. Results 112 strains of E. faecalis and 118 strains of E. faecium HLGR rates were 53. 1% and 73.2%, and HLSR rates were 38. 9% and 59. 8%. The drug resistance of the two species to high-level gentamicin and high

  20. Study on drug resistance of ESBLs-producing Klebsiella pneumoniae to aminoglycosides and genotypes of aminoglycoside modifying enzymes%产ESBLs肺炎克雷伯菌氨基糖苷类耐药性及其修饰酶基因型的研究

    Institute of Scientific and Technical Information of China (English)

    梁彩倩; 张永标; 杨晓燕; 冯亚群; 符永玫

    2012-01-01

    Objective: To explore the resistance of ESBLs - producing Klebsiella pneumoniae to aminoglycosides and the genotypes of aminoglycoside modifying enzymes ( AMEs). Methods; The susceptibility tests of amikacin, gentamicin, tobramycin and netilmicin were done by Kirby - Bauer diffusions method. PCR was used to amplify 6 kinds of AMEs genes, then the PCR positive products were sequenced to identify their genotypeSj Results; Among the 77 ESBLs - producing KPN strains, the resistant rates to amikacin, gentamicin, tobramycin and netilmicin were 22. 1% , 59.7% , 44.2% and 42.9% , respectively. The detection rates of aac(3) - Ⅱ , aac(6') -Ib, ant(3") -Ⅰ and ant(2") -Ⅰ genes were 49.4% , 35.1% , 22.1% and 6.5% respectively, but aac(3) -Ⅰ and aac(6') -Ⅱ gene were not found. Conclusion: ESBLs - producing KPN are highly resistant to aminoglycosides, which is closely related with AMEs. The prevalent principal genotypes are aac(3) - Ⅱ , aac(6') - Ib and ant(3") -Ⅰ genes in these isolates.%目的:了解产超广谱β-内酰胺酶(ESBLs)肺炎克雷伯菌对氨基糖苷类的耐药性,及其氨基糖苷类修饰酶(AMEs)基因型的流行状况.方法:采用K-B纸片法测定阿米卡星、庆大霉素、妥布霉素、奈替米星的敏感性,应用PCR方法扩增6种AMEs基因,并对PCR阳性产物进行测序以确定其基因型.结果:77株产ESBLs肺炎克雷伯菌对阿米卡星、庆大霉素、妥布霉素、奈替米星的耐药率分别为22.1%、59.7%、44.2%、42.9%,aac(3)-Ⅱ、aac (6′)-Ⅰb、ant(3")-Ⅰ、ant(2")-Ⅰ基因检出率分别为49.4%、35.1%、22.1%、6.5%,未检出aac(3)-Ⅰ和aac(6′)-Ⅱ基因.结论:产ESBLs肺炎克雷伯菌对氨基糖苷类高度耐药,其耐药性与AMEs密切相关,流行的AMEs基因型主要为aac(3)-Ⅱ、aac(6′)-Ⅰb和ant(3")-Ⅰ.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Identification of aminoglycoside resistance genes by Triplex PCR in Enterococcus spp. isolated from ICUs.

    Science.gov (United States)

    Mirnejad, Reza; Sajjadi, Nikta; Masoumi Zavaryani, Sara; Piranfar, Vahhab; Hajihosseini, Maryam; Roshanfekr, Maliheh

    2016-09-01

    Early detection of antibiotic-resistant enterococci is an important part of patient treatment. Therefore, the aim of the present study was to evaluate the resistance patterns and simultaneously identify and characterise the resistance genes in Enterococcus spp. using a triplex polymerase chain reaction (PCR) method. In all, 150 consecutive Enterococcus spp were collected from several hospitals in Tehran (Iran) from January to December 2015. The Enterococcus species were identified by standard phenotypic/biochemical tests and PCR. The antimicrobial resistance patterns were determined using a disk diffusion method. The triplex PCR method was designed to identify gentamicin and other aminoglycoside resistance genes. Among the 150 Enterococcus specimens, 87 cases (58%) were Enterococcus faecalis, and 63 cases (42%) were Enterococcus faecium. The highest frequency of resistance was observed for tetracycline while the lowest was found for vancomycin. Among the identified samples, 56.9% contained the aac(6')-Ie-aph(2'')-Ia gene, 22.2% contained the aph(3')-IIIa gene, and 38.8% contained the ant(4')-?a gene. Eight percent of the isolates contained the three aminoglycoside resistance genes. Data analysis showed that there was a significant correlation between the phenotypic gentamicin resistance and the presence of the aminoglycoside resistance genes (18.9%, p Enterococcus strains had increased aminoglycoside resistance. The direct correlation between resistance genes, such as the aminoglycoside resistance factor, and phenotypic resistance was not significant (p > 0.05).

  3. Study on genotype of enzymes associated with aminoglycosides resistance in Klebsiella pneumoniae%肺炎克雷伯菌氨基糖苷类耐药相关酶的基因型研究

    Institute of Scientific and Technical Information of China (English)

    梁彩倩; 张永标; 杨晓燕; 符永玫; 冯亚群

    2013-01-01

    目的:了解肺炎克雷伯菌中氨基糖苷类修饰酶(AMEs)和16S rRNA甲基化酶的基因型,及其对氨基糖苷类(AGs)耐药性的影响.方法:采用琼脂稀释法测定阿米卡星、庆大霉素、妥布霉素的最低抑菌浓度(MICs),应用PCR方法扩增AMEs基因aac (3)-Ⅱ,aac(6')-Ⅰb,ant(3")-Ⅰ,ant(2")-Ⅰ,aac (3)-Ⅰ,aac(6')-Ⅱ和16S rRNA甲基化酶基因armA,rmtA,rmtB,rmtC,rmtD,npmA,并对PCR阳性产物进行测序以确定基因型.结果:162株肺炎克雷伯菌中检测到aac (3)-Ⅱ,aac(6')-Ⅰb,ant(3")-Ⅰ,ant(2")-Ⅰ,armA,rmtB基因,阿米卡星、庆大霉素、妥布霉素对同时携带AMEs和16S rRNA甲基化酶基因菌株的MIC50 、MIC90均高于单纯携带AMEs基因菌株.结论:肺炎克雷伯菌中流行AMEs基因aac (3)-Ⅱ,aac(6')-Ⅰb,ant(3")-Ⅰ,ant(2")-Ⅰ和16S rRNA甲基化酶基因armA,rmtB,同时携带AMEs和16S rRNA甲基化酶基因菌株对AGs的耐药性比单纯携带AMEs基因菌株更为明显.%Objective: To explore the genotype of aminoglycoside modifying enzymes ( AMEs) and 16S rRNA methylases in Klebsiella pneumoniae, and the influence on aminoglycosides ( AGs) resistance. Methods: Minimal inhibitory concentrations ( MICs) of amikacin, gentamicin and tobramycin to K. pneumoniae were detected by agar dilution methods. PCR was used to amplify AMEs genes aac (3) -Ⅱ, aac (6′) -Ⅰb, ant (3") -Ⅰ, ant (2") -Ⅰ, aac (3) -Ⅰ, aac (6′) - Ⅱ and 16S rRNA mehtylases genes armA, rmtA, rmtB, rmtC, rmtD, npmA. The PCR positive products were sequenced to identify their genotype. Results: AMEs genes aac (3) - Ⅱ, aac (6′) - Ⅰb, ant (3") -Ⅰ, ant (2") - Ⅰ and 16S rRNA mehtylases genes armA and rmtB were detected among 162 strains of K. pneumoniae. MIC50 and MIC90 of amikacin, gentamicin and tobramycin for the strains harbouring both AMEs and 16S rRNA methylases genes were higher than that of the strains harbouring AMEs genes only. Conclusion: AMEs genes aac (3) - Ⅱ, aac (6′) - Ib, ant (3") -

  4. Study on epidemiologic characteristics of aminoglycoside modifying enzymes in Klebsiella pneumoniae%肺炎克雷伯菌中氨基糖苷类修饰酶基因流行特征的研究

    Institute of Scientific and Technical Information of China (English)

    梁彩倩; 张永标; 杨晓燕; 冯亚群; 符永玫

    2013-01-01

    目的 了解肺炎克雷伯菌中氨基糖苷类修饰酶(AMEs)基因的流行状况及AMEs与超广谱β-内酰胺酶(ESBLs)的关系,为防治肺炎克雷伯菌感染及指导临床合理用药提供参考.方法 收集2009年10月—2010年12月医院临床分离的无重复肺炎克雷伯菌162株,采用K-B纸片扩散法测定阿米卡星、庆大霉素、妥布霉素、奈替米星的敏感性,并进行ESBLs表型筛选与确证试验;应用PCR方法检测AMEs基因.结果 162株肺炎克雷伯菌中共检出aac(3)-Ⅱ、aac(6′)-Ⅰb 、ant(3″)-Ⅰ、ant(2″)-Ⅰ 4种AMEs基因,检出率分别为30.2%、19.8%、13.6%和4.3%,AMEs基因总检出率为38.3%,ESBLs检出率为47.5%;除阿米卡星外,产ESBLs菌株对庆大霉素、妥布霉素、奈替米星的耐药率高于非产ESBLs菌株(P<0.05);除ant(2″)-Ⅰ基因外,产ESBLs菌株aac(3)-Ⅱ、aac(6)-Ⅰb、ant(3″)-Ⅰ基因的检出率高于非产ESBLs菌株(P<0.05).结论 AMEs基因在肺炎克雷伯菌中广泛流行,并介导氨基糖苷类药物的耐药性,肺炎克雷伯菌常同时产生AMEs和ESBLs.%OBJECTIVE To explore the prevalence of aminoglycoside modifying enzymes (AMEs) genes in Klebsiella pneumoniae (KPN) and analyze the relationship between AMEs and extended spectrum β-1actamases (ESBLs) so as to provide basis for the prevention and treatment of K.pneumoniae infections and to guide the reasonable use of antibiotics.METHODS A total of 162 non-repetitive strains of K.pneumoniae isolated from Oct 2009 to Dec 2010 in the hospital were collected.The drug susceptibility testing of aminoglycosides (amikacin,gentamicin,tobramycin and netilmicin) and confirmatory tests for ESBLs were performed by Kirby-Bauer diffusions method.The AMEs genotypes of KPN were verified by PCR.RESULTS Among the 162 strains,aac(3)-Ⅱ,aac(6′)-Ⅰb,ant(3″)-Ⅰ and ant(2″)-Ⅰ genes were detected with detection rates of 30.2%,19.8%,13.6%,and 4.3%,respectively.The positive

  5. Structural and Molecular Basis for Resistance to Aminoglycoside Antibiotics by the Adenylyltransferase ANT(2″)-Ia

    OpenAIRE

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

    2015-01-01

    ABSTRACT   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...

  6. High Level Aminoglycoside Resistance and Distribution of Aminoglycoside Resistant Genes among Clinical Isolates of Enterococcus Species in Chennai, India

    Directory of Open Access Journals (Sweden)

    Elango Padmasini

    2014-01-01

    Full Text Available Enterococci are nosocomial pathogen with multiple-drug resistance by intrinsic and extrinsic mechanisms. Aminoglycosides along with cell wall inhibitors are given clinically for treating enterococcal infections. 178 enterococcal isolates were analyzed in this study. E. faecalis is identified to be the predominant Enterococcus species, along with E. faecium, E. avium, E. hirae, E. durans, E. dispar and E. gallinarum. High level aminoglycoside resistance (HLAR by MIC for gentamicin (GM, streptomycin (SM and both (GM + SM antibiotics was found to be 42.7%, 29.8%, and 21.9%, respectively. Detection of aminoglycoside modifying enzyme encoding genes (AME in enterococci was identified by multiplex PCR for aac(6′-Ie-aph(2′′-Ia; aph(2′′-Ib; aph(2′′-Ic; aph(2′′-Id and aph(3′-IIIa genes. 38.2% isolates carried aac(6′-Ie-aph(2′′-Ia gene and 40.4% isolates carried aph(3′-IIIa gene. aph(2′′-Ib; aph(2′′-Ic; aph(2′′-Id were not detected among our study isolates. aac(6′-Ie-aph(2′′-Ia and aph(3′-IIIa genes were also observed in HLAR E. durans, E. avium, E. hirae, and E. gallinarum isolates. This indicates that high level aminoglycoside resistance genes are widely disseminated among isolates of enterococci from Chennai.

  7. High level aminoglycoside resistance and distribution of aminoglycoside resistant genes among clinical isolates of Enterococcus species in Chennai, India.

    Science.gov (United States)

    Padmasini, Elango; Padmaraj, R; Ramesh, S Srivani

    2014-01-01

    Enterococci are nosocomial pathogen with multiple-drug resistance by intrinsic and extrinsic mechanisms. Aminoglycosides along with cell wall inhibitors are given clinically for treating enterococcal infections. 178 enterococcal isolates were analyzed in this study. E. faecalis is identified to be the predominant Enterococcus species, along with E. faecium, E. avium, E. hirae, E. durans, E. dispar and E. gallinarum. High level aminoglycoside resistance (HLAR) by MIC for gentamicin (GM), streptomycin (SM) and both (GM + SM) antibiotics was found to be 42.7%, 29.8%, and 21.9%, respectively. Detection of aminoglycoside modifying enzyme encoding genes (AME) in enterococci was identified by multiplex PCR for aac(6')-Ie-aph(2'')-Ia; aph(2'')-Ib; aph(2'')-Ic; aph(2'')-Id and aph(3')-IIIa genes. 38.2% isolates carried aac(6')-Ie-aph(2'')-Ia gene and 40.4% isolates carried aph(3')-IIIa gene. aph(2'')-Ib; aph(2'')-Ic; aph(2'')-Id were not detected among our study isolates. aac(6')-Ie-aph(2'')-Ia and aph(3')-IIIa genes were also observed in HLAR E. durans, E. avium, E. hirae, and E. gallinarum isolates. This indicates that high level aminoglycoside resistance genes are widely disseminated among isolates of enterococci from Chennai.

  8. Antibiotic-Resistant Bacteria Detected in Sewage Spill

    Science.gov (United States)

    ... medlineplus.gov/news/fullstory_160031.html Antibiotic-Resistant Bacteria Detected in Sewage Spill 'People need to be ... News) -- Sewer line breaks can release antibiotic-resistant bacteria that pose a public health threat, a new ...

  9. Crystal structures of antibiotic-bound complexes of aminoglycoside 2''-phosphotransferase IVa highlight the diversity in substrate binding modes among aminoglycoside kinases.

    Science.gov (United States)

    Shi, Kun; Houston, Douglas R; Berghuis, Albert M

    2011-07-19

    Aminoglycoside 2''-phosphotransferase IVa [APH(2'')-IVa] is a member of a family of bacterial enzymes responsible for medically relevant resistance to antibiotics. APH(2'')-IVa confers high-level resistance against several clinically used aminoglycoside antibiotics in various pathogenic Enterococcus species by phosphorylating the drug, thereby preventing it from binding to its ribosomal target and producing a bactericidal effect. We describe here three crystal structures of APH(2'')-IVa, one in its apo form and two in complex with a bound antibiotic, tobramycin and kanamycin A. The apo structure was refined to a resolution of 2.05 Å, and the APH(2'')-IVa structures with tobramycin and kanamycin A bound were refined to resolutions of 1.80 and 2.15 Å, respectively. Comparison among the structures provides insight concerning the substrate selectivity of this enzyme. In particular, conformational changes upon substrate binding, involving rotational shifts of two distinct segments of the enzyme, are observed. These substrate-induced shifts may also rationalize the altered substrate preference of APH(2'')-IVa in comparison to those of other members of the APH(2'') subfamily, which are structurally closely related. Finally, analysis of the interactions between the enzyme and aminoglycoside reveals a distinct binding mode as compared to the intended ribosomal target. The differences in the pattern of interactions can be utilized as a structural basis for the development of improved aminoglycosides that are not susceptible to these resistance factors.

  10. Once-daily aminoglycoside therapy: potential ototoxicity.

    OpenAIRE

    Kirkpatrick, C. M.; Duffull, S. B.; Begg, E J

    1996-01-01

    Current data indicate that once-daily aminoglycoside therapy is as efficacious as traditional multiple daily dosing and equally or less toxic. Our experience with once-daily gentamicin, 6 mg/kg of body weight led to a 10% (3 of 33 patients) occurrence of documented ototoxicity after prolonged aminoglycoside exposure.

  11. 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.

  12. Structural basis for dual nucleotide selectivity of aminoglycoside 2''-phosphotransferase IVa provides insight on determinants of nucleotide specificity of aminoglycoside kinases.

    Science.gov (United States)

    Shi, Kun; Berghuis, Albert M

    2012-04-13

    Enzymatic phosphorylation through a family of enzymes called aminoglycoside O-phosphotransferases (APHs) is a major mechanism by which bacteria confer resistance to aminoglycoside antibiotics. Members of the APH(2″) subfamily are of particular clinical interest because of their prevalence in pathogenic strains and their broad substrate spectra. APH(2″) enzymes display differential preferences between ATP or GTP as the phosphate donor, with aminoglycoside 2″-phosphotransferase IVa (APH(2″)-IVa) being a member that utilizes both nucleotides at comparable efficiencies. We report here four crystal structures of APH(2″)-IVa, two of the wild type enzyme and two of single amino acid mutants, each in complex with either adenosine or guanosine. Together, these structures afford a detailed look at the nucleoside-binding site architecture for this enzyme and reveal key elements that confer dual nucleotide specificity, including a solvent network in the interior of the nucleoside-binding pocket and the conformation of an interdomain linker loop. Steady state kinetic studies, as well as sequence and structural comparisons with members of the APH(2″) subfamily and other aminoglycoside kinases, rationalize the different substrate preferences for these enzymes. Finally, despite poor overall sequence similarity and structural homology, analysis of the nucleoside-binding pocket of APH(2″)-IVa shows a striking resemblance to that of eukaryotic casein kinase 2 (CK2), which also exhibits dual nucleotide specificity. These results, in complement with the multitude of existing inhibitors against CK2, can serve as a structural basis for the design of nucleotide-competitive inhibitors against clinically relevant APH enzymes.

  13. Enzyme

    Science.gov (United States)

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  14. Mitochondrial DNA Mutations Associated with Aminoglycoside Ototoxicity

    Institute of Scientific and Technical Information of China (English)

    GUAN Min-Xin

    2006-01-01

    The mitochondrial 12S rRNA has been shown to be the hot spot for mutations associated with both aminoglycoside-induced and non-syndromic hearing loss. Of all the mutations, the homoplasmic A1555G and C1494T mutations at a highly conserved decoding region in the 12S rRNA have been associated with aminoglycoside-induced and non-syndromic hearing loss in many families worldwide. The A1555G or C1494T mutation is expected to form novel 1494C-G1555 or 1494U-A1555 base-pair at the highly conserved A-site of 12S rRNA. These transitions make the secondary structure of this RNA more closely resemble the corresponding region of bacterial 16S rRNA. Thus, the new U - A or G-C pair in 12S rRNA created by the C1494T or A1555G transition facilitates the binding of aminoglycosides, thereby accounting for the fact that the exposure to aminoglycosides can induce or worsen hearing loss in individuals carrying these mutations. Furthermore, the growth defect and impairment of mitochondrial translation were observed in cell lines carrying the A1555G or C1494T mutation in the presence of high concentration of aminoglycosides. In addition, nuclear modifier genes and mitochondrial haplotypes modulate the phenotypic manifestation of the A1555G and C1494T mutations. These observations provide the direct genetic and biochemical evidences that the A1555G or C1494T mutation is a pathogenic mtDNA mutation associated with aminoglycoside-induced and nonsyndromic hearing loss. Therefore, these data have been providing valuable information and technology to predict which individuals are at risk for ototoxicity, to improve the safety of aminoglycoside antibiotic therapy, and eventually to decrease the incidence of deafness.

  15. Aminoglycoside nephrotoxicity: modeling, simulation, and control.

    Science.gov (United States)

    Rougier, Florent; Claude, Daniel; Maurin, Michel; Sedoglavic, Alexandre; Ducher, Michel; Corvaisier, Stéphane; Jelliffe, Roger; Maire, Pascal

    2003-03-01

    The main constraints on the administration of aminoglycosides are the risks of nephrotoxicity and ototoxicity, which can lead to acute, renal, vestibular, and auditory toxicities. In the present study we focused on nephrotoxicity. No reliable predictor of nephrotoxicity has been found to date. We have developed a deterministic model which describes the pharmacokinetic behavior of aminoglycosides (with a two-compartment model), the kinetics of aminoglycoside accumulation in the renal cortex, the effects of aminoglycosides on renal cells, the resulting effects on renal function by tubuloglomerular feedback, and the resulting effects on serum creatinine concentrations. The pharmacokinetic parameter values were estimated by use of the NPEM program. The estimated pharmacodynamic parameter values were obtained after minimization of the least-squares objective function between the measured and the calculated serum creatinine concentrations. A simulation program assessed the influences of the dosage regimens on the occurrence of nephrotoxicity. We have also demonstrated the relevancy of modeling of the circadian rhythm of the renal function. We have shown the ability of the model to fit with 49 observed serum creatinine concentrations for a group of eight patients treated for endocarditis by comparison with 49 calculated serum creatinine concentrations (r(2) = 0.988; P < 0.001). We have found that for the same daily dose, the nephrotoxicity observed with a thrice-daily administration schedule appears more rapidly, induces a greater decrease in renal function, and is more prolonged than those that occur with less frequent administration schedules (for example, once-daily administration). Moreover, for once-daily administration, we have demonstrated that the time of day of administration can influence the incidence of aminoglycoside nephrotoxicity. The lowest level of nephrotoxicity was observed when aminoglycosides were administered at 1:30 p.m. Clinical application of this

  16. Bacteriophage biosensors for antibiotic-resistant bacteria.

    Science.gov (United States)

    Sorokulova, Irina; Olsen, Eric; Vodyanoy, Vitaly

    2014-03-01

    An increasing number of disease-causing bacteria are resistant to one or more anti-bacterial drugs utilized for therapy. Early and speedy detection of these pathogens is therefore very important. Traditional pathogen detection techniques, that include microbiological and biochemical assays are long and labor-intensive, while antibody or DNA-based methods require substantial sample preparation and purification. Biosensors based on bacteriophages have demonstrated remarkable potential to surmount these restrictions and to offer rapid, efficient and sensitive detection technique for antibiotic-resistant bacteria.

  17. Antibiotic Binding Drives Catalytic Activation of Aminoglycoside Kinase APH(2″)-Ia.

    Science.gov (United States)

    Caldwell, Shane J; Huang, Yue; Berghuis, Albert M

    2016-06-01

    APH(2″)-Ia is a widely disseminated resistance factor frequently found in clinical isolates of Staphylococcus aureus and pathogenic enterococci, where it is constitutively expressed. APH(2″)-Ia confers high-level resistance to gentamicin and related aminoglycosides through phosphorylation of the antibiotic using guanosine triphosphate (GTP) as phosphate donor. We have determined crystal structures of the APH(2″)-Ia in complex with GTP analogs, guanosine diphosphate, and aminoglycosides. These structures collectively demonstrate that aminoglycoside binding to the GTP-bound kinase drives conformational changes that bring distant regions of the protein into contact. These changes in turn drive a switch of the triphosphate cofactor from an inactive, stabilized conformation to a catalytically competent active conformation. This switch has not been previously reported for antibiotic kinases or for the structurally related eukaryotic protein kinases. This catalytic triphosphate switch presents a means by which the enzyme can curtail wasteful hydrolysis of GTP in the absence of aminoglycosides, providing an evolutionary advantage to this enzyme.

  18. [PK/PD modeling of aminoglycoside nephrotoxicity].

    Science.gov (United States)

    Rougier, F; Corvaisier, S; Ducher, M; Claude, D; Jelliffe, R W; Maire, P

    2003-06-01

    Aminoglycosides are bactericidial antibiotics with a serum concentration-dependent activity. They are mainly eliminated by the kidneys and the main difficulty arising in clinical use is their uptake by the renal cortex which leads to nephrotoxicity. An ototoxicity is also reported. We propose a PK/PD modelling of aminoglycoside nephrotoxicity which unifies more fourty years of physiological knowledge. This deterministic model successively describes the pharmacokinetics of aminoglycosides, their storage into renal cortex, their effect on renal cells, their consequences on the renal function through tubuloglomerular feedback and the changes in the serum concentrations of creatinine that is considered as a toxicity marker. The simulation of the model displays the leading effect of the shape and daily-time of administration schedule on the search for minimizing toxicity.

  19. Aminoglycoside resistance rates, phenotypes, and mechanisms of Gram-negative bacteria from infected patients in upper Egypt.

    Directory of Open Access Journals (Sweden)

    Gamal F Gad

    Full Text Available With the re-emergence of older antibiotics as valuable choices for treatment of serious infections, we studied the aminoglycoside resistance of Gram-negative bacteria isolated from patients with ear, urinary tract, skin, and gastrointestinal tract infections at Minia university hospital in Egypt. Escherichia coli (mainly from urinary tract and gastrointestinal tract infections was the most prevalent isolate (28.57%, followed by Pseudomonas aeruginosa (25.7% (mainly from ear discharge and skin infections. Isolates exhibited maximal resistance against streptomycin (83.4%, and minimal resistance against amikacin (17.7% and intermediate degrees of resistance against neomycin, kanamycin, gentamicin, and tobramycin. Resistance to older aminoglycosides was higher than newer aminoglycosides. The most common aminoglycoside resistance phenotype was that of streptomycin resistance, present as a single phenotype or in combination, followed by kanamycin-neomycin as determined by interpretative reading. The resistant Pseudomonas aeruginosa strains were capable of producing aminoglycoside-modifying enzymes and using efflux as mechanisms of resistance. Using checkerboard titration method, the most frequently-observed outcome in combinations of aminoglycosides with β-lactams or quinolones was synergism. The most effective combination was amikacin with ciprofloxacin (100% Synergism, whereas the least effective combination was gentamicin with amoxicillin (53.3% Synergistic, 26.7% additive, and 20% indifferent FIC indices. Whereas the studied combinations were additive and indifferent against few of the tested strains, antagonism was never observed. The high resistance rates to aminoglycosides exhibited by Gram-negative bacteria in this study could be attributed to the selective pressure of aminoglycoside usage which could be controlled by successful implementation of infection control measures.

  20. 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

    2015-01-01

    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

  1. 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

    2015-01-01

    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

  2. Audiological Management of Patients Receiving Aminoglycoside Antibiotics

    Science.gov (United States)

    Konrad-Martin, Dawn; Wilmington, Debra J.; Gordon, Jane S.; Reavis, Kelly M.; Fausti, Stephen A.

    2005-01-01

    Aminoglycoside antibiotics, commonly prescribed for adults and children to treat a wide range of bacterial infections, are potentially ototoxic, often causing irreversible damage to the auditory and vestibular systems. Ototoxic hearing loss usually begins at the higher frequencies and can progress to lower frequencies necessary for understanding…

  3. Physiological and Molecular Pathology of Aminoglycoside Ototoxicity

    Science.gov (United States)

    Sha, Su-Hua

    2005-01-01

    The problem of aminoglycoside-induced ototoxicity, which was recognized within a year of the discovery of streptomycin to combat tuberculosis in 1944, is still of great concern due to the widespread use of these powerful antibacterial agents. These drugs can damage to varying degrees the cochlea and vestibular system. Their primary targets are the…

  4. Incidence of antimicrobial-resistance genes and integrons in antibiotic-resistant bacteria isolated from eels and aquaculture ponds.

    Science.gov (United States)

    Lin, Mao; Wu, Xiaomei; Yan, Qingpi; Ma, Ying; Huang, Lixing; Qin, Yingxue; Xu, Xiaojin

    2016-07-01

    The overuse of antimicrobials in aquaculture has promoted the selection of antimicrobial-resistant bacteria. Here we investigated the abundance of antimicrobial-resistance genes and integrons in 108 strains of antibiotic-resistant bacteria isolated from eels and aquaculture ponds in China. Conventional PCR was implemented to examine common antibiotic-resistance genes, integrons, and their gene cassette arrays. The results showed that the antibiotic-resistance genes blaTEM, tetC, sulI, aadA, floR, and qnrB were detected at high percentages, as were a number of other resistance genes. Class I integrons were present in 79.63% of the strains, and 10 out of 108 isolates carried class II integrons. Class III integrons were not detected. Three strains carried both class I and class II integrons, and 73.26% of the class I integron-positive isolates contained the qacEΔ1/sul1 gene. Fourteen types of integron cassette arrays were found among class I integron-positive isolates. A new array, dfrB4-catB3-blaOXA-10-aadA1, was discovered in this study. The gene cassette array dfrA12-orfF-aadA2 was the most widely distributed. In summary, 23 different gene cassettes encoding resistance to 8 classes of antibiotics were identified in the class I integrons, and the main cassettes contained genes encoding resistance to aminoglycosides (aad) and trimethoprim (dfr). All class II integron-positive strains had only a single gene cassette array, viz. dfrA1-catB2-sat2-aadA1. High levels of antimicrobial-resistance genes and integrons in eels and auqauculture ponds suggest that the overuse of antimicrobials should be strictly controlled and that the levels of bacterial antimicrobial-resistance genes in aquaculture should be monitored. PMID:27409235

  5. Antibiotic-Resistant Vibrios in Farmed Shrimp

    Directory of Open Access Journals (Sweden)

    Renata Albuquerque Costa

    2015-01-01

    Full Text Available Antimicrobial susceptibility pattern was determined in 100 strains of Vibrio isolated from the Litopenaeus vannamei shrimp and identified phenotypically. A high antibiotic-resistance index (75% was observed, with the following phenotypic profiles: monoresistance (n=42, cross-resistance to β-lactams (n=20 and multiple resistance (n=13. Plasmid resistance was characterized for penicillin (n=11, penicillin + ampicillin (n = 1, penicillin + aztreonam (n = 1, and ampicillin (n = 1. Resistance to antimicrobial drugs by the other strains (n=86 was possibly mediated by chromosomal genes. The findings of this study support the conclusion that the cultured shrimps can be vehicles of vibrios resistant to β-lactam and tetracycline.

  6. Study of Klebsiella pneumoniae producing extended-spectrum β-lactamases against aminoglycosides

    Institute of Scientific and Technical Information of China (English)

    WEI FENG SHI; SU JIAN WANG; JIAN PING QIN

    2007-01-01

    Klebsiella pneumoniae ( K. pneumoniae) is one of the main gram-negative bacilli in clinical practice. Nosocomial infections caused by K. pneumoniae producing extended-spectrum β-lactamases (ESBLs) are very difficult to treat. This paper investigated the resistant characteristics of K. pneumoniae producing ESBLs and their aminoglycoside-modifying enzyme gene expressions including Nacetyltransferases and O-adenyhransferases. Bacteria identification and ESBLs confirmatory tests were performed by Phoenix TM-100 system. And minimum inhibitory concentrations (MICs) of gentamicin,amikacin, kanamycin, tobramycin, netilmicin and neomycin in 53 K. pneumoniae isolates were detected by agar dilution. In addition, six aminoglycoside-modifying enzyme genes were amplified by polymerase chain reaction (PCR) and verified by DNA sequencer. It was found that imipenem and meropenem against 120 K. pneumoniae isolates produced powerful antimicrobial activities. The resistant rates of gentamicin and amikacin were 55.0% and 46.7%, respectively. Except neomycin,MIC50 and MIC90 of gentamicin, amikacin, kanamycin, tobramycin and netilmicin in 53 K. pneumoniae were all > 128 μg/ml, and the resistant rates were 83.0%, 52.3%, 75.5%, 81. 1% and 69.8%, respectively. However, neomycin was only 39.6%. In addition, five modifying enzyme genes, including aac(3)- Ⅰ , aac(3)-Ⅱ, aac(6′) - Ⅰ b, ant(3″) - Ⅰ, ant(2″) - Ⅰ genes, were found in 53 isoahes except aac (6′)-Ⅱ, and their positive rates were 11.3%, 67.9%, 47.2%,1.9 % and 39.6 %, respectively. It was also confirmed by nucleotide sequence analysis that the above resistant genes shared nearly 100% identities with GenBank published genes. The results obtained in the present study indicated that K. pneumoniae producing ESBLs strains are rapidly spreading in our hospital, and their resistance to aminoglycosides may be associated with aminoglycoside-modifying enzyme gene expressions.

  7. Synthesis of 4′-aminopantetheine and derivatives to probe aminoglycoside N-6′-acetyltransferase

    OpenAIRE

    Yan, Xuxu; Akinnusi, T. Olukayode; Larsen, Aaron T.; Auclair, Karine

    2011-01-01

    A convenient synthesis of 4′-aminopantetheine from commercial D-pantethine is reported. The amino group was introduced by reductive amination in order to avoid substitution at a sterically congested position. Derivatives of 4′-aminopantetheine were also prepared to evaluate the effect of O-to-N substitution on inhibitors of the resistance-causing enzyme aminoglycoside N-6′-acetyltransferase. The biological results combined with docking studies indicate that in spite of its reported unusual fl...

  8. Synergistic ototoxicity due to noise exposure and aminoglycoside antibiotics.

    Science.gov (United States)

    Li, Hongzhe; Steyger, Peter S

    2009-01-01

    Acoustic exposure to high intensity and/or prolonged noise causes temporary or permanent threshold shifts in auditory perception, reflected by reversible or irreversible damage in the cochlea. Aminoglycoside antibiotics, used for treating or preventing life-threatening bacterial infections, also induce cytotoxicity in the cochlea. Combined noise and aminoglycoside exposure, particularly in neonatal intensive care units, can lead to auditory threshold shifts greater than simple summation of the two insults. The synergistic toxicity of acoustic exposure and aminoglycoside antibiotics is not limited to simultaneous exposures. Prior acoustic insult which does not result in permanent threshold shifts potentiates aminoglycoside ototoxicity. In addition, exposure to subdamaging doses of aminoglycosides aggravates noise-induced cochlear damage. The mechanisms by which aminoglycosides cause auditory dysfunction are still being unraveled, but likely include the following: 1) penetration into the endolymphatic fluid of the scala media, 2) permeation of nonselective cation channels on the apical surface of hair cells, and 3) generation of toxic reactive oxygen species and interference with other cellular pathways. Here we discuss the effect of combined noise and aminoglycoside exposure to identify pivotal synergistic events that can potentiate ototoxicity, in addition to a current understanding of aminoglycoside trafficking within the cochlea. Preventing the ototoxic synergy of noise and aminoglycosides is best achieved by using non-ototoxic bactericidal drugs, and by attenuating perceived noise intensity when life-saving aminoglycoside therapy is required.

  9. DNA-Aptamers Binding Aminoglycoside Antibiotics

    OpenAIRE

    Nadia Nikolaus; Beate Strehlitz

    2014-01-01

    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 aminog...

  10. 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.

  11. Probing minority population of antibiotic-resistant bacteria.

    Science.gov (United States)

    Huang, Tianxun; Zheng, Yan; Yan, Ya; Yang, Lingling; Yao, Yihui; Zheng, Jiaxin; Wu, Lina; Wang, Xu; Chen, Yuqing; Xing, Jinchun; Yan, Xiaomei

    2016-06-15

    The evolution and spread of antibiotic-resistant pathogens has become a major threat to public health. Advanced tools are urgently needed to quickly diagnose antibiotic-resistant infections to initiate appropriate treatment. Here we report the development of a highly sensitive flow cytometric method to probe minority population of antibiotic-resistant bacteria via single cell detection. Monoclonal antibody against TEM-1 β-lactamase and Alexa Fluor 488-conjugated secondary antibody were used to selectively label resistant bacteria green, and nucleic acid dye SYTO 62 was used to stain all the bacteria red. A laboratory-built high sensitivity flow cytometer (HSFCM) was applied to simultaneously detect the side scatter and dual-color fluorescence signals of single bacteria. By using E. coli JM109/pUC19 and E. coli JM109 as the model systems for antibiotic-resistant and antibiotic-susceptible bacteria, respectively, as low as 0.1% of antibiotic-resistant bacteria were accurately quantified. By monitoring the dynamic population change of a bacterial culture with the administration of antibiotics, we confirmed that under the antimicrobial pressure, the original low population of antibiotic-resistant bacteria outcompeted susceptible strains and became the dominant population after 5hours of growth. Detection of antibiotic-resistant infection in clinical urine samples was achieved without cultivation, and the bacterial load of susceptible and resistant strains can be faithfully quantified. Overall, the HSFCM-based quantitative method provides a powerful tool for the fundamental studies of antibiotic resistance and holds the potential to provide rapid and precise guidance in clinical therapies. PMID:26852201

  12. Synthesis of 4′-aminopantetheine and derivatives to probe aminoglycoside N-6′-acetyltransferase

    Science.gov (United States)

    Yan, Xuxu; Akinnusi, T. Olukayode; Larsen, Aaron T.; Auclair, Karine

    2011-01-01

    Summary A convenient synthesis of 4′-aminopantetheine from commercial D-pantethine is reported. The amino group was introduced by reductive amination in order to avoid substitution at a sterically congested position. Derivatives of 4′-aminopantetheine were also prepared to evaluate the effect of O-to-N substitution on inhibitors of the resistance-causing enzyme aminoglycoside N-6′-acetyltransferase. The biological results combined with docking studies indicate that in spite of its reported unusual flexibility and ability to adopt different folds, this enzyme is highly specific for AcCoA. PMID:21225062

  13. Synthesis of 4'-aminopantetheine and derivatives to probe aminoglycoside N-6'-acetyltransferase.

    Science.gov (United States)

    Yan, Xuxu; Akinnusi, T Olukayode; Larsen, Aaron T; Auclair, Karine

    2011-03-01

    A convenient synthesis of 4'-aminopantetheine from commercial D-pantethine is reported. The amino group was introduced by reductive amination in order to avoid substitution at a sterically congested position. Derivatives of 4'-aminopantetheine were also prepared to evaluate the effect of O-to-N substitution on inhibitors of the resistance-causing enzyme aminoglycoside N-6'-acetyltransferase. The biological results combined with docking studies indicate that in spite of its reported unusual flexibility and ability to adopt different folds, this enzyme is highly specific for AcCoA. PMID:21225062

  14. 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......, which are thought to be a source of extracellular DNA at sites of infections, increases the tolerance of P. aeruginosa biofilms toward aminoglycosides. Although biofilm-associated aminoglycoside tolerance recently has been linked to extracellular DNA-mediated activation of the pmr genes, we demonstrate...

  15. Aminoglycosides resistance in clinical isolates of Staphylococcus aureus from a University Hospital in Bialystok, Poland.

    Directory of Open Access Journals (Sweden)

    Katarzyna Kaczyńska

    2008-06-01

    Full Text Available Staphylococcus aureus obtained from a University Hospital in Poland were characterized in relation to resistance to aminoglycoside antibiotics and the distribution of the genes encoding the most clinically relevant aminoglycoside modifying enzymes (AMEs. Of a total of 118 S. aureus, 45 (38.1% isolates were found to be resistant to at least one of the tested antibiotics. All aminoglycoside resistant isolates except one 44 (97.8% were resistant to kanamycin. The majority of strains 37 (82.2% and 32 (71.1% expressed resistance to neomycin and tobramycin, respectively. Eleven strains (24.4% were resistant to gentamicin or amikacin. All S. aureus strains were sensitive to netilmicin. The most prevalent resistance gene was aac(6'-Ie+aph(2' found in 13 (28.9% strains and 12 (26.7% isolates carried ant(4'-Ia gene, whilst aph(3'-IIIa gene was detected in only 7 (15.6% isolates. Additionally, the ant(6-Ia and str genes were detected in 14 (31.1% and 2 (4.4% strains, respectively. Ten (22.2% strains resistant to amikacin, tobramycin, kanamycin or neomycin did not harbor any of the above-noted genes.

  16. Worldwide Disseminated armA Aminoglycoside Resistance Methylase Gene Is Borne by Composite Transposon Tn1548

    OpenAIRE

    Galimand, M.; Sabtcheva, S.; Courvalin, P; Lambert, T.

    2005-01-01

    The armA (aminoglycoside resistance methylase) gene, which confers resistance to 4,6-disubstituted deoxystreptamines and fortimicin, was initially found in Klebsiella pneumoniae BM4536 on IncL/M plasmid pIP1204 of ca. 90 kb which also encodes the extended-spectrum β-lactamase CTX-M-3. Thirty-four enterobacteria from various countries that were likely to produce a CTX-M enzyme since they were more resistant to cefotaxime than to ceftazidime were studied. The armA gene was detected in 12 clinic...

  17. 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...

  18. 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.

  19. Resurrecting the intestinal microbiota to combat antibiotic-resistant pathogens.

    Science.gov (United States)

    Pamer, Eric G

    2016-04-29

    The intestinal microbiota, which is composed of diverse populations of commensal bacterial species, provides resistance against colonization and invasion by pathogens. Antibiotic treatment can damage the intestinal microbiota and, paradoxically, increase susceptibility to infections. Reestablishing microbiota-mediated colonization resistance after antibiotic treatment could markedly reduce infections, particularly those caused by antibiotic-resistant bacteria. Ongoing studies are identifying commensal bacterial species that can be developed into next-generation probiotics to reestablish or enhance colonization resistance. These live medicines are at various stages of discovery, testing, and production and are being subjected to existing regulatory gauntlets for eventual introduction into clinical practice. The development of next-generation probiotics to reestablish colonization resistance and eliminate potential pathogens from the gut is warranted and will reduce health care-associated infections caused by highly antibiotic-resistant bacteria. PMID:27126035

  20. High throughput LSPR and SERS analysis of aminoglycoside antibiotics.

    Science.gov (United States)

    McKeating, Kristy S; Couture, Maxime; Dinel, Marie-Pier; Garneau-Tsodikova, Sylvie; Masson, Jean-Francois

    2016-08-15

    Aminoglycoside antibiotics are used in the treatment of infections caused by Gram-negative bacteria, and are often dispensed only in severe cases due to their adverse side effects. Patients undergoing treatment with these antibiotics are therefore commonly subjected to therapeutic drug monitoring (TDM) to ensure a safe and effective personalised dosage. The ability to detect these antibiotics in a rapid and sensitive manner in human fluids is therefore of the utmost importance in order to provide effective monitoring of these drugs, which could potentially allow for a more widespread use of this class of antibiotics. Herein, we report on the detection of various aminoglycosides, by exploiting their ability to aggregate gold nanoparticles. The number and position of the amino groups of aminoglycoside antibiotics controlled the aggregation process. We investigated the complementary techniques of surface enhanced Raman spectroscopy (SERS) and localized surface plasmon resonance (LSPR) for dual detection of these aminoglycoside antibiotics and performed an in-depth study of the feasibility of carrying out TDM of tobramycin using a platform amenable to high throughput analysis. Herein, we also demonstrate dual detection of tobramycin using both LSPR and SERS in a single platform and within the clinically relevant concentration range needed for TDM of this particular aminoglycoside. Additionally we provide evidence that tobramycin can be detected in spiked human serum using only functionalised nanoparticles and SERS analysis. PMID:27412506

  1. 阴沟肠杆菌16S rRNA甲基化酶基因及氨基糖苷类修饰酶基因研究%Analysis on 16S rRNA methylase genes and aminoglycoside modifying enzymes genes in Enterobacter cloacae in China

    Institute of Scientific and Technical Information of China (English)

    黄支密; 单浩; 糜祖煌; 杨海燕; 仵蕾; 储秋菊; 秦玲

    2008-01-01

    目的 了解临床分离的40株阴沟肠杆菌中16S rRNA甲基化酶基因及氨基糖苷类修饰酶(AMEs)基因存在状况.方法 在2003年9月至2004年11月从解放军第98医院住院患者中分离40株阴沟肠杆菌,采用聚合酶链反应(PCR)及序列分析的方法分析5种16S rRNA甲基化酶基因(armA、rmtA、rmtB、rmtC和rmtD)和9种AMEs基因[aac(3)-Ⅰ、aac(3)-Ⅱ、aac(3)-Ⅲ、aac(3)-Ⅳ、aac(6')-Ⅰ b、aac(6')-Ⅱ、ant(3'')-Ⅰ、ant(2'')-Ⅰ和aph(3')-Ⅵ].结果 40株阴沟肠杆菌中,6种基因rmtB、aac(3)-Ⅱ、aac(6')-Ⅰ b、ant(3'')-Ⅰ、ant(2'')-Ⅰ和aph(3')-Ⅵ的阳性株数分别为5株(12.5%)、11株(27.5%)、29株(72.5%)、13株(32.5%)、2株(5.0%)和2株(5.0%);其余8种基因均阴性;AMEs基因总阳性率为85.0%(34/40).对29株aac(6')-Ⅰ b基因PCR阳性产物进行测序,证实有7株(24.1%)单独携带aac(6')-Ⅰ b-cr双功能酶基因(GenBank:EF375620、EU159121),18株(62.1%)单独携带aac(6')-Ⅰ b-Snzhou(苏州型,EU085533),3株(10.3%)同时携带aac(6')-Ⅰ b-Suzhou及aac(6')-Ⅰ b-cr 2种亚型,仅1株(3.4%)为aac(6')-Ⅰ b经典型.结论 临床分离的40株阴沟肠杆菌中16S rRNA甲基化酶基因阳性率较低而AMEs基因阳性率很高,至少存在5种AMEs基因.%Objective To investigate the 16S rRNA methylase genes and Aminoglycoside modifying enzymes(AMEs)genes in Enterobacter cloacae isolated from the People's Liberation Army 98th Hospital,Huzhou district,Zhejiang province,China.Methods 40 strains of Enterobacter cloacae were isolated from the inpatients between September,2003 and November,2004.5 kinds of 16S rRNA methylase gene (including armA,rmtA,rmtB,rmtC and rmtD)and 9 kinds of AMEs gene[including aac(3)-Ⅰ,aac(3)-Ⅱ,aac(3)-Ⅲ,aac(3)-Ⅳ,aac(6')-Ⅰ b,aac(6')-Ⅱ,ant(3'')-Ⅰ,ant(2'')-Ⅰ and aph(3')-Ⅵ]were analyzed by PCR and verificated by DNA sequencing.Results In 40 strains of Enterobacter cloacae,the positive rates of genes of rmtB,aac(3)-Ⅱ,aac(6')-Ⅰ b,ant(3'')-

  2. Evaluation of Aminoglycoside and Non-Aminoglycoside Compounds for Stop-Codon Readthrough Therapy in Four Lysosomal Storage Diseases.

    Directory of Open Access Journals (Sweden)

    Marta Gómez-Grau

    Full Text Available Nonsense mutations are quite prevalent in inherited diseases. Readthrough drugs could provide a therapeutic option for any disease caused by this type of mutation. Geneticin (G418 and gentamicin were among the first to be described. Novel compounds have been generated, but only a few have shown improved results. PTC124 is the only compound to have reached clinical trials. Here we first investigated the readthrough effects of gentamicin on fibroblasts from one patient with Sanfilippo B, one with Sanfilippo C, and one with Maroteaux-Lamy. We found that ARSB activity (Maroteaux-Lamy case resulted in an increase of 2-3 folds and that the amount of this enzyme within the lysosomes was also increased, after treatment. Since the other two cases (Sanfilippo B and Sanfilippo C did not respond to gentamicin, the treatments were extended with the use of geneticin and five non-aminoglycoside (PTC124, RTC13, RTC14, BZ6 and BZ16 readthrough compounds (RTCs. No recovery was observed at the enzyme activity level. However, mRNA recovery was observed in both cases, nearly a two-fold increase for Sanfilippo B fibroblasts with G418 and around 1.5 fold increase for Sanfilippo C cells with RTC14 and PTC124. Afterwards, some of the products were assessed through in vitro analyses for seven mutations in genes responsible for those diseases and, also, for Niemann-Pick A/B. Using the coupled transcription/translation system (TNT, the best results were obtained for SMPD1 mutations with G418, reaching a 35% recovery at 0.25 μg/ml, for the p.W168X mutation. The use of COS cells transfected with mutant cDNAs gave positive results for most of the mutations with some of the drugs, although to a different extent. The higher enzyme activity recovery, of around two-fold increase, was found for gentamicin on the ARSB p.W146X mutation. Our results are promising and consistent with those of other groups. Further studies of novel compounds are necessary to find those with more

  3. 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 ...

  4. Cymbopogon citratus protects against the renal injury induced by toxic doses of aminoglycosides in rabbits

    Directory of Open Access Journals (Sweden)

    N Ullah

    2013-01-01

    Full Text Available Renal injury is the most common side-effect of aminoglycosides. These antimicrobial drugs are particularly effective against Gram-negative microorganisms. The present study was conducted to investigate the renal protective activity of Cymbopogon citratus in gentamicin-induced nephrotoxicity. Male rabbits were divided into four groups (n=6 including group 1 (0.9% saline treated, group 2 (80 mg/kg/day gentamicin-treated, group 3 (200 mg/kg/day Cymbopogon citratus treated and group 4 (80 mg/kg/day gentamicin and 200 mg/kg/day Cymbopogon citratus treated. Biochemical kidney functioning parameters, urinary enzymes and histopathological examination were performed. The results of the present study showed that simultaneous administration of Cymbopogon citrates and gentamicin significantly protected alteration in body weight, blood urea nitrogen, serum creatinine, creatinine clearance, serum uric acid, serum electrolytes, urinary volume, urinary protein, urinary lactate dehydrogenase and urinary alkaline phosphatase induced by gentamicin. Histological examination of the kidney also suggested the same. It is concluded from the current study that co-administration of Cymbopogon citratus with gentamicin for 3 weeks successfully prevented renal damage associated with aminoglycosides.

  5. Irrigation waters and pipe-based biofilms as sources for antibiotic-resistant bacteria

    Science.gov (United States)

    The presence of antibiotic-resistant bacteria in environmental surface waters has gained recent attention. Wastewater- and drinking water distribution systems are known to disseminate antibiotic-resistant bacteria, with the biofilms that form on the inner-surfaces of the pipeline as a hotspot for pr...

  6. In vitro read-through of phenylalanine hydroxylase (PAH) nonsense mutations using aminoglycosides: a potential therapy for phenylketonuria.

    Science.gov (United States)

    Ho, Gladys; Reichardt, Juergen; Christodoulou, John

    2013-11-01

    Phenylketonuria (PKU, OMIM 261600) is an autosomal recessive inborn error of phenylalanine metabolism, predominantly caused by mutations in the phenylalanine hydroxylase (PAH) gene. Approximately 10% of patients carry a nonsense mutation, which results in an inactive or unstable truncated protein. In some genetic disorders, including cystic fibrosis and Duchenne muscular dystrophy, restoration of full-length protein has been achieved by aminoglycoside antibiotics, such as gentamicin and G-418 (Geneticin). More recently, nonsense read-through has been induced at greater rates using a non-aminoglycoside drug, PTC124 (Ataluren), which has the advantage of being non-toxic in contrast to the antibiotics. The efficacy of read-through induced by three compounds, aminoglycosides G418 and gentamicin, and PTC124 were evaluated for four nonsense mutations of PAH in an in vitro expression system in two mammalian cell lines (COS-7 and HEK293). The production of full-length PAH was investigated using western blotting and the functionality confirmed by enzyme activity. Gentamicin and G-418 induced read-through of nonsense PAH mutations in HEK293 cells. The read-through product partially restored enzymatic activity, which was significantly less than that of wild-type, but comparable to a missense mutation of PAH associated with less severe forms of PKU. Treatment with PTC124 up to 100 μM did not result in full-length PAH polypeptide. Nonsense read-through drugs are a potential form of treatment for PKU, although the high dosage of aminoglycosides used is not appropriate in a clinical setting. In vitro studies with new non-toxic read-through agents as well as in vivo studies would also be essential to determine the extent of read-through required to restore normal phenylalanine levels. PMID:23532445

  7. 30S Subunit-Dependent Activation of the Sorangium cellulosum So ce56 Aminoglycoside Resistance-Conferring 16S rRNA Methyltransferase Kmr

    Science.gov (United States)

    Savic, Miloje; Sunita, S.; Zelinskaya, Natalia; Desai, Pooja M.; Macmaster, Rachel; Vinal, Kellie

    2015-01-01

    Methylation of bacterial 16S rRNA within the ribosomal decoding center confers exceptionally high resistance to aminoglycoside antibiotics. This resistance mechanism is exploited by aminoglycoside producers for self-protection while functionally equivalent methyltransferases have been acquired by human and animal pathogenic bacteria. Here, we report structural and functional analyses of the Sorangium cellulosum So ce56 aminoglycoside resistance-conferring methyltransferase Kmr. Our results demonstrate that Kmr is a 16S rRNA methyltransferase acting at residue A1408 to confer a canonical aminoglycoside resistance spectrum in Escherichia coli. Kmr possesses a class I methyltransferase core fold but with dramatic differences in the regions which augment this structure to confer substrate specificity in functionally related enzymes. Most strikingly, the region linking core β-strands 6 and 7, which forms part of the S-adenosyl-l-methionine (SAM) binding pocket and contributes to base flipping by the m1A1408 methyltransferase NpmA, is disordered in Kmr, correlating with an exceptionally weak affinity for SAM. Kmr is unexpectedly insensitive to substitutions of residues critical for activity of other 16S rRNA (A1408) methyltransferases and also to the effects of by-product inhibition by S-adenosylhomocysteine (SAH). Collectively, our results indicate that adoption of a catalytically competent Kmr conformation and binding of the obligatory cosubstrate SAM must be induced by interaction with the 30S subunit substrate. PMID:25733511

  8. Can chlorination co-select antibiotic-resistance genes?

    Science.gov (United States)

    Lin, Wenfang; Zhang, Menglu; Zhang, Shenghua; Yu, Xin

    2016-08-01

    Selective pressures, such as chemical or heavy metal pollution, may co-select for bacterial antibiotic resistance in the environment. However, whether chlorination in water treatment can co-select antibiotic-resistant bacteria is controversial. In this study, high capacity quantitative polymerase chain reaction (qPCR) analysis was applied to target almost all known antibiotic-resistance genes (ARGs) (282 types) and 13 mobile genetic elements (MGEs) in bacteria detected in secondary effluents from a municipal wastewater treatment plant after chlorination. The results revealed that 125 unique ARGs were detected in non-chlorinated samples, and the number decreased (79-91 types) as the chlorine concentration was increased. Moreover, 7.49 × 10(4)-3.92 × 10(7) copies/100 ml water reduction of ARGs occurred with 4 mg Cl2/l. Considering the relative abundance of ARGs (i.e., ARG copies normalized to 16S rRNA gene copies), 119 ARGs decreased in response to chlorination, whereas only six ARGs, such as dfrA1, tetPB-03, tetPA, ampC-04, tetA-02, and erm(36), were potentially enriched by 10.90-, 10.06-, 8.63-, 6.86-, 3.77-, and 1.09-fold, respectively. Furthermore, the relative abundance of 12 detected MGEs was lower after chlorination. Therefore, chlorination was effective in reducing ARGs and MGEs rather than co-selecting them. PMID:27192478

  9. Study of the aminoglycoside subsistence phenotype of bacteria residing in the gut of humans and zoo animals

    Directory of Open Access Journals (Sweden)

    Teresita De Jesus eBello Gonzalez

    2016-01-01

    Full Text Available Recent studies indicate that next to antibiotic resistance, bacteria are able to subsist on antibiotics as a carbon source. Here we evaluated the potential of gut bacteria from healthy human volunteers and zoo animals to subsist on antibiotics. Nine gut isolates of Escherichia coli and Cellulosimicrobium spp. displayed increases in colony forming units during incubations in minimal medium with only antibiotics added, i.e. the antibiotic subsistence phenotype. Furthermore, laboratory strains of E. coli and Pseudomonas putida equipped with the aminoglycoside 3’phosphotransferase II gene also displayed the subsistence phenotype on aminoglycosides. In order to address which endogenous genes could be involved in these subsistence phenotypes, the broad-range glycosyl-hydrolase inhibiting iminosugar deoxynojirimycin (DNJ was used. Addition of DNJ to minimal medium containing glucose showed initial growth retardation of resistant E. coli, which was rapidly recovered to normal growth. In contrast, addition of DNJ to minimal medium containing kanamycin arrested resistant E. coli growth, suggesting that glycosyl-hydrolases were involved in the subsistence phenotype. However, antibiotic degradation experiments showed no reduction in kanamycin, even though the number of colony forming units increased. Although antibiotic subsistence phenotypes are readily observed in bacterial species, and are even found in susceptible laboratory strains carrying standard resistance genes, we conclude there is a discrepancy between the observed antibiotic subsistence phenotype and actual antibiotic degradation. Based on these results we can hypothesise that aminoglycoside modifying enzymes might first inactivate the antibiotic (i.e. by acetylation of amino groups, modification of hydroxyl groups by adenylation and phosphorylation respectively, before the subsequent action of catabolic enzymes. Even though we do not dispute that antibiotics could be used as a single carbon

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

    OpenAIRE

    Shrestha, Sanjib K.; Cheng-Wei T Chang; Meissner, Nicole; Oblad, John; Shrestha, Jaya P.; Sorensen, Kevin N.; Michelle M. Grilley; Jon Y Takemoto

    2014-01-01

    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 de...

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

    OpenAIRE

    Shrestha, Sanjib K.; Cheng-Wei Tom Chang; Nicole eMeissner; John eOblad; Shrestha, Jaya P.; Sorensen, Kevin N.; Michelle M. Grilley; Jon Y Takemoto

    2014-01-01

    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 d...

  12. DETERMINATION OF AMINOGLYCOSIDES IN FOOD BY FLUORESCENCE POLARIZATION IMMUNOASSAY

    Directory of Open Access Journals (Sweden)

    FARAFONOVA O.V.

    2015-01-01

    Full Text Available The methodic for quantitative determination of aminoglycoside antibiotics (gentamicin, kanamycin, streptomycin, amikacin, neomycin in food by polarization fluorescent immunoassay (FPIA is developed. The size and structure influence of a fluorescent molecule on a fluorescence polarization degree is analyzed. Affinity constants of antibodies to compounds and tracers were estimated, optimized working concentration of tracers and antibodies that provide the maximum value of analytical signal. Methods were tested in the antibiotics identification in milk, eggs and chicken.

  13. Determination of aminoglycoside resistance in Staphylococcus aureus by DNA hybridization.

    OpenAIRE

    Dickgiesser, N; Kreiswirth, B N

    1986-01-01

    A method is described for identification of the genes conferring aminoglycoside resistance in Staphylococcus aureus by dot-blot and Southern blot techniques. As radioactive probes, fragments of plasmids pAT48, pUBH2, and pH13, carrying the genes for an aminocyclitol-3'-phosphotransferase, an aminocyclitol-4'-adenylyltransferase, and an aminocyclitol-2''-phosphotransferase-aminocyclitol-6'-acetyltransferase, respectively, were used.

  14. Purification, Crystallization And Preliminary X-Ray Analysis of Aminoglycoside-2 ''-Phosphotransferase-Ic [APH(2 '')-Ic] From Enterococcus Gallinarum

    Energy Technology Data Exchange (ETDEWEB)

    Byrnes, L.J.; /SLAC, SSRL; Badarau, A.; Vakulenko, S.B.; /Notre Dame U.; Smith, C.A.; /SLAC, SSRL

    2009-04-30

    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{double_prime}-phosphotransferase-Ic [APH(2{double_prime})-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{double_prime})-Ic variants were crystallized in the presence of 14-20%(w/v) PEG 4000, 0.25 M MgCl{sub 2}, 0.1 M Tris-HCl pH 8.5 and 1 mM Mg{sub 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 {angstrom}, {beta} = 108.8{sup o}. X-ray diffraction data were collected to approximately 2.15 {angstrom} resolution from an F108L crystal at beamline BL9-2 at SSRL, Stanford, California, USA.

  15. Carriage of antibiotic-resistant bacteria by healthy children.

    Science.gov (United States)

    Millar, M R; Walsh, T R; Linton, C J; Zhang, S; Leeming, J P; Bennett, P M

    2001-05-01

    The frequency of carriage of antibiotic-resistant bacteria in healthy 7- and 8-year-old children in Bristol was studied. Children born in Avon between 1 April 1991 and 31 December 1992, attending the Avon Longitudinal Study of Pregnancy and Childhood (ALSPAC) 7 year follow-up clinic, formed the study population. Carriage was estimated using mouth and stool samples. None of 105 children on whom information was available had received tetracycline, chloramphenicol, ciprofloxacin or an extended-spectrum cephalosporin in the previous year. Staphylococcus aureus was isolated from mouthwashes from 200 (37.1%) of 539 children sampled. Six (3%) of the isolates were resistant to chloramphenicol or tetracycline and four (2%) were methicillin resistant. Haemophilus spp. were isolated from 369 (72%) of 513 samples and 63 (17%) were ampicillin resistant, 49 (13.3%) were erythromycin resistant and seven (1.9%) were tetracycline resistant. Branhamella catarrhalis was isolated from 333 (74%) of 450 samples. Twenty-eight (8.4%) were erythromycin resistant and 14 (4.2%) strains were tetracycline resistant. Group A beta-haemolytic streptococci were isolated from 17 of 507 children sampled. One (5.9%) was tetracycline resistant. Stool samples were returned from 335 (62%) of 539 children from whom they were requested. Eleven per cent of samples yielded Gram-negative bacilli with high-level resistance to chloramphenicol, which was frequently linked to resistance to ampicillin, spectinomycin and streptomycin. Isolates demonstrating resistance to the third-generation cephalosporin ceftazidime were recovered from 17 subjects (3.2%). Six (35%) of 17 isolates possessed extended-spectrum beta-lactamases. Healthy children carry bacteria resistant to antibiotics to which children are not usually exposed. Resistance to ceftazidime, chloramphenicol and tetracycline may be co-selected by exposure to other antibiotics used in children or may be acquired from family members, pets, other children or

  16. 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. PMID:27472455

  17. 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...

  18. A random sequential mechanism of aminoglycoside acetylation by Mycobacterium tuberculosis Eis protein.

    Directory of Open Access Journals (Sweden)

    Oleg V Tsodikov

    Full Text Available An important cause of bacterial resistance to aminoglycoside antibiotics is the enzymatic acetylation of their amino groups by acetyltransferases, which abolishes their binding to and inhibition of the bacterial ribosome. Enhanced intracellular survival (Eis protein from Mycobacterium tuberculosis (Mt is one of such acetyltransferases, whose upregulation was recently established as a cause of resistance to aminoglycosides in clinical cases of drug-resistant tuberculosis. The mechanism of aminoglycoside acetylation by MtEis is not completely understood. A systematic analysis of steady-state kinetics of acetylation of kanamycin A and neomycin B by Eis as a function of concentrations of these aminoglycosides and the acetyl donor, acetyl coenzyme A, reveals that MtEis employs a random-sequential bisubstrate mechanism of acetylation and yields the values of the kinetic parameters of this mechanism. The implications of these mechanistic properties for the design of inhibitors of Eis and other aminoglycoside acetyltransferases are discussed.

  19. Photodynamic inactivation of antibiotic-resistant bacteria and biofilms by hematoporphyrin monomethyl ether.

    Science.gov (United States)

    Liu, Chengcheng; Hu, Min; Ma, Dandan; Lei, Jin'e; Xu, Jiru

    2016-02-01

    The worldwide increase in bacterial antibiotic resistance has led to a search for alternative antibacterial therapies. A promising approach to killing antibiotic-resistant bacteria is photodynamic antimicrobial chemotherapy, which uses light in combination with a photosensitizer to induce a phototoxic reaction. We evaluated the photodynamic inactivation (PDI) efficiency of hematoporphyrin monomethyl ether (HMME) on antibiotic-resistant bacteria and biofilms. HMME exhibited no significant dark toxicity and provided dose-dependent inactivation of antibiotic-resistant bacteria and biofilms. After incubation with 100-μM HMME and irradiation with 72-J cm(-2) white light, 4.19-7.59 log10 reductions in survival were achieved in planktonic suspension. Antibiotic-resistant strains were as susceptible to PDI in biofilms as in planktonic suspensions, but the inactivation of bacterial cells in biofilms was attenuated. In addition, gram-positive bacterial strains and biofilms were more susceptible than gram-negative strains and biofilms to the PDI effect of HMME. Thus, HMME is a promising photosensitizer for the treatment of infectious diseases caused by antibiotic-resistant bacteria, especially gram-positive bacteria.

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

    Energy Technology Data Exchange (ETDEWEB)

    Byrnes, Laura J. [Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CA 94025 (United States); Badarau, Adriana; Vakulenko, Sergei B. [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)

    2008-02-01

    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{sub 2}, 0.1 M Tris–HCl pH 8.5 and 1 mM Mg{sub 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.

  1. 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.

  2. In70 of plasmid pAX22, a bla(VIM-1)-containing integron carrying a new aminoglycoside phosphotransferase gene cassette.

    Science.gov (United States)

    Riccio, M L; Pallecchi, L; Fontana, R; Rossolini, G M

    2001-04-01

    An Achromobacter xylosoxydans strain showing broad-spectrum resistance to beta-lactams (including carbapenems) and aminoglycosides was isolated at the University Hospital of Verona (Verona, Italy). This strain was found to produce metallo-beta-lactamase activity and to harbor a 30-kb nonconjugative plasmid, named pAX22, carrying a bla(VIM-1) determinant inserted into a class 1 integron. Characterization of this integron, named In70, revealed an original array of four gene cassettes containing, respectively, the bla(VIM-1) gene and three different aminoglycoside resistance determinants, including an aacA4 allele, a new aph-like gene named aphA15, and an aadA1 allele. The aphA15 gene is the first example of an aph-like gene carried on a mobile gene cassette, and its product exhibits close similarity to the APH(3')-IIa aminoglycoside phosphotransferase encoded by Tn5 (36% amino acid identity) and to an APH(3')-IIb enzyme from Pseudomonas aeruginosa (38% amino acid identity). Expression of the cloned aphA15 gene in Escherichia coli reduced the susceptibility to kanamycin and neomycin as well as (slightly) to amikacin, netilmicin, and streptomycin. Characterization of the 5' and 3' conserved segments of In70 and of their flanking regions showed that In70 belongs to the group of class 1 integrons associated with defective transposon derivatives originating from Tn402-like elements. The structure of the 3' conserved segment indicates the closest ancestry with members of the In0-In2 lineage. In70, with its array of cassette-borne resistance genes, can mediate broad-spectrum resistance to most beta-lactams and aminoglycosides. PMID:11257042

  3. 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

  4. 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.

  5. Cp*Rh-based indicator-displacement assays for the identification of amino sugars and aminoglycosides.

    Science.gov (United States)

    Zaubitzer, Friederike; Buryak, Andrey; Severin, Kay

    2006-05-01

    Indicator-displacement assays based on the organometallic complex [{Cp*RhCl2}2] (Cp*=pentamethylcyclopentadienyl) and the dye gallocyanine were used to sense amino sugars and aminoglycosides in buffered aqueous solution by conducting UV-visible spectroscopy. The data of three assays at pH 7.0, 8.0, and 9.0 were sufficient to distinguish between the amino sugars galactosamine, glucosamine, mannosamine and the aminoglycosides kanamycin A, kanamycin B, amikacin, apramycin, paromomycin, and streptomycin. Furthermore, the assays were used to characterize mixtures of aminoglycosides and obtain quantitative information about the respective analytes. PMID:16521137

  6. 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....... Here, we show that aminoglycosides cause cytosolic protein misfolding and that chaperonin GroEL/GroES overexpression counters this defect. During aminoglycoside exposure to exponential cultures, chaperonin overexpression protected the bacterial membrane potential, rescued cell growth, and facilitated...... bacteria cope during early exposure to these drugs....

  7. Study of Pseudomonas Aeroginosa resistance to Penicillines, Cephalosporins and Aminoglycosides

    Directory of Open Access Journals (Sweden)

    Maleknezhad P

    1998-07-01

    Full Text Available Drug therapy and prophylaxy in infectious diseases, from hygienic and economical point of view, are very important. Infections caused by pseudomonas aeroginosa were particularly severe, with high mortality rates. In the recent years pseudomonas aeroginosa continued to cause the most severe, life-thereating infections in burned patients, in spite of the introduction of a wide variety of antibiotics advised specifically for their anti pseudomonal activity. The aim of this study, in which many cases of ps.aeroginosa infections are assessed is to identify the drug resistance of this bacteria to penicillines, cephalosporins and aminoglycosides by antibiotic sensitivity test (disk ager diffusion. Results as percent of resistance to each antibiotic were 89% to carbenicillin, 55% to piperacillin, 89% to mezlocillin, 89.5% to ticarcillin+clavulonic acid, 85% to ceftriaxone, 95% to tobramycin, 5% of all isolates were not sensitive to any antibiotics.

  8. 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.

  9. Environmental waters as a source of antibiotic-resistant Enterococcus species in Belgrade, Serbia.

    Science.gov (United States)

    Veljović, Katarina; Popović, Nikola; Vidojević, Amarela Terzić; Tolinački, Maja; Mihajlović, Sanja; Jovčić, Branko; Kojić, Milan

    2015-09-01

    Despite the number of studies on antibiotic-resistant enterococci from Serbian clinical settings, there are no data about environmental contamination with these bacteria. Thus, this study investigated the prevalence of antibiotic-resistant enterococci in Belgrade, Serbia. Enterococcus species collected from ten surface water sites, including a lake, two major river systems, and springs, were tested. Among enterococci, we found single (21.7 %), double (17.4 %), and multiple antibiotic resistance patterns (56.3 %). Vancomycin-resistant strains were not found, indicating that their abundance in Belgrade is tightly linked to clinical settings. The multiple drug-resistant strains Enterococcus faecalis, Enterococcus faecium, and Enterococcus mundtii were frequently detected in the lake during the swimming season and in the rivers near industrial zones. We confirmed the presence of ermB, ermC, ant(6)-Ia, tetM, and tetL and mutations in gyrA genes. The phylogenetic analysis of 16S rRNA gene of E. faecium isolates that harbor esp gene classified them into two groups based on high-bootstraps scores in the tree analysis. Pulsed-field gel electrophoresis analysis of antibiotic-resistant enterococci revealed genomic similarity ranging from 75 to 100 %. This study indicates the importance of anthropogenic impact to the spread of antibiotic-resistant enterococci in environmental waters of Belgrade, Serbia.

  10. Secular Trends in Nosocomial Bloodstream Infections : Antibiotic-Resistant Bacteria Increase the Total Burden of Infection

    NARCIS (Netherlands)

    Ammerlaan, H. S. M.; Harbarth, S.; Buiting, A. G. M.; Crook, D. W.; Fitzpatrick, F.; Hanberger, H.; Herwaldt, L. A.; van Keulen, P. H. J.; Kluytmans, J. A. J. W.; Kola, A.; Kuchenbecker, R. S.; Lingaas, E.; Meessen, N.; Morris-Downes, M. M.; Pottinger, J. M.; Rohner, P.; dos Santos, R. P.; Seifert, H.; Wisplinghoff, H.; Ziesing, S.; Walker, A. S.; Bonten, M. J. M.

    2013-01-01

    Background. It is unknown whether rising incidence rates of nosocomial bloodstream infections (BSIs) caused by antibiotic-resistant bacteria (ARB) replace antibiotic-susceptible bacteria (ASB), leaving the total BSI rate unaffected. Methods. We investigated temporal trends in annual incidence densit

  11. Fe-S cluster biosynthesis controls uptake of aminoglycosides in a ROS-less death pathway.

    Science.gov (United States)

    Ezraty, Benjamin; Vergnes, Alexandra; Banzhaf, Manuel; Duverger, Yohann; Huguenot, Allison; Brochado, Ana Rita; Su, Shu-Yi; Espinosa, Leon; Loiseau, Laurent; Py, Béatrice; Typas, Athanasios; Barras, Frédéric

    2013-06-28

    All bactericidal antibiotics were recently proposed to kill by inducing reactive oxygen species (ROS) production, causing destabilization of iron-sulfur (Fe-S) clusters and generating Fenton chemistry. We find that the ROS response is dispensable upon treatment with bactericidal antibiotics. Furthermore, we demonstrate that Fe-S clusters are required for killing only by aminoglycosides. In contrast to cells, using the major Fe-S cluster biosynthesis machinery, ISC, cells using the alternative machinery, SUF, cannot efficiently mature respiratory complexes I and II, resulting in impendence of the proton motive force (PMF), which is required for bactericidal aminoglycoside uptake. Similarly, during iron limitation, cells become intrinsically resistant to aminoglycosides by switching from ISC to SUF and down-regulating both respiratory complexes. We conclude that Fe-S proteins promote aminoglycoside killing by enabling their uptake.

  12. Characterization of a C3 Deoxygenation Pathway Reveals a Key Branch Point in Aminoglycoside Biosynthesis.

    Science.gov (United States)

    Lv, Meinan; Ji, Xinjian; Zhao, Junfeng; Li, Yongzhen; Zhang, Chen; Su, Li; Ding, Wei; Deng, Zixin; Yu, Yi; Zhang, Qi

    2016-05-25

    Apramycin is a clinically interesting aminoglycoside antibiotic (AGA) containing a highly unique bicyclic octose moiety, and this octose is deoxygenated at the C3 position. Although the biosynthetic pathways for most 2-deoxystreptamine-containing AGAs have been well characterized, the pathway for apramycin biosynthesis, including the C3 deoxygenation process, has long remained unknown. Here we report detailed investigation of apramycin biosynthesis by a series of genetic, biochemical and bioinformatical studies. We show that AprD4 is a novel radical S-adenosyl-l-methionine (SAM) enzyme, which uses a noncanonical CX3CX3C motif for binding of a [4Fe-4S] cluster and catalyzes the dehydration of paromamine, a pseudodisaccharide intermediate in apramycin biosynthesis. We also show that AprD3 is an NADPH-dependent reductase that catalyzes the reduction of the dehydrated product from AprD4-catalyzed reaction to generate lividamine, a C3' deoxygenated product of paromamine. AprD4 and AprD3 do not form a tight catalytic complex, as shown by protein complex immunoprecipitation and other assays. The AprD4/AprD3 enzyme system acts on different pseudodisaccharide substrates but does not catalyze the deoxygenation of oxyapramycin, an apramycin analogue containing a C3 hydroxyl group on the octose moiety, suggesting that oxyapramycin and apramycin are partitioned into two parallel pathways at an early biosynthetic stage. Functional dissection of the C6 dehydrogenase AprQ shows the crosstalk between different AGA biosynthetic gene clusters from the apramycin producer Streptomyces tenebrarius, and reveals the remarkable catalytic versatility of AprQ. Our study highlights the intriguing chemistry in apramycin biosynthesis and nature's ingenuity in combinatorial biosynthesis of natural products. PMID:27120352

  13. Characterization of a C3 Deoxygenation Pathway Reveals a Key Branch Point in Aminoglycoside Biosynthesis.

    Science.gov (United States)

    Lv, Meinan; Ji, Xinjian; Zhao, Junfeng; Li, Yongzhen; Zhang, Chen; Su, Li; Ding, Wei; Deng, Zixin; Yu, Yi; Zhang, Qi

    2016-05-25

    Apramycin is a clinically interesting aminoglycoside antibiotic (AGA) containing a highly unique bicyclic octose moiety, and this octose is deoxygenated at the C3 position. Although the biosynthetic pathways for most 2-deoxystreptamine-containing AGAs have been well characterized, the pathway for apramycin biosynthesis, including the C3 deoxygenation process, has long remained unknown. Here we report detailed investigation of apramycin biosynthesis by a series of genetic, biochemical and bioinformatical studies. We show that AprD4 is a novel radical S-adenosyl-l-methionine (SAM) enzyme, which uses a noncanonical CX3CX3C motif for binding of a [4Fe-4S] cluster and catalyzes the dehydration of paromamine, a pseudodisaccharide intermediate in apramycin biosynthesis. We also show that AprD3 is an NADPH-dependent reductase that catalyzes the reduction of the dehydrated product from AprD4-catalyzed reaction to generate lividamine, a C3' deoxygenated product of paromamine. AprD4 and AprD3 do not form a tight catalytic complex, as shown by protein complex immunoprecipitation and other assays. The AprD4/AprD3 enzyme system acts on different pseudodisaccharide substrates but does not catalyze the deoxygenation of oxyapramycin, an apramycin analogue containing a C3 hydroxyl group on the octose moiety, suggesting that oxyapramycin and apramycin are partitioned into two parallel pathways at an early biosynthetic stage. Functional dissection of the C6 dehydrogenase AprQ shows the crosstalk between different AGA biosynthetic gene clusters from the apramycin producer Streptomyces tenebrarius, and reveals the remarkable catalytic versatility of AprQ. Our study highlights the intriguing chemistry in apramycin biosynthesis and nature's ingenuity in combinatorial biosynthesis of natural products.

  14. Sensitivity of ribosomes of the hyperthermophilic bacterium Aquifex pyrophilus to aminoglycoside antibiotics.

    OpenAIRE

    Bocchetta, M; Huber, R.; Cammarano, P

    1996-01-01

    A poly(U)-programmed cell-free system from the hyperthermophilic bacterium Aquifex pyrophilus has been developed, and the susceptibility of Aquifex ribosomes to the miscoding-inducing and inhibitory actions of all known classes of aminoglycoside antibiotics has been assayed at temperatures (75 to 80 degrees C) close to the physiological optimum for cell growth. Unlike Thermotoga maritima ribosomes, which are systematically refractory to all known classes of aminoglycoside compounds (P. Londei...

  15. Toggled RNA Aptamers Against Aminoglycosides Allowing Facile Detection of Antibiotics Using Gold Nanoparticle Assays

    OpenAIRE

    Derbyshire, Nicola; White, Simon J.; Bunka, David H. J.; Song, Lei; Stead, Sara; Tarbin, Jonathan; Sharman, Matthew; Zhou, Dejian; Stockley, Peter G.

    2012-01-01

    We have used systematic evolution of ligands by exponential enrichment (SELEX) to isolate RNA aptamers against aminoglycoside antibiotics. The SELEX rounds were toggled against four pairs of aminoglycosides with the goal of isolating reagents that recognize conserved structural features. The resulting aptamers bind both of their selection targets with nanomolar affinities. They also bind the less structurally related targets, although they show clear specificity for this class of antibiotics....

  16. The aac(6'Ib gene in Proteus mirabilis strains resistant to aminoglycosides.

    Directory of Open Access Journals (Sweden)

    Jerzy Ratajczak

    2009-01-01

    Full Text Available The aim of this study was to evaluate the presence of aac(6'-Ib gene conferring resistance to aminoglycosides in Proteus mirabilis strains. Five isolates had aac(6'-Ib gene. In one case the gene was no-expressed. Three isolates were resistant to all aminoglycosides and minimum inhibitory concentrations were > or = 256 microg/ml. Additionally, all positive strains were resistant to tetracycline and ciprofloxacin.

  17. Once Daily Dosing of Aminoglycosides in Pediatric Cystic Fibrosis Patients: A Review of the Literature

    OpenAIRE

    Wassil, Sarah K.; Fox, Kristie M.; White, James W.

    2008-01-01

    Patients with cystic fibrosis receive many courses of antibiotic therapy throughout their lifetime. Dosing aminoglycosides once daily has become common practice in many of these individuals. Due to ease of home administration, decreased nursing time, and improved quality of life, this regimen is being increasingly explored in the cystic fibrosis population. Because patients with cystic fibrosis have increased aminoglycoside clearance, once daily dosing may result in a prolonged time during th...

  18. Toxicity modulation, resistance enzyme evasion, and A-site X-ray structure of broad-spectrum antibacterial neomycin analogs.

    Science.gov (United States)

    Maianti, Juan Pablo; Kanazawa, Hiroki; Dozzo, Paola; Matias, Rowena D; Feeney, Lee Ann; Armstrong, Eliana S; Hildebrandt, Darin J; Kane, Timothy R; Gliedt, Micah J; Goldblum, Adam A; Linsell, Martin S; Aggen, James B; Kondo, Jiro; Hanessian, Stephen

    2014-09-19

    Aminoglycoside antibiotics are pseudosaccharides decorated with ammonium groups that are critical for their potent broad-spectrum antibacterial activity. Despite over three decades of speculation whether or not modulation of pKa is a viable strategy to curtail aminoglycoside kidney toxicity, there is a lack of methods to systematically probe amine-RNA interactions and resultant cytotoxicity trends. This study reports the first series of potent aminoglycoside antibiotics harboring fluorinated N1-hydroxyaminobutyryl acyl (HABA) appendages for which fluorine-RNA contacts are revealed through an X-ray cocrystal structure within the RNA A-site. Cytotoxicity in kidney-derived cells was significantly reduced for the derivative featuring our novel β,β-difluoro-HABA group, which masks one net charge by lowering the pKa without compromising antibacterial potency. This novel side-chain assists in evasion of aminoglycoside-modifying enzymes, and it can be easily transferred to impart these properties onto any number of novel analogs.

  19. Mechanistic studies of copper(II)-aminoglycoside mediated DNA damage and magnesium catalyzed nuclease activity of hammerhead ribozyme

    Science.gov (United States)

    Patwardhan, Anjali A.

    The antibacterial activity of aminoglycosides stems from their high affinity binding to the 16S rRNA in bacteria resulting in inhibition of protein synthesis. Used to treat acute bacterial infections these antibiotics have limited applications due to their high dosage requirements and the emergence of resistant strains. We have synthesized and characterized Cu(II) derivatives of the aminoglycosides, kanamycin A, tobramycin, neamine, kanamycin B, neomycin B, and paromomycin. The first three exhibit preferential and tight binding to Cu(II) as against neomycin B and kanamycin B and paromomycin. EPR of frozen solutions and UV-visible spectroscopy suggest a change in geometry around the Cu(II) but the stabilities of the complexes in water differ. These copper derivatives efficiently cleave plasmid DNA at micromolar concentrations (hydrolytic) and at nanomolar concentrations in the presence co-reactants like hydrogen peroxide or ascorbic acid. Hydrolysis is multi turnover and exhibits Michelis-Menten kinetics with enzyme-like behavior whereas oxidative cleavage is highly specific with C-4' H abstraction resulting in characteristic base propenal and nucleotide base products. Hydroxyl radicals generated are copper based and are generated in close proximity of the substrate. Hammerhead ribozymes are selectively hydrolyzed in the presence of divalent ions with Mg2+ being the metal ion of choice in vivo . Our studies with complex ions like cobalt hexaammine and fac-triamminetriaquochromium(III) establish outer sphere interactions of Mg2+ with the hammerhead in the catalytic site. There are two sets of sites, one structural and one catalytic. Complex ions in the catalytic site and divalent ions in the structural site result in a slow but active hammerhead ribozyme suggesting that the complex ions are not inhibitory, contrary to what was suggested previously.

  20. A surprising dipolar cycloaddition provides ready access to aminoglycosides.

    Science.gov (United States)

    Dahl, Russell S; Finney, Nathaniel S

    2004-07-14

    This contribution describes the results of a new research effort in our laboratory aimed at the synthesis of novel aminoglycosides and amino-C-glycosides. Despite the importance of such compounds, and the previous development of some methodological solutions, this remains an important area of research. Notable features of our approach, which is distinct from and complementary to previous efforts, are the following: (1) Reliance on a surprising and unprecedented formation of glycal triazolines via an inverse electron demand dipolar cycloaddition of glucal. We believe this desirable transformation has not previously been discovered because of the unusual selection of substrates and solvent required. (2) Very mild reaction conditions. An initial thermal cycloaddition is carried out in an inert solvent, the triazoline generated is photochemically converted to a reactive aziridine, and the crude aziridine undergoes ring opening at room temperature in the presence of a nucleophile and a mild Lewis acid catalyst. (3) Formation of products lacking an N-acyl group, allowing ready synthesis of novel glucosamine derivatives. PMID:15237974

  1. 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.

  2. Aminoglycoside-mediated relaxation of the ductus arteriosus in sepsis-associated PDA.

    Science.gov (United States)

    Vucovich, Megan M; Cotton, Robert B; Shelton, Elaine L; Goettel, Jeremy A; Ehinger, Noah J; Poole, Stanley D; Brown, Naoko; Wynn, James L; Paria, Bibhash C; Slaughter, James C; Clark, Reese H; Rojas, Mario A; Reese, Jeff

    2014-09-01

    Sepsis is strongly associated with patency of the ductus arteriosus (PDA) in critically ill newborns. Inflammation and the aminoglycoside antibiotics used to treat neonatal sepsis cause smooth muscle relaxation, but their contribution to PDA is unknown. We examined whether: 1) lipopolysaccharide (LPS) or inflammatory cytokines cause relaxation of the ex vivo mouse DA; 2) the aminoglycosides gentamicin, tobramycin, or amikacin causes DA relaxation; and 3) newborn infants treated with aminoglycosides have an increased risk of symptomatic PDA (sPDA). Changes in fetal mouse DA tone were measured by pressure myography in response to LPS, TNF-α, IFN-γ, macrophage-inflammatory protein 2, IL-15, IL-13, CXC chemokine ligand 12, or three aminoglycosides. A clinical database of inborn patients of all gestations was analyzed for association between sPDA and aminoglycoside treatment. Contrary to expectation, neither LPS nor any of the inflammatory mediators caused DA relaxation. However, each of the aminoglycosides caused concentration-dependent vasodilation in term and preterm mouse DAs. Pretreatment with indomethacin and N-(G)-nitro-L-arginine methyl ester did not prevent gentamicin-induced DA relaxation. Gentamicin-exposed DAs developed less oxygen-induced constriction than unexposed DAs. Among 488,349 infants who met the study criteria, 40,472 (8.3%) had sPDA. Confounder-adjusted odds of sPDA were higher in gentamicin-exposed infants, 32 wk. Together, these findings suggest that factors other than inflammation contribute to PDA. Aminoglycoside-induced vasorelaxation and inhibition of oxygen-induced DA constriction support the paradox that antibiotic treatment of sepsis may contribute to DA relaxation. This association was also found in newborn infants, suggesting that antibiotic selection may be an important consideration in efforts to reduce sepsis-associated PDA. PMID:24993047

  3. Quantifying Cost-Effectiveness of Controlling Nosocomial Spread of Antibiotic-Resistant Bacteria : The Case of MRSA

    NARCIS (Netherlands)

    Wassenberg, Marjan W. M.; de Wit, G. Ardine; van Hout, Ben A.; Bonten, Marc J. M.

    2010-01-01

    Background: The costs and benefits of controlling nosocomial spread of antibiotic-resistant bacteria are unknown. Methods: We developed a mathematical algorithm to determine cost-effectiveness of infection control programs and explored the dynamical interactions between different epidemiological var

  4. Occurrence and distribution of multiple antibiotic-resistant bacteria of Enterobacteriaceae family in waters of Veraval coast, India

    Digital Repository Service at National Institute of Oceanography (India)

    Maloo, A.; Borade, S.; Dhawde, R.; Gajbhiye, S.N.; Dastager, S.G.

    Current investigation was aimed to the assess occurrence and distribution of multiple antibiotic-resistant bacteria of the Enterobacteriaceae family in surface and bottom waters along the Veraval coast. Comparative prevalence of drug...

  5. Prevalence of Antibiotic-Resistant Bacteria on Rectal Swabs and Factors Affecting Resistance to Antibiotics in Patients Undergoing Prostate Biopsy

    OpenAIRE

    Kim, Jong Beom; Jung, Seung Il; Hwang, Eu Chang; Kwon, Dong Deuk

    2014-01-01

    Purpose The prevalence of antibiotic-resistant bacteria on rectal swabs in patients undergoing transrectal ultrasound (TRUS)-guided prostate biopsy and the factors affecting resistance to antibiotics were evaluated. Materials and Methods Two hundred twenty-three men who underwent TRUS-guided prostate biopsy from November 2011 to December 2012 were retrospectively evaluated. Rectal swabs were cultured on MacConkey agar to identify antibiotic-resistant bacteria in rectal flora before TRUS-guide...

  6. A Multifunctional Subphthalocyanine Nanosphere for Targeting, Labeling, and Killing of Antibiotic-Resistant Bacteria.

    Science.gov (United States)

    Roy, Indranil; Shetty, Dinesh; Hota, Raghunandan; Baek, Kangkyun; Kim, Jeesu; Kim, Chulhong; Kappert, Sandro; Kim, Kimoon

    2015-12-01

    Developing a material that can combat antibiotic-resistant bacteria, a major global health threat, is an urgent requirement. To tackle this challenge, we synthesized a multifunctional subphthalocyanine (SubPc) polymer nanosphere that has the ability to target, label, and photoinactivate antibiotic-resistant bacteria in a single treatment with more than 99 % efficiency, even with a dose as low as 4.2 J cm(-2) and a loading concentration of 10 nM. The positively charged nanosphere shell composed of covalently linked SubPc units can increase the local concentration of photosensitizers at therapeutic sites. The nanosphere shows superior performance compared to corresponding monomers presumably because of their enhanced water dispersibility, higher efficiency of singlet-oxygen generation, and phototoxicity. In addition, this material is useful in fluorescence labeling of living cells and shows promise in photoacoustic imaging of bacteria in vivo.

  7. Irrigation waters and pipe-based biofilms as sources for antibiotic-resistant bacteria.

    Science.gov (United States)

    Blaustein, Ryan A; Shelton, Daniel R; Van Kessel, Jo Ann S; Karns, Jeffrey S; Stocker, Matthew D; Pachepsky, Yakov A

    2016-01-01

    The presence of antibiotic-resistant bacteria in environmental surface waters has gained recent attention. Wastewater and drinking water distribution systems are known to disseminate antibiotic-resistant bacteria, with the biofilms that form on the inner-surfaces of the pipeline as a hot spot for proliferation and gene exchange. Pipe-based irrigation systems that utilize surface waters may contribute to the dissemination of antibiotic-resistant bacteria in a similar manner. We conducted irrigation events at a perennial stream on a weekly basis for 1 month, and the concentrations of total heterotrophic bacteria, total coliforms, and fecal coliforms, as well as the concentrations of these bacterial groups that were resistant to ampicillin and tetracycline, were monitored at the intake water. Prior to each of the latter three events, residual pipe water was sampled and 6-in. sections of pipeline (coupons) were detached from the system, and biofilm from the inner-wall was removed and analyzed for total protein content and the above bacteria. Isolates of biofilm-associated bacteria were screened for resistance to a panel of seven antibiotics, representing five antibiotic classes. All of the monitored bacteria grew substantially in the residual water between irrigation events, and the biomass of the biofilm steadily increased from week to week. The percentages of biofilm-associated isolates that were resistant to antibiotics on the panel sometimes increased between events. Multiple-drug resistance was observed for all bacterial groups, most often for fecal coliforms, and the distributions of the numbers of antibiotics that the total coliforms and fecal coliforms were resistant to were subject to change from week to week. Results from this study highlight irrigation waters as a potential source for antibiotic-resistant bacteria, which can subsequently become incorporated into and proliferate within irrigation pipe-based biofilms. PMID:26703979

  8. Antibiotic-Resistant Gram Negative Bacilli in Meals Delivered at a General Hospital, Italy

    OpenAIRE

    Maria Rosa Anna Plano; Anna Maria Di Noto; Alberto Firenze; Sonia Sciortino; Caterina Mammina

    2009-01-01

    This study aimed at detecting the presence of antibiotic-resistant Gram-negatives in samples of meals delivered at the University General Hospital of Palermo, Italy. Antibiotic resistant Gram negatives were isolated in July—September 2007 ffrom cold dishes and food contact surfaces and utensils. Bacterial strains were submitted to susceptibility test and subtyped by random amplification of polymorphic DNA (RAPD). Forty-six of 55 (83.6%) food samples and 14 of 17 (82.3%) environmental swabs ...

  9. Nasopharyngeal carriage of community-acquired, antibiotic-resistant Streptococcus pneumoniae in a Zambian paediatric population.

    OpenAIRE

    Woolfson, A; Huebner, R.; Wasas, A; Chola, S.; Godfrey-Faussett, P.; Klugman, K.

    1997-01-01

    The emergence of antibiotic-resistant Streptococcus pneumoniae is an international health problem. Apart from South Africa few data on pneumococcal resistance are available for sub-Saharan Africa. This study examines the nasopharyngeal carriage and prevalence of antibiotic resistance in pneumococci isolated from 260 Zambian children aged < 6 years. Pneumococci were isolated from 71.9% of the children; the odds of carrying organisms were twice as high among children < 2 years of age compared w...

  10. Assessment of altered binding specificity of bacteriophage for ciprofloxacin-induced antibiotic-resistant Salmonella Typhimurium.

    Science.gov (United States)

    Kim, Jeongjin; Jo, Ara; Ding, Tian; Lee, Hyeon-Yong; Ahn, Juhee

    2016-08-01

    This study describes a new effort toward understanding the interaction mechanisms between antibiotic-resistant Salmonella Typhimurium and phages. The antibiotic susceptibility, β-lactamase activity, bacterial motility, gene expression, and lytic activity were evaluated in ciprofloxacin-induced antibiotic-sensitive Salmonella Typhimurium (ASST(CIP)) and ciprofloxacin-induced antibiotic-resistant S. Typhimurium (ARST(CIP)), which were compared to the wild-type strains (ASST(WT) and ARST(WT)). The MIC values of ampicillin, norfloxacin, chloramphenicol, and tetracycline were significantly increased to > 512, 16, 16, and 256 μg/ml, respectively, in the ARST(CIP). The lowest and highest extracellular lactamase activities were observed in ASST(WT) (6.85 μmol/min/ml) and ARST(CIP) (48.83 μmol/min/ml), respectively. The acrA, lpfE, and hilA genes were significantly upregulated by more than tenfold in both ASST(CIP) and ARST(CIP). The induction of multiple antibiotic resistance resulted from the increased efflux pump activity (AcrAB-TolC). The highest phage adsorption rates were more than 95 % for ASST(WT), ASST(CIP), and ARST(WT), while the lowest adsorption rate was 52 % for ARST(CIP) at 15 min of infection. The least lytic activity of phage was 20 % against the ARST(CIP), followed by ASST(CIP) (30 %). The adsorption rate of phage against ARST(CIP) was 52 % at 15 min of infection, which resulted in the decrease in lytic activity (12 %). Understanding the interaction of phage and bacteria is essential for the practical application of phage to control and detect antibiotic-resistant bacteria. The results provide useful information for understanding the binding specificity of phages for multiple antibiotic-resistant pathogens. PMID:27000396

  11. Effective Phages as Green Antimicrobial Agents Against Antibiotic-Resistant Hospital Escherichia coli

    OpenAIRE

    Rahmani, Rana; Zarrini, Gholamreza; Sheikhzadeh, Farzam; Aghamohammadzadeh, Naser

    2015-01-01

    Background: Bacteriophages are viruses that attack bacteria and lead to their lysis in an efficient and highly specific manner. These natural enemies of bacteria were used as therapeutic agents before the advent of antibiotics. Currently, with the rapid spread of multi-drug resistant bacteria, phage therapy can be an effective alternative treatment for antibiotic resistant bacteria. Objectives: This study evaluated the effectiveness of bacteriophages in removing antibiotic-resistant clinical ...

  12. Irrigation waters and pipe-based biofilms as sources for antibiotic-resistant bacteria.

    Science.gov (United States)

    Blaustein, Ryan A; Shelton, Daniel R; Van Kessel, Jo Ann S; Karns, Jeffrey S; Stocker, Matthew D; Pachepsky, Yakov A

    2016-01-01

    The presence of antibiotic-resistant bacteria in environmental surface waters has gained recent attention. Wastewater and drinking water distribution systems are known to disseminate antibiotic-resistant bacteria, with the biofilms that form on the inner-surfaces of the pipeline as a hot spot for proliferation and gene exchange. Pipe-based irrigation systems that utilize surface waters may contribute to the dissemination of antibiotic-resistant bacteria in a similar manner. We conducted irrigation events at a perennial stream on a weekly basis for 1 month, and the concentrations of total heterotrophic bacteria, total coliforms, and fecal coliforms, as well as the concentrations of these bacterial groups that were resistant to ampicillin and tetracycline, were monitored at the intake water. Prior to each of the latter three events, residual pipe water was sampled and 6-in. sections of pipeline (coupons) were detached from the system, and biofilm from the inner-wall was removed and analyzed for total protein content and the above bacteria. Isolates of biofilm-associated bacteria were screened for resistance to a panel of seven antibiotics, representing five antibiotic classes. All of the monitored bacteria grew substantially in the residual water between irrigation events, and the biomass of the biofilm steadily increased from week to week. The percentages of biofilm-associated isolates that were resistant to antibiotics on the panel sometimes increased between events. Multiple-drug resistance was observed for all bacterial groups, most often for fecal coliforms, and the distributions of the numbers of antibiotics that the total coliforms and fecal coliforms were resistant to were subject to change from week to week. Results from this study highlight irrigation waters as a potential source for antibiotic-resistant bacteria, which can subsequently become incorporated into and proliferate within irrigation pipe-based biofilms.

  13. 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.

  14. Practical survey on antibiotic-resistant bacterial communities in livestock manure and manure-amended soil.

    Science.gov (United States)

    Yang, Qingxiang; Wang, Ruifei; Ren, Siwei; Szoboszlay, Marton; Moe, Luke A

    2016-01-01

    Through livestock manure fertilization, antibiotics, antibiotic-resistant bacteria and genes are transferred to agricultural soils, resulting in a high prevalence of antibiotic-resistant bacteria in the soil. It is not clear, however, whether a correlation exists between resistant bacterial populations in manure and manure-amended soil. In this work, we demonstrate that the prevalence of cephalexin-, amoxicillin-, kanamycin- and gentamicin-resistant bacteria as well as bacteria simultaneously resistant to all four antibiotics was much higher in manure-amended soils than in manure-free soil. 454-pyrosequencing indicated that the ARB and multiple antibiotic-resistant bacteria (MARB) in swine or chicken manure and manure-amended soil were mainly distributed among Sphingobacterium, Myroides, Enterococcus, Comamonas and unclassified Flavobacteriaceae. The genus Sphingobacterium was highly prevalent among ARB from swine manure and manure-amended soil, and was also the most dominant genus among MARB from chicken manure and manure-amended soil. Other dominant genera among ARB or MARB populations in manure samples, including Myroides, Enterococcus and Comamonas, could not be detected or were detected at very low relative abundance in manure-amended soil. The present study suggests the possibility of transfer of ARBs from livestock manures to soils and persistence of ARB in these environments. PMID:26513264

  15. In vitro effect of levofloxacin and vancomycin combination against high level aminoglycoside-resistant enterococci.

    Science.gov (United States)

    Erdem, Ilknur; Cicek-Senturk, Gonul; Yucesoy-Dede, Behiye; Yuksel-Kocdogan, Funda; Yuksel, Saim; Karagul, Emin

    2004-01-01

    The in vitro effects of levofloxacin and vancomycin in combination were evaluated against high level aminoglycoside-resistant (HLAR) enterococci using chequerboard and time-kill curve techniques. We examined 28 strains of enterococci comprising 17 Enterococcus faecalis, 10 E. faecium and one E. durans. The combination of vancomycin and levofloxacin had indifferent activity against all isolates according to chequerboard microdilution method, but was synergistic for two isolates, one E. faecium and one E. faecalis, using the time-kill curve method. Both strains were levofloxacin resistant and had high level aminoglycoside resistance to gentamicin and streptomycin. Antagonism was not detected in any strain. The results of this study suggested that the combination of vancomycin with levofloxacin does not often show synergistic effect against high level aminoglycoside-resistant enterococci.

  16. Aminoglycoside resistance pattern and genetic background in multi-drug resistant acinetobacter baumannii%多药耐药鲍氏不动杆菌氨基糖苷类药物耐药遗传学背景研究

    Institute of Scientific and Technical Information of China (English)

    陈月馨; 周惠芬; 钟育红; 吴润香; 黄烈; 陈智睿

    2011-01-01

    OBJECTIVE To investigate the background of the Aminoglycoside resistance pattern and genetic type in multidrug-resistant Acinetobacter baumannii (MDR-ABA). METHODS From Apr 2009 to Aug 2009, twenty MDRABA strains were isolated from The Third Affiliated Hospital of Sun Yat-sen University. Drug susceptibility test to 10 kinds of antimicrobial agents was detected by K-B disk diffusion tests. Then, resistant genes and genetic markers were analyzed by PCR and verified by DNA sequencing, including 8 kinds of aminoglycoside modifying enzyme genes(aac(3)- Ⅰ ,aac(3)- Ⅱ ,aac(6′)- Ⅰ ad,aac(6′)- Ⅰ b,aac(6′)- Ⅱ ,ant(3″)- Ⅰ ,ant(2″)- Ⅰ , aph(3′)- Ⅰ ),and 6 kinds of 16S rRNA methylase genes (rmtA, rmtB, rmtC, rmtD, armA, npmA). RESULTS A total of 4 kinds of aminoglycoside modifying enzyme genes of MDR-ABA were detected, including aac(3)-Ⅰ , aac(6′)-Ⅰ b, ant (3″)- Ⅰ , aph(3′)- Ⅰ , and 6 kinds of 16S rRNA methylase genes in twenty MDR-ABA strains were not detected.CONCLUSIONS There is a very high positive rate of aminoglycoside modifying enzyme genes in MDR-ABA isolated from inpatients; The aminoglycosides-resistant MDR-ABA is mainly related to aminoglycoside modifying enzyme genes; The mobile genetic element is the main factor for MDR-ABA to acquire aminoglycoside modifying enzyme genes.%目的 了解临床分离的多药耐药鲍氏不动杆菌(MDR-ABA)氨基糖苷类药物耐药遗传学背景.方法 从2009年4-8月中山大学附属第三医院住院患者中分离20株MDR-ABA,用K-B法测定鲍氏不动杆菌对10种抗菌药物的敏感性,采用PCR及序列分析的方法分析氨基糖苷类修饰酶基因.结果 20株MDR-ABA检出aac(3)-Ⅰ、aac(6′)-Ⅰb、ant(3′)-Ⅰ、aph(3′)-Ⅰ4种基因阳性,6种16S rRNA甲基化酶基因未检出.结论 临床分离的MDR-ABA中氨基糖苷类修饰酶基因阳性率较高,MDR-ABA氨基糖苷类抗菌药物耐药主要与氨基糖苷类修饰酶基因有关;通过可移动遗传元

  17. Characterization of aminoglycoside resistance and virulence genes among Enterococcus spp. isolated from a hospital in China.

    Science.gov (United States)

    Li, Wanxiang; Li, Jing; Wei, Quhao; Hu, Qingfeng; Lin, Xiaowei; Chen, Mengquan; Ye, Renji; Lv, Huoyang

    2015-03-11

    This study investigated the aminoglycoside resistance phenotypes and genotypes, as well as the prevalence of virulence genes, in Enterococcus species isolated from clinical patients in China. A total of 160 enterococcal isolates from various clinical samples collected from September 2013 to July 2014 were identified to the species level using the VITEK-2 COMPACT system. The antimicrobial susceptibilities of the identified Enterococcus strains were determined by the Kirby-Bauer (K-B) disc diffusion method. PCR-based assays were used to detect the aminoglycoside resistance and virulence genes in all enterococcal isolates. Of 160 Enterococcus isolates, 105 were identified as E. faecium, 35 as E. faecalis, and 20 isolates were classified as "other" Enterococcus species. High-level aminoglycoside resistance (HLAR) for gentamicin, streptomycin, and both antibiotics was identified in 58.8, 50, and 34.4% of strains, respectively. The most common virulence gene (50.6% of isolates) was efaA, followed by asa1 (28.8%). The most prevalent aminoglycoside resistance genes were aac(6')-Ie-aph(2''), aph(2')-Id, aph(3')-IIIa, and ant(6')-Ia, present in 49.4%, 1.3%, 48.8% and 31.3% of strains, respectively. Overall, E. faecium and E. faecalis were most frequently associated with hospital-acquired enterococcal infections in Zhejiang Province. All aminoglycoside resistance genes, except aph(2'')-Id, were significantly more prevalent in HLAR strains than amongst high level aminoglycoside susceptible (HLAS) strains, while there was no significant difference between HLAR and HLAS strains in regard to the prevalence of virulence genes, apart from esp, therefore, measures should be taken to manage infections caused by multi-drug resistant Enterococcus species.

  18. Study of acquired aminoglycosides resistance genes in Enterobacter aerogenes%产气肠杆菌氨基糖苷类药物获得性耐药基因研究

    Institute of Scientific and Technical Information of China (English)

    刘鹏; 许小敏; 许兆军

    2014-01-01

    目的:研究医院产气肠杆菌临床分离株氨基糖苷类药物获得性耐药机制,了解该细菌对氨基糖苷类药物的耐药性,为临床资料提供参考依据。方法24株产气肠杆菌分离自2012年1月-2012年12月住院患者,采用VITEK-2 Compact分析系统的药敏卡AST-GN13及K-B法测定抗菌药物的敏感性,聚合酶链反应(PCR)检测6种氨基糖苷类修饰酶基因和两种16SrRNA甲基化酶基因。结果24株产气肠杆菌对头孢替坦100.0%耐药,有16株对头孢曲松的耐药率为66.7%,14株对环丙沙星的耐药率为58.3%;PCR检出氨基糖苷类修饰酶基因 aac(3)-Ⅱ1株,阳性率为4.2%,aac(6′)-Ⅰb 6株阳性率为25.0%,其余4种氨基糖苷类修饰酶基因未检出。结论氨基糖苷类修饰酶基因检出阳性率与产气肠杆菌对氨基糖苷类药物的耐药率基本相符。%OBJECTIVE To study the mechanism of acquired aminoglycosides resistance genes in Enterobacter aero-genes isolated from clinical and understand the drug resistance for the bacterial to aminoglycosides ,so as to provide reference to clinic .METHODS A total of 24 strains of Enterobacter aerogenes were isolated from the inpatients during Jan .2012 to Dec .2012 .The antimicrobial susceptibility was detected by VITEK2-compact assay system card AST-GN13 and K-B tests ;6 kinds of aminoglycoside modifying enzyme genes and 2 16SrRNA methyltrans-ferase genes were detected by polymerase chain reaction (PCR) .RESULTS The 24 strains E .aerogenes were all resistant to cefotetan ,16 strains were resistant to cefatriaxone ,14 strains were resistant to ciprofloxacin ,and the resistance rates were 100% ,66 .7% and 58 .3% ,respectively .Aminoglycoside modifying enzyme gene aac(3)-Ⅱand aac(6′)-Ⅰb were detected in 1 and 6 strains E .aerogenes ,the positive rates were 4 .2% and 25% ,respective-ly .The other 4 kinds of aminoglycoside modifying enzyme genes were not detected

  19. Tracking Down Antibiotic-Resistant Pseudomonas aeruginosa Isolates in a Wastewater Network

    Science.gov (United States)

    Slekovec, Céline; Plantin, Julie; Cholley, Pascal; Thouverez, Michelle; Talon, Daniel; Bertrand, Xavier; Hocquet, Didier

    2012-01-01

    The Pseudomonas aeruginosa-containing wastewater released by hospitals is treated by wastewater treatment plants (WWTPs), generating sludge, which is used as a fertilizer, and effluent, which is discharged into rivers. We evaluated the risk of dissemination of antibiotic-resistant P. aeruginosa (AR-PA) from the hospital to the environment via the wastewater network. Over a 10-week period, we sampled weekly 11 points (hospital and urban wastewater, untreated and treated water, sludge) of the wastewater network and the river upstream and downstream of the WWTP of a city in eastern France. We quantified the P. aeruginosa load by colony counting. We determined the susceptibility to 16 antibiotics of 225 isolates, which we sorted into three categories (wild-type, antibiotic-resistant and multidrug-resistant). Extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs) were identified by gene sequencing. All non-wild-type isolates (n = 56) and a similar number of wild-type isolates (n = 54) were genotyped by pulsed-field gel electrophoresis and multilocus sequence typing. Almost all the samples (105/110, 95.5%) contained P. aeruginosa, with high loads in hospital wastewater and sludge (≥3×106 CFU/l or/kg). Most of the multidrug-resistant isolates belonged to ST235, CC111 and ST395. They were found in hospital wastewater and some produced ESBLs such as PER-1 and MBLs such as IMP-29. The WWTP greatly reduced P. aeruginosa counts in effluent, but the P. aeruginosa load in the river was nonetheless higher downstream than upstream from the WWTP. We conclude that the antibiotic-resistant P. aeruginosa released by hospitals is found in the water downstream from the WWTP and in sludge, constituting a potential risk of environmental contamination. PMID:23284623

  20. Tracking down antibiotic-resistant Pseudomonas aeruginosa isolates in a wastewater network.

    Directory of Open Access Journals (Sweden)

    Céline Slekovec

    Full Text Available The Pseudomonas aeruginosa-containing wastewater released by hospitals is treated by wastewater treatment plants (WWTPs, generating sludge, which is used as a fertilizer, and effluent, which is discharged into rivers. We evaluated the risk of dissemination of antibiotic-resistant P. aeruginosa (AR-PA from the hospital to the environment via the wastewater network. Over a 10-week period, we sampled weekly 11 points (hospital and urban wastewater, untreated and treated water, sludge of the wastewater network and the river upstream and downstream of the WWTP of a city in eastern France. We quantified the P. aeruginosa load by colony counting. We determined the susceptibility to 16 antibiotics of 225 isolates, which we sorted into three categories (wild-type, antibiotic-resistant and multidrug-resistant. Extended-spectrum β-lactamases (ESBLs and metallo-β-lactamases (MBLs were identified by gene sequencing. All non-wild-type isolates (n = 56 and a similar number of wild-type isolates (n = 54 were genotyped by pulsed-field gel electrophoresis and multilocus sequence typing. Almost all the samples (105/110, 95.5% contained P. aeruginosa, with high loads in hospital wastewater and sludge (≥3×10(6 CFU/l or/kg. Most of the multidrug-resistant isolates belonged to ST235, CC111 and ST395. They were found in hospital wastewater and some produced ESBLs such as PER-1 and MBLs such as IMP-29. The WWTP greatly reduced P. aeruginosa counts in effluent, but the P. aeruginosa load in the river was nonetheless higher downstream than upstream from the WWTP. We conclude that the antibiotic-resistant P. aeruginosa released by hospitals is found in the water downstream from the WWTP and in sludge, constituting a potential risk of environmental contamination.

  1. 21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

    Science.gov (United States)

    2010-04-01

    ... genetically modified cotton, oilseed rape, and tomatoes in accordance with the following prescribed conditions... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Aminoglycoside 3â²-phospho- transferase II. 573.130 Section 573.130 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  2. 21 CFR 173.170 - Aminoglycoside 3′-phospho-trans-ferase II.

    Science.gov (United States)

    2010-04-01

    ... development of genetically modified cotton, oilseed rape, and tomatoes in accordance with the following... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Aminoglycoside 3â²-phospho-trans-ferase II. 173.170 Section 173.170 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...

  3. Extracellular DNA Acidifies Biofilms and Induces Aminoglycoside Resistance in Pseudomonas aeruginosa.

    Science.gov (United States)

    Wilton, Mike; Charron-Mazenod, Laetitia; Moore, Richard; Lewenza, Shawn

    2015-11-09

    Biofilms consist of surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, exopolysaccharides, and proteins. Extracellular DNA (eDNA) has a structural role in the formation of biofilms, can bind and shield biofilms from aminoglycosides, and induces antimicrobial peptide resistance mechanisms. Here, we provide evidence that eDNA is responsible for the acidification of Pseudomonas aeruginosa planktonic cultures and biofilms. Further, we show that acidic pH and acidification via eDNA constitute a signal that is perceived by P. aeruginosa to induce the expression of genes regulated by the PhoPQ and PmrAB two-component regulatory systems. Planktonic P. aeruginosa cultured in exogenous 0.2% DNA or under acidic conditions demonstrates a 2- to 8-fold increase in aminoglycoside resistance. This resistance phenotype requires the aminoarabinose modification of lipid A and the production of spermidine on the bacterial outer membrane, which likely reduce the entry of aminoglycosides. Interestingly, the additions of the basic amino acid L-arginine and sodium bicarbonate neutralize the pH and restore P. aeruginosa susceptibility to aminoglycosides, even in the presence of eDNA. These data illustrate that the accumulation of eDNA in biofilms and infection sites can acidify the local environment and that acidic pH promotes the P. aeruginosa antibiotic resistance phenotype.

  4. Emergence of aminoglycoside resistance genes aadA and aadE in the genus Campylobacter.

    OpenAIRE

    Pinto-Alphandary, H; Mabilat, C; Courvalin, P

    1990-01-01

    Resistance to streptomycin or spectinomycin or both in five Campylobacter coli strains, two Campylobacter jejuni strains, and a Campylobacter-like strain was studied by enzymatic assays and dot blot hybridization. Resistance was due to 6- or 3",9-aminoglycoside adenylyltransferases and to new types of phospho- and adenylyltransferases.

  5. Extracellular DNA Acidifies Biofilms and Induces Aminoglycoside Resistance in Pseudomonas aeruginosa.

    Science.gov (United States)

    Wilton, Mike; Charron-Mazenod, Laetitia; Moore, Richard; Lewenza, Shawn

    2016-01-01

    Biofilms consist of surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, exopolysaccharides, and proteins. Extracellular DNA (eDNA) has a structural role in the formation of biofilms, can bind and shield biofilms from aminoglycosides, and induces antimicrobial peptide resistance mechanisms. Here, we provide evidence that eDNA is responsible for the acidification of Pseudomonas aeruginosa planktonic cultures and biofilms. Further, we show that acidic pH and acidification via eDNA constitute a signal that is perceived by P. aeruginosa to induce the expression of genes regulated by the PhoPQ and PmrAB two-component regulatory systems. Planktonic P. aeruginosa cultured in exogenous 0.2% DNA or under acidic conditions demonstrates a 2- to 8-fold increase in aminoglycoside resistance. This resistance phenotype requires the aminoarabinose modification of lipid A and the production of spermidine on the bacterial outer membrane, which likely reduce the entry of aminoglycosides. Interestingly, the additions of the basic amino acid L-arginine and sodium bicarbonate neutralize the pH and restore P. aeruginosa susceptibility to aminoglycosides, even in the presence of eDNA. These data illustrate that the accumulation of eDNA in biofilms and infection sites can acidify the local environment and that acidic pH promotes the P. aeruginosa antibiotic resistance phenotype. PMID:26552982

  6. 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.

  7. Structural Analysis of a Putative Aminoglycoside N-Acetyltransferase from Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Klimecka, Maria M.; Chruszcz, Maksymilian; Font, Jose; Skarina, Tatiana; Shumilin, Igor; Onopryienko, Olena; Porebski, Przemyslaw J.; Cymborowski, Marcin; Zimmerman, Matthew D.; Hasseman, Jeremy; Glomski, Ian J.; Lebioda, Lukasz; Savchenko, Alexei; Edwards, Aled; Minor, Wladek (SC); (Toronto); (UV)

    2012-02-15

    For the last decade, worldwide efforts for the treatment of anthrax infection have focused on developing effective vaccines. Patients that are already infected are still treated traditionally using different types of standard antimicrobial agents. The most popular are antibiotics such as tetracyclines and fluoroquinolones. While aminoglycosides appear to be less effective antimicrobial agents than other antibiotics, synthetic aminoglycosides have been shown to act as potent inhibitors of anthrax lethal factor and may have potential application as antitoxins. Here, we present a structural analysis of the BA2930 protein, a putative aminoglycoside acetyltransferase, which may be a component of the bacterium's aminoglycoside resistance mechanism. The determined structures revealed details of a fold characteristic only for one other protein structure in the Protein Data Bank, namely, YokD from Bacillus subtilis. Both BA2930 and YokD are members of the Antibiotic-NAT superfamily (PF02522). Sequential and structural analyses showed that residues conserved throughout the Antibiotic-NAT superfamily are responsible for the binding of the cofactor acetyl coenzyme A. The interaction of BA2930 with cofactors was characterized by both crystallographic and binding studies.

  8. A new aspect of aminoglycoside ototoxicity : impairment of cochlear dopamine release

    NARCIS (Netherlands)

    Gáborján, A; Halmos, G; Répássy, G; Vizi, E S

    2001-01-01

    Aminoglycoside ototoxicity is a well-documented process via several pathophysiological pathways. The protective role of cochlear dopamine, released from the lateral olivocochlear efferents, was implicated previously in case of ischemia or acoustic trauma, as it postsynaptically inhibits the effect o

  9. Role of aromatic rings in the molecular recognition of aminoglycoside antibiotics: implications for drug design.

    Science.gov (United States)

    Vacas, Tatiana; Corzana, Francisco; Jiménez-Osés, Gonzalo; González, Carlos; Gómez, Ana M; Bastida, Agatha; Revuelta, Julia; Asensio, Juan Luis

    2010-09-01

    Aminoglycoside antibiotics participate in a large variety of binding processes involving both RNA and proteins. The description, in recent years, of several clinically relevant aminoglycoside/receptor complexes has greatly stimulated the structural-based design of new bioactive derivatives. Unfortunately, design efforts have frequently met with limited success, reflecting our incomplete understanding of the molecular determinants for the antibiotic recognition. Intriguingly, aromatic rings of the protein/RNA receptors seem to be key actors in this process. Indeed, close inspection of the structural information available reveals that they are frequently involved in CH/pi stacking interactions with sugar/aminocyclitol rings of the antibiotic. While the interaction between neutral carbohydrates and aromatic rings has been studied in detail during past decade, little is known about these contacts when they involve densely charged glycosides. Herein we report a detailed experimental and theoretical analysis of the role played by CH/pi stacking interactions in the molecular recognition of aminoglycosides. Our study aims to determine the influence that the antibiotic polycationic character has on the stability, preferred geometry, and dynamics of these particular contacts. With this purpose, different aminoglycoside/aromatic complexes have been selected as model systems. They varied from simple bimolecular interactions to the more stable intramolecular CH/pi contacts present in designed derivatives. The obtained results highlight the key role played by electrostatic forces and the desolvation of charged groups in the molecular recognition of polycationic glycosides and have clear implications for the design of improved antibiotics.

  10. Enzymatic method for inactivation of aminoglycosides during measurement of postantibiotic effect

    NARCIS (Netherlands)

    J.G. den Hollander (Jan); J.W. Mouton (Johan); I.A.J.M. Bakker-Woudenberg (Irma); F.P. Vleggaar (Frank); M.P.J. van Goor (Marie-Louise); H.A. Verbrugh (Henri)

    1996-01-01

    textabstractTo determine the postantibiotic effect of aminoglycosides, two methods are currently being used to remove the test drug: repeated washing and dilution. An enzymatic inactivation method of removing gentamicin and tobramycin was developed and compared with the dilution me

  11. Natural Bizbenzoquinoline Derivatives Protect Zebrafish Lateral Line Sensory Hair Cells from Aminoglycoside Toxicity.

    Science.gov (United States)

    Kruger, Matthew; Boney, Robert; Ordoobadi, Alexander J; Sommers, Thomas F; Trapani, Josef G; Coffin, Allison B

    2016-01-01

    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. PMID:27065807

  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

    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. 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 skimm

  14. 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...

  15. Network-assisted investigation of virulence and antibiotic-resistance systems in Pseudomonas aeruginosa

    Science.gov (United States)

    Hwang, Sohyun; Kim, Chan Yeong; Ji, Sun-Gou; Go, Junhyeok; Kim, Hanhae; Yang, Sunmo; Kim, Hye Jin; Cho, Ara; Yoon, Sang Sun; Lee, Insuk

    2016-05-01

    Pseudomonas aeruginosa is a Gram-negative bacterium of clinical significance. Although the genome of PAO1, a prototype strain of P. aeruginosa, has been extensively studied, approximately one-third of the functional genome remains unknown. With the emergence of antibiotic-resistant strains of P. aeruginosa, there is an urgent need to develop novel antibiotic and anti-virulence strategies, which may be facilitated by an approach that explores P. aeruginosa gene function in systems-level models. Here, we present a genome-wide functional network of P. aeruginosa genes, PseudomonasNet, which covers 98% of the coding genome, and a companion web server to generate functional hypotheses using various network-search algorithms. We demonstrate that PseudomonasNet-assisted predictions can effectively identify novel genes involved in virulence and antibiotic resistance. Moreover, an antibiotic-resistance network based on PseudomonasNet reveals that P. aeruginosa has common modular genetic organisations that confer increased or decreased resistance to diverse antibiotics, which accounts for the pervasiveness of cross-resistance across multiple drugs. The same network also suggests that P. aeruginosa has developed mechanism of trade-off in resistance across drugs by altering genetic interactions. Taken together, these results clearly demonstrate the usefulness of a genome-scale functional network to investigate pathogenic systems in P. aeruginosa.

  16. High-throughput screening of antibiotic-resistant bacteria in picodroplets.

    Science.gov (United States)

    Liu, X; Painter, R E; Enesa, K; Holmes, D; Whyte, G; Garlisi, C G; Monsma, F J; Rehak, M; Craig, F F; Smith, C A

    2016-04-26

    The prevalence of clinically-relevant bacterial strains resistant to current antibiotic therapies is increasing and has been recognized as a major health threat. For example, multidrug-resistant tuberculosis and methicillin-resistant Staphylococcus aureus are of global concern. Novel methodologies are needed to identify new targets or novel compounds unaffected by pre-existing resistance mechanisms. Recently, water-in-oil picodroplets have been used as an alternative to conventional high-throughput methods, especially for phenotypic screening. Here we demonstrate a novel microfluidic-based picodroplet platform which enables high-throughput assessment and isolation of antibiotic-resistant bacteria in a label-free manner. As a proof-of-concept, the system was used to isolate fusidic acid-resistant mutants and estimate the frequency of resistance among a population of Escherichia coli (strain HS151). This approach can be used for rapid screening of rare antibiotic-resistant mutants to help identify novel compound/target pairs. PMID:27033300

  17. Rapid analysis of aminoglycoside antibiotics in bovine tissues using disposable pipette extraction and ultrahigh performance liquid chromatography - tandem mass spectrometry

    Science.gov (United States)

    A high-throughput qualitative screening and identification method for 9 aminoglycosides of regulatory interest has been developed, validated, and implemented for bovine kidney, liver, and muscle tissues. The method involves extraction at previously validated conditions, cleanup using disposable pip...

  18. 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......-term tolerance, respectively, to this drug class. Here, we show that chaperonin GroEL/GroES over-expression accelerates acquisition of streptomycin resistance and reduces susceptibility to several other antibiotics following sub-lethal streptomycin antibiotic exposure. Chaperonin buffering could provide a novel...

  19. Assessment on the adverse effects of Aminoglycosides and Flouroquinolone on sperm parameters and male reproductive tissue: A systematic review

    OpenAIRE

    Arash Khaki

    2015-01-01

    Background: Antibiotic therapies used in treatment of many diseases have adverse effects on fertility. This review analyzes previous comparative studies that surveyed the effects of two common groups of antibiotics on male fertility. Objective: To evaluate histo-pathological effects of fluoroquinolones and aminoglycosides on sperm parameters and male reproductive tissue. Materials and Methods: Articles about the effects of aminoglycosides and fluoroquinolones on male infertility, sperm parame...

  20. Influence of Chicken Manure Fertilization on Antibiotic-Resistant Bacteria in Soil and the Endophytic Bacteria of Pakchoi.

    Science.gov (United States)

    Yang, Qingxiang; Zhang, Hao; Guo, Yuhui; Tian, Tiantian

    2016-01-01

    Animal manure is commonly used as fertilizer for agricultural crops worldwide, even though it is believed to contribute to the spread of antibiotic resistance from animal intestines to the soil environment. However, it is unclear whether and how there is any impact of manure fertilization on populations and community structure of antibiotic-resistant endophytic bacteria (AREB) in plant tissues. To investigate the effect of manure and organic fertilizer on endophytic bacterial communities, pot experiments were performed with pakchoi grown with the following treatments: (1) non-treated; (2) chicken manure-treated and (3) organic fertilizer-treated. Manure or organic fertilizer significantly increased the abundances of total cultivable endophytic bacteria (TCEB) and AREB in pakchoi, and the effect of chicken manure was greater than that of organic fertilizer. Further, 16S rDNA sequencing and the phylogenetic analysis indicated that chicken manure or organic fertilizer application increased the populations of multiple antibiotic-resistant bacteria (MARB) in soil and multiple antibiotic-resistant endophytic bacteria (MAREB) in pakchoi. The identical multiple antibiotic-resistant bacterial populations detected in chicken manure, manure- or organic fertilizer-amended soil and the vegetable endophytic system were Brevundimonas diminuta, Brachybacterium sp. and Bordetella sp., suggesting that MARB from manure could enter and colonize the vegetable tissues through manure fertilization. The fact that some human pathogens with multiple antibiotic resistance were detected in harvested vegetables after growing in manure-amended soil demonstrated a potential threat to human health. PMID:27376311

  1. Influence of Chicken Manure Fertilization on Antibiotic-Resistant Bacteria in Soil and the Endophytic Bacteria of Pakchoi.

    Science.gov (United States)

    Yang, Qingxiang; Zhang, Hao; Guo, Yuhui; Tian, Tiantian

    2016-01-01

    Animal manure is commonly used as fertilizer for agricultural crops worldwide, even though it is believed to contribute to the spread of antibiotic resistance from animal intestines to the soil environment. However, it is unclear whether and how there is any impact of manure fertilization on populations and community structure of antibiotic-resistant endophytic bacteria (AREB) in plant tissues. To investigate the effect of manure and organic fertilizer on endophytic bacterial communities, pot experiments were performed with pakchoi grown with the following treatments: (1) non-treated; (2) chicken manure-treated and (3) organic fertilizer-treated. Manure or organic fertilizer significantly increased the abundances of total cultivable endophytic bacteria (TCEB) and AREB in pakchoi, and the effect of chicken manure was greater than that of organic fertilizer. Further, 16S rDNA sequencing and the phylogenetic analysis indicated that chicken manure or organic fertilizer application increased the populations of multiple antibiotic-resistant bacteria (MARB) in soil and multiple antibiotic-resistant endophytic bacteria (MAREB) in pakchoi. The identical multiple antibiotic-resistant bacterial populations detected in chicken manure, manure- or organic fertilizer-amended soil and the vegetable endophytic system were Brevundimonas diminuta, Brachybacterium sp. and Bordetella sp., suggesting that MARB from manure could enter and colonize the vegetable tissues through manure fertilization. The fact that some human pathogens with multiple antibiotic resistance were detected in harvested vegetables after growing in manure-amended soil demonstrated a potential threat to human health.

  2. Aminoglycoside Efflux in Pseudomonas aeruginosa: Involvement of Novel Outer Membrane Proteins

    OpenAIRE

    Jo, James T. H.; Brinkman, Fiona S.L.; Hancock, Robert E W

    2003-01-01

    The expression of tripartite multidrug efflux pumps such as MexA-MexB-OprM in Pseudomonas aeruginosa contributes to intrinsic resistance to a wide variety of antimicrobials, including β-lactams, chloramphenicol, macrolides, quinolones, and tetracycline. The MexX-MexY linker-pump combination has been shown to be involved in intrinsic resistance to aminoglycosides, but the identity of the cognate outer membrane channel component remains under debate. Fourteen uncharacterized OprM homologs ident...

  3. Molecular Epidemiology of Aminoglycosides Resistance in Acinetobacter Spp. with Emergence of Multidrug-Resistant Strains

    Directory of Open Access Journals (Sweden)

    MH Nazem Shirazi

    2010-06-01

    Full Text Available Background: Acinetobacter spp. is characterized as an important nosocomial pathogen and increasing antimicrobial resistance. Our aim was to evaluate antimicrobial susceptibility and aminoglycosides resistance genes of Acinetobacter spp. isolated from hospitalized patients.Methods: Sixty isolates were identified as Acinetobacter species. The isolates were tested for antibiotic resistance by disc diffusion method for 12 antimicrobials. The presence of aphA6, aacC1 aadA1, and aadB genes were detected using PCR.Results: From the isolated Acinetobacter spp. the highest resistance rate showed against amikacin, tobramycin, and ceftazidim, respectively; while isolated bacteria were more sensitive to ampicillic/subactam. More than 66% of the isolates were resistant to at least three classes of antibiotics, and 27.5% of MDR strains were resistant to all seven tested classes of antimicrobials. The higher MDR rate presented in bacteria isolated from the ICU and blood samples. More than 60% of the MDR bacteria were resistance to amikacin, ceftazidim, ciprofloxacin, piperacillin/tazobactam, doxycycline, tobramycin and levofloxacin. Also, more than 60% of the isolates contained phosphotransferase aphA6, and acetyltransferase genes aacC1, but adenylyltransferase genes aadA1 (41.7%, and aadB (3.3% were less prominent. 21.7% of the strains contain three aminoglycoside resistance genes (aphA6, aacC1 and aadA1.Conclusion: The rising trend of resistance to aminoglycosides poses an alarming threat to treatment of such infections. The findings showed that clinical isolates of Acinetobacter spp. in our hospital carrying various kinds of aminoglycoside resistance genes.

  4. Rescue of non-sense mutated p53 tumor suppressor gene by aminoglycosides

    OpenAIRE

    Floquet, Célia; Deforges, Jules; Rousset, Jean-Pierre; Bidou, Laure

    2010-01-01

    Mutation-based treatments are a new development in genetic medicine, in which the nature of the mutation dictates the therapeutic strategy. Interest has recently focused on diseases caused by premature termination codons (PTCs). Drugs inducing the readthrough of these PTCs restore the production of a full-length protein. In this study, we explored the possibility of using aminoglycoside antibiotics to induce the production of a full-length functional p53 protein from a gene carrying a PTC. We...

  5. Sodium-Glucose Transporter-2 (SGLT2; SLC5A2) Enhances Cellular Uptake of Aminoglycosides

    OpenAIRE

    Meiyan Jiang; Qi Wang; Takatoshi Karasawa; Ja-Won Koo; Hongzhe Li; Steyger, Peter S.

    2014-01-01

    Aminoglycoside antibiotics, like gentamicin, continue to be clinically essential worldwide to treat life-threatening bacterial infections. Yet, the ototoxic and nephrotoxic side-effects of these drugs remain serious complications. A major site of gentamicin uptake and toxicity resides within kidney proximal tubules that also heavily express electrogenic sodium-glucose transporter-2 (SGLT2; SLC5A2) in vivo. We hypothesized that SGLT2 traffics gentamicin, and promotes cellular toxicity. We conf...

  6. Synergistic Effect of Oleanolic Acid on Aminoglycoside Antibiotics against Acinetobacter baumannii.

    Directory of Open Access Journals (Sweden)

    Bora Shin

    Full Text Available Difficulties involved in treating drug-resistant pathogens have created a need for new therapies. In this study, we investigated the possibility of using oleanolic acid (OA, a natural pentacyclic triterpenoid, as a natural adjuvant for antibiotics against Acinetobacter baumannii. High concentrations of OA can kill cells, partly because it generates reactive oxygen species. Measurement of the fractional inhibitory concentration (FIC for OA and time-kill experiments demonstrated that it only synergizes with aminoglycoside antibiotics (e.g., gentamicin, kanamycin. Other classes of antibiotics (e.g., ampicillin, rifampicin, norfloxacin, chloramphenicol, and tetracycline have no interactions with OA. Microarray and quantitative reverse transcription-PCR analysis indicated that genes involved in ATP synthesis and cell membrane permeability, the gene encoding glycosyltransferase, peptidoglycan-related genes, phage-related genes, and DNA repair genes were upregulated under OA. OA highly induces the expression of adk, which encodes an adenylate kinase, and des6, which encodes a linoleoyl-CoA desaturase, and deletion of these genes increased FICs; these observations indicate that adk and des6 are involved in the synergism of OA with aminoglycosides. Data obtained using 8-anilino-1-naphthalenesulfonic acid, fluorescence-conjugated gentamicin, and membrane fatty acid analysis indicates that adk and des6 are involved in changes in membrane permeability. Proton-motive force and ATP synthesis tests show that those genes are also involved in energy metabolism. Taken together, our data show that OA boosts aminoglycoside uptake by changing membrane permeability and energy metabolism in A. baumannii.

  7. LOWER DOSE OF AMINOGLYCOSIDE OTOTOXIC EXPOSURE CAUSES PRESYNAPTIC ALTERATIONS ASSOICATED WITH HEARING LOSS

    Institute of Scientific and Technical Information of China (English)

    LIU Ke; WANG Xiaoyu; LI Sijun; TANG Siquan; XU Yice; WANG Xuefeng; SUN Jianhe; YANG Weiyan; YANG Shiming

    2014-01-01

    Objective To study presynaptic alternations of cochlear ribbons arising from aminoglycoside ototoxic stimuli in C57BL/6J mice. Methods Animals were injected with low dose gentamicin (100 mg/kg/day) for 14 days, From the 14th to 28th days, the mice were maintained free of gentamicin treatment. Immunohisto-chemistry labeling was employed to trace RIBEYE, a major presynaptic componment of ribbon synapses. RIBEYE/CtBP2 expression levels were assessed and compared with hearing threshold shifts. Auditory func-tion was assessed by auditory brainstem responses. The stereocilia of outer hair cells (OHCs) and IHCs was examined by scanning electron microscopy (SEM). Results Hearing thresholds were elevated with peak hearing loss observed on the 7th day after gentamicin exposure, followed by improvement after the 7th day. RIBEYE/CtBP2 expression directly correlated with observed hearing threshold shifts. Strikingly, we did not see any obvious changes in stereocilia in both OHCs and IHCs until the 28th day. Mild changes in stereocil-ia were only observed in OHCs on the 28th day. Conclusions These findings indicate that presynapse co-chlear ribbons, rather than stereocilia, may be sensitive to aminoglycoside ototoxic exposure in mice cochle-ae. A pattern of RIBEYE/CtBP2 expression changes seems to parallel hearing threshold shifts and suggests presynaptic response properties to lower dosage of aminoglycoside ototoxic stimuli.

  8. [Is it possible to reduce the incidence of aminoglycoside-induced nephrotoxicity?].

    Science.gov (United States)

    Fillastre, J P

    1999-01-01

    The incidence of nephrotoxicity due to aminoglycosides should be sharply reduced. The indications for prescribing these antibiotics should be limited to infectious disorders induced by aerobic Gram-negative bacteria and by some Gram-positive bacteria requiring treatment in specialized hospital units using an association of aminoglycosides and another antibiotic. Daily doses should not exceed those indicated by the manufacturer, and the length of treatment should be as short as possible, with a relay to other antibiotics that are not or are less nephrotoxic. The possibilities for reducing the incidence of nephrotoxicity are few. It is not possible to prevent the antibiotic from entering the renal tubular cell or from producing deleterious effects therein. However, by using short-term intravenous infusion as the administration route, prolonged contact between the antibiotic and its receptors on the brush borders of the proximal tubular cells can be avoided, particularly since the process of cellular absorption is saturable. Essentially, doses should be adapted according to the age and the glomerular filtration of the patient, since renal function usually decreases with age. Volemic and hydroelectrolytic disorders favour nephrotoxicity and should be corrected. Associations with other nephrotoxic drugs should either be avoided or used with increased caution. The same is true in special situations such as endotoxaemia, severe renal parenchymatous infections and cholestasis. In any case, given the well-known insidious onset of nephropathy, aminoglycoside treatment always requires laboratory follow-up consisting of repeated testing of creatinemia during the two weeks of treatment. PMID:10465001

  9. Antibacterial Efficacy of Lytic Bacteriophages against Antibiotic-Resistant Klebsiella Species

    Directory of Open Access Journals (Sweden)

    M. Khajeh Karamoddini

    2011-01-01

    Full Text Available Bacterial resistance to antibiotics is a leading and highly prevalent problem in the treatment of infectious diseases. Bacteriophages (phages appear to be effective and safe alternatives for the treatment of resistant infections because of their specificity for bacterial species and lack of infectivity in eukaryotic cells. The present study aimed to isolate bacteriophages against Klebsiella spp. and evaluate their efficacy against antibiotic-resistant species. Seventy-two antibiotic-resistant Klebsiella spp. were isolated from samples of patients who referred to the Ghaem Hospital (Mashhad, Iran. Lytic bacteriophages against Klebsiella spp. were isolated from wastewater of the septic tank of the same hospital. Bactericidal activity of phages against resistant Klebsiella spp. was tested in both liquid (tube method; after 1 and 24 h of incubation and solid (double-layer agar plate method; after 24 h of incubation phases. In each method, three different concentrations of bacteriophages (low: 107 PFU/mL were used. Bacteriophages showed promising bactericidal activity at all assessed concentrations, regardless of the test method and duration of incubation. Overall, bactericidal effects were augmented at higher concentrations. In the tube method, higher activity was observed after 24 h of incubation compared to the 1-h incubation. The bactericidal effects were also higher in the tube method compared to the double-layer agar plate method after 24 h of incubation. The findings of the present study suggest that bacteriophages possess effective bactericidal activity against resistant Klebsiella spp. These bactericidal activities are influenced by phage concentration, duration of incubation, and test method.

  10. Prevalence of antibiotic-resistant bacteria in three different aquatic environments over three seasons.

    Science.gov (United States)

    Mohanta, Tandra; Goel, Sudha

    2014-08-01

    The objective of this study was to evaluate the impact of urbanization and seasonal changes on the prevalence of antibiotic-resistant bacteria in different aqueous environments. To this end, bacteria were isolated from three different water sources: the River Hooghly in Kolkata, River Kangsabati and groundwater from Kharagpur, West Bengal over three seasons: post-monsoon, winter and summer in 2012-2013. A total of 163 Gram-negative bacteria were isolated from the River Hooghly (n = 138), River Kangsabati (n = 13) and groundwater (n = 12). Antibiotic susceptibility testing was done using 12 antibiotic discs. The percentages of multiple antibiotic-resistant (MAR) bacteria at the three sampling locations were found to be 71.01 % (98/138) for River Hooghly, 15.38 % (2/13) for River Kangsabati and 8.33 % (1/12) for groundwater. Prevalence of MAR bacteria with respect to the three seasons were the following: 73.58 % in post-monsoon, 59.26 % in winter and 53.57 % in summer. Antibiotic resistance index (ARI) was calculated for each location and each season. In general, ARI values for all the River Hooghly samples were >0.2 while those for the River Kangsabati and groundwater in Kharagpur were always resistance in bacteria from the River Hooghly compared to the other two locations. In addition, percentage of MAR and ARI values followed the trend: post-monsoon > winter > summer. This may be due to the additional terrestrial resistants that get swept along with surface runoff during the monsoons.

  11. Bacteriophage therapy for membrane biofouling in membrane bioreactors and antibiotic-resistant bacterial biofilms.

    Science.gov (United States)

    Bhattacharjee, Ananda Shankar; Choi, Jeongdong; Motlagh, Amir Mohaghegh; Mukherji, Sachiyo T; Goel, Ramesh

    2015-08-01

    To demonstrate elimination of bacterial biofilm on membranes to represent wastewater treatment as well as biofilm formed by antibiotic-resistant bacterial (ARB) to signify medical application, an antibiotic-resistant bacterium and its lytic bacteriophage were isolated from a full-scale wastewater treatment plant. Based on gram staining and complete 16 S rDNA sequencing, the isolated bacterium showed a more than 99% homology with Delftia tsuruhatensis, a gram-negative bacterium belonging to β-proteobacteria. The Delftia lytic phage's draft genome revealed the phage to be an N4-like phage with 59.7% G + C content. No transfer RNAs were detected for the phage suggesting that the phage is highly adapted to its host Delftia tsuruhatensis ARB-1 with regard to codon usage, and does not require additional tRNAs of its own. The gene annotation of the Delftia lytic phage found three different components of RNA polymerase (RNAP) in the genome, which is a typical characteristic of N4-like phages. The lytic phage specific to D. tsuruhatensis ARB-1 could successfully remove the biofilm formed by it on a glass slide. The water flux through the membrane of a prototype lab-scale membrane bioreactor decreased from 47 L/h m(2) to ∼15 L/h m(2) over 4 days due to a biofilm formed by D. tsuruhatensis ARB-1. However, the flux increased to 70% of the original after the lytic phage application. Overall, this research demonstrated phage therapy's great potential to solve the problem of membrane biofouling, as well as the problems posed by pathogenic biofilms in external wounds and on medical instruments.

  12. Antibiotic-Resistant Neisseria gonorrhoeae Spread Faster with More Treatment, Not More Sexual Partners.

    Science.gov (United States)

    Fingerhuth, Stephanie M; Bonhoeffer, Sebastian; Low, Nicola; Althaus, Christian L

    2016-05-01

    The sexually transmitted bacterium Neisseria gonorrhoeae has developed resistance to all antibiotic classes that have been used for treatment and strains resistant to multiple antibiotic classes have evolved. In many countries, there is only one antibiotic remaining for empirical N. gonorrhoeae treatment, and antibiotic management to counteract resistance spread is urgently needed. Understanding dynamics and drivers of resistance spread can provide an improved rationale for antibiotic management. In our study, we first used antibiotic resistance surveillance data to estimate the rates at which antibiotic-resistant N. gonorrhoeae spread in two host populations, heterosexual men (HetM) and men who have sex with men (MSM). We found higher rates of spread for MSM (0.86 to 2.38 y-1, mean doubling time: 6 months) compared to HetM (0.24 to 0.86 y-1, mean doubling time: 16 months). We then developed a dynamic transmission model to reproduce the observed dynamics of N. gonorrhoeae transmission in populations of heterosexual men and women (HMW) and MSM. We parameterized the model using sexual behavior data and calibrated it to N. gonorrhoeae prevalence and incidence data. In the model, antibiotic-resistant N. gonorrhoeae spread with a median rate of 0.88 y-1 in HMW and 3.12 y-1 in MSM. These rates correspond to median doubling times of 9 (HMW) and 3 (MSM) months. Assuming no fitness costs, the model shows the difference in the host population's treatment rate rather than the difference in the number of sexual partners explains the differential spread of resistance. As higher treatment rates result in faster spread of antibiotic resistance, treatment recommendations for N. gonorrhoeae should carefully balance prevention of infection and avoidance of resistance spread. PMID:27196299

  13. Antibiotic-Resistant Neisseria gonorrhoeae Spread Faster with More Treatment, Not More Sexual Partners.

    Directory of Open Access Journals (Sweden)

    Stephanie M Fingerhuth

    2016-05-01

    Full Text Available The sexually transmitted bacterium Neisseria gonorrhoeae has developed resistance to all antibiotic classes that have been used for treatment and strains resistant to multiple antibiotic classes have evolved. In many countries, there is only one antibiotic remaining for empirical N. gonorrhoeae treatment, and antibiotic management to counteract resistance spread is urgently needed. Understanding dynamics and drivers of resistance spread can provide an improved rationale for antibiotic management. In our study, we first used antibiotic resistance surveillance data to estimate the rates at which antibiotic-resistant N. gonorrhoeae spread in two host populations, heterosexual men (HetM and men who have sex with men (MSM. We found higher rates of spread for MSM (0.86 to 2.38 y-1, mean doubling time: 6 months compared to HetM (0.24 to 0.86 y-1, mean doubling time: 16 months. We then developed a dynamic transmission model to reproduce the observed dynamics of N. gonorrhoeae transmission in populations of heterosexual men and women (HMW and MSM. We parameterized the model using sexual behavior data and calibrated it to N. gonorrhoeae prevalence and incidence data. In the model, antibiotic-resistant N. gonorrhoeae spread with a median rate of 0.88 y-1 in HMW and 3.12 y-1 in MSM. These rates correspond to median doubling times of 9 (HMW and 3 (MSM months. Assuming no fitness costs, the model shows the difference in the host population's treatment rate rather than the difference in the number of sexual partners explains the differential spread of resistance. As higher treatment rates result in faster spread of antibiotic resistance, treatment recommendations for N. gonorrhoeae should carefully balance prevention of infection and avoidance of resistance spread.

  14. Antibiotic-Resistant Neisseria gonorrhoeae Spread Faster with More Treatment, Not More Sexual Partners.

    Science.gov (United States)

    Fingerhuth, Stephanie M; Bonhoeffer, Sebastian; Low, Nicola; Althaus, Christian L

    2016-05-01

    The sexually transmitted bacterium Neisseria gonorrhoeae has developed resistance to all antibiotic classes that have been used for treatment and strains resistant to multiple antibiotic classes have evolved. In many countries, there is only one antibiotic remaining for empirical N. gonorrhoeae treatment, and antibiotic management to counteract resistance spread is urgently needed. Understanding dynamics and drivers of resistance spread can provide an improved rationale for antibiotic management. In our study, we first used antibiotic resistance surveillance data to estimate the rates at which antibiotic-resistant N. gonorrhoeae spread in two host populations, heterosexual men (HetM) and men who have sex with men (MSM). We found higher rates of spread for MSM (0.86 to 2.38 y-1, mean doubling time: 6 months) compared to HetM (0.24 to 0.86 y-1, mean doubling time: 16 months). We then developed a dynamic transmission model to reproduce the observed dynamics of N. gonorrhoeae transmission in populations of heterosexual men and women (HMW) and MSM. We parameterized the model using sexual behavior data and calibrated it to N. gonorrhoeae prevalence and incidence data. In the model, antibiotic-resistant N. gonorrhoeae spread with a median rate of 0.88 y-1 in HMW and 3.12 y-1 in MSM. These rates correspond to median doubling times of 9 (HMW) and 3 (MSM) months. Assuming no fitness costs, the model shows the difference in the host population's treatment rate rather than the difference in the number of sexual partners explains the differential spread of resistance. As higher treatment rates result in faster spread of antibiotic resistance, treatment recommendations for N. gonorrhoeae should carefully balance prevention of infection and avoidance of resistance spread.

  15. Antibacterial efficacy of lytic bacteriophages against antibiotic-resistant Klebsiella species.

    Science.gov (United States)

    Karamoddini, M Khajeh; Fazli-Bazzaz, B S; Emamipour, F; Ghannad, M Sabouri; Jahanshahi, A R; Saed, N; Sahebkar, A

    2011-07-07

    Bacterial resistance to antibiotics is a leading and highly prevalent problem in the treatment of infectious diseases. Bacteriophages (phages) appear to be effective and safe alternatives for the treatment of resistant infections because of their specificity for bacterial species and lack of infectivity in eukaryotic cells. The present study aimed to isolate bacteriophages against Klebsiella spp. and evaluate their efficacy against antibiotic-resistant species. Seventy-two antibiotic-resistant Klebsiella spp. were isolated from samples of patients who referred to the Ghaem Hospital (Mashhad, Iran). Lytic bacteriophages against Klebsiella spp. were isolated from wastewater of the septic tank of the same hospital. Bactericidal activity of phages against resistant Klebsiella spp. was tested in both liquid (tube method; after 1 and 24 h of incubation) and solid (double-layer agar plate method; after 24 h of incubation) phases. In each method, three different concentrations of bacteriophages (low: 10(7) PFU/mL) were used. Bacteriophages showed promising bactericidal activity at all assessed concentrations, regardless of the test method and duration of incubation. Overall, bactericidal effects were augmented at higher concentrations. In the tube method, higher activity was observed after 24 h of incubation compared to the 1-h incubation. The bactericidal effects were also higher in the tube method compared to the double-layer agar plate method after 24 h of incubation. The findings of the present study suggest that bacteriophages possess effective bactericidal activity against resistant Klebsiella spp. These bactericidal activities are influenced by phage concentration, duration of incubation, and test method.

  16. A rapid method for detection of five known mutations associated with aminoglycoside-induced deafness

    Directory of Open Access Journals (Sweden)

    Greinwald John H

    2009-01-01

    Full Text Available Abstract Background South Africa has one of the highest incidences of multidrug-resistant tuberculosis (MDR-TB in the world. Concomitantly, aminoglycosides are commonly used in this country as a treatment against MDR-TB. To date, at least five mutations are known to confer susceptibility to aminoglycoside-induced hearing loss. The aim of the present study was to develop a rapid screening method to determine whether these mutations are present in the South African population. Methods A multiplex method using the SNaPshot technique was used to screen for five mutations in the MT-RNR1 gene: A1555G, C1494T, T1095C, 961delT+C(n and A827G. A total of 204 South African control samples, comprising 98 Mixed ancestry and 106 Black individuals were screened for the presence of the five mutations. Results A robust, cost-effective method was developed that detected the presence of all five sequence variants simultaneously. In this pilot study, the A1555G mutation was identified at a frequency of 0.9% in the Black control samples. The 961delT+C(n variant was present in 6.6% of the Black controls and 2% of the Mixed ancestry controls. The T1095C, C1494T and A827G variants were not identified in any of the study participants. Conclusion The frequency of 0.9% for the A1555G mutation in the Black population in South Africa is of concern given the high incidence of MDR-TB in this particular ethnic group. Future larger studies are warranted to determine the true frequencies of the aminoglycoside deafness mutations in the general South African population. The high frequencies of the 961delT+C(n variant observed in the controls suggest that this change is a common non-pathogenic polymorphism. This genetic method facilitates the identification of individuals at high risk of developing hearing loss prior to the start of aminoglycoside therapy. This is important in a low-resource country like South Africa where, despite their adverse side-effects, aminoglycosides will

  17. 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.

  18. 16S rRNA甲基化介导的氨基糖苷类耐药%Resistance mechanism against aminoglycosides mediated by 16S rRNA methylation

    Institute of Scientific and Technical Information of China (English)

    张晓文

    2012-01-01

    Aminoglycosid.es have been used for the treatment of a broad range of life -threatening Gram-positive and Grarrmeg-ative bacterial infections. These agents bind to the A site of the 16S rRNA of the bacterial 30S ribosomal subunit and subsequently block its growth through interference with its protein synthesis . 16S rRNA methylation is capable of conferring an extraordinarily high level of resistance against most of the clinically important aminoglycosides . Previous research has shown that this phenomenon is media -ted by some 16S rRNA methylase. Because of the clinical importance of these enzymes , further global dissemination of 16S rRNA methylase genes among pathogenic bacilli will be a cause of great concern in the near future . This article presents an overview on the action mechanism, origin, classification and genetic environment of 16S rRNA methylase.%氨基糖苷类抗生素在治疗革兰阳性和阴性细菌引起的感染中起着重要的作用,可通过与细菌30S核糖体亚基的16S rRNA的A位点结合而阻碍蛋白质的合成.16S rRNA甲基化作用可导致细菌对氨基糖苷类药物高水平耐药,大量研究显示这一现象是由一类16S rRNA甲基化酶所介导的.由于16S rRNA甲基化酶在临床上的重要性,为引起医务人员的重视,文中将从此类酶的作用机制、起源、分类以及基因环境等方面作一综述.

  19. Draft Genome Sequence of an Antibiotic-Resistant Propionibacterium acnes Strain, PRP-38, from the Novel Type IC Cluster

    OpenAIRE

    McDowell, Andrew; Hunyadkürti, Judit; Horváth, Balázs; Vörös, Andrea; Barnard, Emma; Patrick, Sheila; Nagy, István

    2012-01-01

    Propionibacterium acnes, a non-spore-forming, anaerobic Gram-positive bacterium, is most notably recognized for its association with acne vulgaris (I. Kurokawa et al., Exp. Dermatol. 18:821–832, 2009). We now present the draft genome sequence of an antibiotic-resistant P. acnes strain, PRP-38, isolated from an acne patient in the United Kingdom and belonging to the novel type IC cluster.

  20. Kunstige Enzymer

    DEFF Research Database (Denmark)

    Bols, Mikael; Bjerre, Jeannette; Marinescu, Lavinia

    2007-01-01

    Enzymer har en enestående evne til at accelerere kemiske processer. Der forskes målrettet i at optimere enzymer baseret på cyclodextrin.......Enzymer har en enestående evne til at accelerere kemiske processer. Der forskes målrettet i at optimere enzymer baseret på cyclodextrin....

  1. Impacts of urbanization on the prevalence of antibiotic-resistant Escherichia coli in the Chaophraya River and its tributaries.

    Science.gov (United States)

    Honda, Ryo; Watanabe, Toru; Sawaittayotin, Variga; Masago, Yoshifumi; Chulasak, Rungnapa; Tanong, Kulchaya; Chaminda, G Tushara; Wongsila, Krison; Sienglum, Chawala; Sunthonwatthanaphong, Varisara; Poonnotok, Anupong; Chiemchaisri, Wilai; Chiemchaisri, Chart; Furumai, Hiroaki; Yamamoto, Kazuo

    2016-01-01

    River water samples were taken from 32 locations around the basin of Chaophraya River and its four major tributaries in Thailand to investigate resistance ratios of Escherichia coli isolates to eight antibiotic agents of amoxicillin, sulfamethoxazole/trimethoprim, tetracycline, doxytetracycline, ciprofloxacin, levofloxacin, norfloxacin and ofloxacin. Principal component analysis was performed to characterize resistance patterns of the samples. Relevancy of the obtained principal components with urban land use and fecal contamination of the river were examined. The ratio of antibiotic-resistant bacteria is likely to increase when urban land use near the sampling site exceeds a certain ratio. The resistance ratio to fluoroquinolones tends to be high in a highly populated area. Meanwhile, no significant contribution of fecal contamination was found to increase the resistance ratio. These results suggest that an antibiotic-resistance ratio is dependent on conditions of local urbanization rather than the upstream conditions, and that the major sources of antibiotic-resistant bacteria in the Chaophraya River basin are possibly point sources located in the urban area which contains a high ratio of resistant bacteria. PMID:26819392

  2. Prevalence of Antibiotic-Resistant Fecal Escherichia coli Isolates from Penned Broiler and Scavenging Local Chickens in Arusha, Tanzania.

    Science.gov (United States)

    Rugumisa, Bernadether T; Call, Douglas R; Mwanyika, Gaspary O; Mrutu, Rehema I; Luanda, Catherine M; Lyimo, Beatus M; Subbiah, Murugan; Buza, Joram J

    2016-08-01

    We compared the prevalence of antibiotic-resistant Escherichia coli isolates from household-level producers of broiler (commercial source breeds) and local chickens in the Arusha District of Tanzania. Households were composed of a single dwelling or residence with independent, penned broiler flocks. Free-range, scavenging chickens were mixed breed and loosely associated with individual households. A total of 1,800 E. coli isolates (1,200 from broiler and 600 from scavenging local chickens) from 75 chickens were tested for their susceptibility against 11 antibiotics by using breakpoint assays. Isolates from broiler chickens harbored a higher prevalence of antibiotic-resistant E. coli relative to scavenging local chickens, including sulfamethoxazole (80.3 versus 34%), followed by trimethoprim (69.3 versus 27.7%), tetracycline (56.8 versus 20%), streptomycin (52.7 versus 24.7%), amoxicillin (49.6 versus 17%), ampicillin (49.1 versus 16.8%), ciprofloxacin (21.9 versus 1.7%), and chloramphenicol (1.5 versus 1.2%). Except for resistance to chloramphenicol, scavenging local chickens harbored fewer resistant E. coli isolates (P < 0.05). Broiler chickens harbored more isolates that were resistant to ≥7 antibiotics (P < 0.05). The higher prevalence of antibiotic-resistant E. coli from broiler chickens correlated with the reported therapeutic and prophylactic use of antibiotics in this poultry population. We suggest that improved biosecurity measures and increased vaccination efforts would reduce reliance on antibiotics by these households.

  3. Comparative analysis of bacterial community and antibiotic-resistant strains in different developmental stages of the housefly (Musca domestica).

    Science.gov (United States)

    Wei, Ting; Hu, Jun; Miyanaga, Kazuhiko; Tanji, Yasunori

    2013-02-01

    The housefly (Musca domestica) is an important host for a variety of bacteria, including some pathogenic and antibiotic-resistant strains. To further investigate the relationship between the housefly and the bacteria it harbors, it is necessary to understand the fate of microorganisms during the larval metamorphosis. The major bacterial communities in three developmental stages of the housefly (maggot, pupa, and adult fly) were investigated by a culture-independent method, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of 16S rRNA genes. The bacteria that were identified using DGGE analysis spanned phyla Proteobacteria, Firmicutes, and Bacteroidetes. Changes in the predominant genera were observed during the housefly development. Bacteroides, Koukoulia, and Schineria were detected in maggots, Neisseria in pupae, and Macrococcus, Lactococcus, and Kurthia in adult flies. Antibiotic-resistant bacteria were screened using a selective medium and tested for antibiotic susceptibility. Most resistant isolates from maggots and pupae were classified as Proteus spp., while those from adult flies were much more diverse and spanned 12 genera. Among 20 tested strains across the three stages, 18 were resistant to at least two antibiotics. Overall, we demonstrated that there are changes in the major bacterial communities and antibiotic-resistant strains as the housefly develops. PMID:22526786

  4. Comparative analysis of bacterial community and antibiotic-resistant strains in different developmental stages of the housefly (Musca domestica).

    Science.gov (United States)

    Wei, Ting; Hu, Jun; Miyanaga, Kazuhiko; Tanji, Yasunori

    2013-02-01

    The housefly (Musca domestica) is an important host for a variety of bacteria, including some pathogenic and antibiotic-resistant strains. To further investigate the relationship between the housefly and the bacteria it harbors, it is necessary to understand the fate of microorganisms during the larval metamorphosis. The major bacterial communities in three developmental stages of the housefly (maggot, pupa, and adult fly) were investigated by a culture-independent method, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of 16S rRNA genes. The bacteria that were identified using DGGE analysis spanned phyla Proteobacteria, Firmicutes, and Bacteroidetes. Changes in the predominant genera were observed during the housefly development. Bacteroides, Koukoulia, and Schineria were detected in maggots, Neisseria in pupae, and Macrococcus, Lactococcus, and Kurthia in adult flies. Antibiotic-resistant bacteria were screened using a selective medium and tested for antibiotic susceptibility. Most resistant isolates from maggots and pupae were classified as Proteus spp., while those from adult flies were much more diverse and spanned 12 genera. Among 20 tested strains across the three stages, 18 were resistant to at least two antibiotics. Overall, we demonstrated that there are changes in the major bacterial communities and antibiotic-resistant strains as the housefly develops.

  5. Phytochemical screening and synergistic interactions between aminoglycosides, selected antibiotics and extracts from the bryophyte Octoblepharum albidum Hedw (Calymperaceae

    Directory of Open Access Journals (Sweden)

    Vidal C.A.S.

    2012-01-01

    Full Text Available This work is the first to describe the modulation of antibiotic activity of the bryophyte Octoblepharum albidum Hedw extract. The antibacterial activity of ethanolic extract of O. albidum (EEOa, alone and in association with aminoglycosides, was determined against six bacterial strains by a microdilution test. The results showed a similar inhibitory activity of EEOa against Escherichia coli ATCC 25922 and Klebsiella pneumoniae ATCC 33018 (MICs 512 μg/mL. The synergistic effect of the extracts and aminoglycosides was also verified. The most pronounced effects were obtained with EEOa + gentamicin against E. coli and EEOa + kanamycin against K. pneumoniae with MICs reduction (128 to 32 μg/mL. The data from this study are indicative of the antibacterial activity of the bryophyte O. albidum extracts and its potential in modifying the resistance of aminoglycosides analyzed.

  6. Correction of ATM gene function by aminoglycoside-induced read-through of premature termination codons

    OpenAIRE

    Lai, Chih-Hung; Chun, Helen H.; Nahas, Shareef A.; Mitui, Midori; Gamo, Kristin M.; Du, Liutao; Gatti, Richard A.

    2004-01-01

    Approximately 14% of genetic mutations in patients with ataxia-telangiectsia (A-T) are single-nucleotide changes that result in primary premature termination codons (PTCs), either UAA, UAG, or UGA. The purpose of this study was to explore a potential therapeutic approach for this subset of patients by using aminoglycosides to induce PTC read-through, thereby restoring levels of full-length ATM (A-T mutated) protein. In experiments using a modified in vitro cDNA coupled transcription/translati...

  7. Berberine Is a Novel Type Efflux Inhibitor Which Attenuates the MexXY-Mediated Aminoglycoside Resistance in Pseudomonas aeruginosa

    Science.gov (United States)

    Morita, Yuji; Nakashima, Ken-ichi; Nishino, Kunihiko; Kotani, Kenta; Tomida, Junko; Inoue, Makoto; Kawamura, Yoshiaki

    2016-01-01

    The emergence and spread of multidrug-resistant P. aeruginosa infections is of great concern, as very few agents are effective against strains of this species. Methanolic extracts from the Coptidis Rhizoma (the rhizomes of Coptis japonica var. major Satake) or Phellodendri Cortex (the bark of Phellodendron chinense Schneider) markedly reduced resistance to anti-pseudomonal aminoglycosides (e.g., amikacin) in multidrug-resistant P. aeruginosa strains. Berberine, the most abundant benzylisoquinoline alkaloid in the two extracts, reduced aminoglycoside resistance of P. aeruginosa via a mechanism that required the MexXY multidrug efflux system; berberine also reduced aminoglycoside MICs in Achromobacter xylosoxidans and Burkholderia cepacia, two species that harbor intrinsic multidrug efflux systems very similar to the MexXY. Furthermore this compound inhibited MexXY-dependent antibiotic resistance of other classes including cephalosporins (cefepime), macrolides (erythromycin), and lincosamides (lincomycin) demonstrated using a pseudomonad lacking the four other major Mex pumps. Although phenylalanine-arginine beta-naphthylamide (PAβN), a well-known efflux inhibitor, antagonized aminoglycoside in a MexXY-dependent manner, a lower concentration of berberine was sufficient to reduce amikacin resistance of P. aeruginosa in the presence of PAβN. Moreover, berberine enhanced the synergistic effects of amikacin and piperacillin (and vice versa) in multidrug-resistant P. aeruginosa strains. Thus, berberine appears to be a novel type inhibitor of the MexXY-dependent aminoglycoside efflux in P. aeruginosa. As aminoglycosides are molecules of choice to treat severe infections the clinical impact is potentially important. PMID:27547203

  8. Berberine Is a Novel Type Efflux Inhibitor Which Attenuates the MexXY-Mediated Aminoglycoside Resistance in Pseudomonas aeruginosa.

    Science.gov (United States)

    Morita, Yuji; Nakashima, Ken-Ichi; Nishino, Kunihiko; Kotani, Kenta; Tomida, Junko; Inoue, Makoto; Kawamura, Yoshiaki

    2016-01-01

    The emergence and spread of multidrug-resistant P. aeruginosa infections is of great concern, as very few agents are effective against strains of this species. Methanolic extracts from the Coptidis Rhizoma (the rhizomes of Coptis japonica var. major Satake) or Phellodendri Cortex (the bark of Phellodendron chinense Schneider) markedly reduced resistance to anti-pseudomonal aminoglycosides (e.g., amikacin) in multidrug-resistant P. aeruginosa strains. Berberine, the most abundant benzylisoquinoline alkaloid in the two extracts, reduced aminoglycoside resistance of P. aeruginosa via a mechanism that required the MexXY multidrug efflux system; berberine also reduced aminoglycoside MICs in Achromobacter xylosoxidans and Burkholderia cepacia, two species that harbor intrinsic multidrug efflux systems very similar to the MexXY. Furthermore this compound inhibited MexXY-dependent antibiotic resistance of other classes including cephalosporins (cefepime), macrolides (erythromycin), and lincosamides (lincomycin) demonstrated using a pseudomonad lacking the four other major Mex pumps. Although phenylalanine-arginine beta-naphthylamide (PAβN), a well-known efflux inhibitor, antagonized aminoglycoside in a MexXY-dependent manner, a lower concentration of berberine was sufficient to reduce amikacin resistance of P. aeruginosa in the presence of PAβN. Moreover, berberine enhanced the synergistic effects of amikacin and piperacillin (and vice versa) in multidrug-resistant P. aeruginosa strains. Thus, berberine appears to be a novel type inhibitor of the MexXY-dependent aminoglycoside efflux in P. aeruginosa. As aminoglycosides are molecules of choice to treat severe infections the clinical impact is potentially important. PMID:27547203

  9. Silver nanoparticle-embedded polymersome nanocarriers for the treatment of antibiotic-resistant infections

    Science.gov (United States)

    Geilich, Benjamin M.; van de Ven, Anne L.; Singleton, Gloria L.; Sepúlveda, Liuda J.; Sridhar, Srinivas; Webster, Thomas J.

    2015-02-01

    The rapidly diminishing number of effective antibiotics that can be used to treat infectious diseases and associated complications in a physician's arsenal is having a drastic impact on human health today. This study explored the development and optimization of a polymersome nanocarrier formed from a biodegradable diblock copolymer to overcome bacterial antibiotic resistance. Here, polymersomes were synthesized containing silver nanoparticles embedded in the hydrophobic compartment, and ampicillin in the hydrophilic compartment. Results showed for the first time that these silver nanoparticle-embedded polymersomes (AgPs) inhibited the growth of Escherichia coli transformed with a gene for ampicillin resistance (bla) in a dose-dependent fashion. Free ampicillin, AgPs without ampicillin, and ampicillin polymersomes without silver nanoparticles had no effect on bacterial growth. The relationship between the silver nanoparticles and ampicillin was determined to be synergistic and produced complete growth inhibition at a silver-to-ampicillin ratio of 1 : 0.64. In this manner, this study introduces a novel nanomaterial that can effectively treat problematic, antibiotic-resistant infections in an improved capacity which should be further examined for a wide range of medical applications.

  10. Antibiotic surgical prophylaxis increases nasal carriage of antibiotic-resistant staphylococci.

    Science.gov (United States)

    McMurray, Claire L; Hardy, Katherine J; Verlander, Neville Q; Hawkey, Peter M

    2015-12-01

    Staphylococci are a significant cause of hospital-acquired infection. Nasal carriage of Staphylococcus aureus is an important risk factor for infection in surgical patients and coagulase-negative staphylococci (CNS) are a major cause of prosthetic joint infections. The impact that antibiotic surgical prophylaxis has on the nasal carriage of staphylococci has not been studied. Daily nasal swabs were taken from 63 patients who received antibiotic surgical prophylaxis and 16 patients who received no antibiotics. Total aerobic bacterial count, S. aureus and CNS were enumerated by culture from nasal swabs. Representative isolates were typed by staphylococcal interspersed repeat units (SIRU) typing and PFGE, and MICs to nine antibiotics were determined. After antibiotic administration, there was a reduction in S. aureus counts (median - 2.3 log(10)c.f.u. ml(- 1)) in 64.0 % of S. aureus carriers, compared with only a 0.89 log(10)c.f.u. ml(- 1) reduction in 75.0 % of S. aureus carriers who did not receive antibiotics. A greater increase in the nasal carriage rate of meticillin-resistant CNS was observed after antibiotic surgical prophylaxis compared with hospitalization alone, with increases of 16.4 and 4.6 %, respectively. Antibiotic-resistant S. epidermidis carriage rate increased by 16.6 % after antibiotic administration compared with 7.5 % with hospitalization alone. Antibiotic surgical prophylaxis impacts the nasal carriage of both S. aureus and CNS.

  11. Antibiotic concentration and antibiotic-resistant bacteria in two shallow urban lakes after stormwater event.

    Science.gov (United States)

    Zhang, Songhe; Pang, Si; Wang, PeiFang; Wang, Chao; Han, Nini; Liu, Bin; Han, Bing; Li, Yi; Anim-Larbi, Kwaku

    2016-05-01

    Stormwater runoff is generally characterized as non-point source pollution. In the present study, antibiotic concentration and antibiotic susceptibilities of cultivable heterotrophic bacteria were investigated in two small shallow urban lakes before and after strong storm event. Several antibiotics, lactose-fermenting bacteria and cultivable heterotrophic bacteria concentrations increased in surface water and/or surface sediment of two small urban lakes (Lake Xuanwu and Wulongtan) after strong storm event. In general, the frequencies of bacteria showing resistance to nine antibiotics increased after storm event. Based on the 16S rRNA genes of 50 randomly selected isolates from each water sample of two lakes, Aeromonas and Bacillus were dominant genera in samples from two lakes, while genera Proteus and Lysinibacillus were the third abundant genera in Lake Xuanwu and Wulongtu, respectively. Presences of nine antibiotic resistance genes (ARGs) in the 100 isolates were detected and most of these isolates harbored at least two ARGs with different functions. The detection frequency of ARGs in Gram-negative isolates was higher than that in Gram-positive isolates. The most prevalent integron in 100 isolates was int(II) (n = 28), followed by int(I) (n = 17) and int(III) (n = 17). Our results indicate that strong storm events potentially contribute to the transfer of ARGs and antibiotic-resistant bacteria from land-sewer system to the urban Lakes. PMID:26865482

  12. Selection of antibiotic-resistant standard plate count bacteria during water treatment.

    Science.gov (United States)

    Armstrong, J L; Calomiris, J J; Seidler, R J

    1982-08-01

    Standard plate count (SPC) bacteria were isolated from a drinking-water treatment facility and from the river supplying the facility. All isolates were identified and tested for their resistance to six antibiotics to determine if drug-resistant bacteria were selected for as a consequence of water treatment. Among the isolates surviving our test procedures, there was a significant selection (P less than 0.05) of gram-negative SPC organisms resistant to two or more of the test antibiotics. These bacteria were isolated from the flash mix tank, where chlorine, alum, and lime are added to the water. Streptomycin resistance in particular was more frequent in this population as compared with bacteria in the untreated river water (P less than 0.01). SPC bacteria from the clear well, which is a tank holding the finished drinking water at the treatment facility, were also more frequently antibiotic resistant than were the respective river water populations. When 15.8 and 18.2% of the river water bacteria were multiply antibiotic resistant, 57.1 and 43.5%, respectively, of the SPC bacteria in the clear well were multiply antibiotic resistant. Selection for bacteria exhibiting resistance to streptomycin was achieved by chlorinating river water in the laboratory. We concluded that the selective factors operating in the aquatic environment of a water treatment facility can act to increase the proportion of antibiotic-resistant members of the SPC bacterial population in treated drinking water.

  13. Isolated deafness following recovery from neurologic injury and adult respiratory distress syndrome. A sequela of intercurrent aminoglycoside and diuretic use.

    Science.gov (United States)

    Lynn, A M; Redding, G J; Morray, J P; Tyler, D C

    1985-05-01

    We report two children who survived neurologic injury (near-drowning and Reye's syndrome) and adult respiratory distress syndrome and who required prolonged ventilatory support. Follow-up examination in both children showed steady neurologic recovery, but five months following discharge from their acute illness, profound hearing loss was diagnosed in both children. A review of the literature is reported and the hypothesis that combined aminoglycoside antibiotic and loop diuretic therapy caused the hearing loss is presented. Recommendation is made for audiologic assessment within six months of recovery from critical illness of pediatric patients in whom therapy has included loop diuretic and aminoglycoside antibiotic therapy.

  14. 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.

  15. Introduction of biocides into clinical practice and the impact on antibiotic-resistant bacteria.

    Science.gov (United States)

    Russell, A D

    2002-01-01

    Biocides and other antimicrobial agents have been employed for centuries. Much later, iodine found use as a wound disinfectant, chlorine water in obstetrics, alcohol as a hand disinfectant and phenol as a wound dressing and in antiseptic surgery. In the early part of the twentieth century, other chlorine-releasing agents (CRAs), and acridine and other dyes were introduced, as were some quaternary ammonium compounds (QACs, although these were only used as biocides from the 1930s). Later still, various phenolics and alcohols, formaldehyde and hydrogen peroxide were introduced and subsequently (although some had actually been produced at an earlier date) biguanides, iodophors, bisphenols, aldehydes, diamidines, isocyanurates, isothiazolones and peracetic acid. Antibiotics were introduced clinically in the 1940s, although sulphonamides had been synthesized and used previously. After penicillin came streptomycin and other aminoglycosides-aminocyclitols, tetracyclines, chloramphenicol, macrolides, semi-synthetic beta-lactams, glycopeptides, lincosamides, 4-quinolones and diaminopyrimidines. Bacterial resistance to antibiotics is causing great concern. Mechanisms of such resistance include cell impermeability, target site mutation, drug inactivation and drug efflux. Bacterial resistance to biocides was described in the 1950s and 1960s and is also apparently increasing. Of the biocides listed above, cationic agents (QACs, chlorhexidine, diamidines, acridines) and triclosan have been implicated as possible causes for the selection and persistence of bacterial strains with low-level antibiotic resistance. It has been claimed that the chronological emergence of qacA and qacB determinants in clinical isolates of Staphylococcus aureus mirrors the introduction and usage of cationic biocides.

  16. Indigenous and acquired modifications in the aminoglycoside binding sites of Pseudomonas aeruginosa rRNAs

    DEFF Research Database (Denmark)

    Gutierrez, Belen; Douthwaite, Stephen Roger; Gonzalez-Zorn, Bruno

    2013-01-01

    of 16S rRNA helix 44 with a secondary target in 23S rRNA helix 69. Here, we have mapped P. aeruginosa rRNAs using MALDI mass spectrometry and reverse transcriptase primer extension to identify nucleotide modifications that could influence aminoglycoside interactions. Helices 44 and 45 contain...... indigenous (housekeeping) modifications at m (4)Cm1402, m (3)U1498, m (2)G1516, m (6) 2A1518, and m (6) 2A1519; helix 69 is modified at m (3)Ψ1915, with m (5)U1939 and m (5)C1962 modification in adjacent sequences. All modifications were close to stoichiometric, with the exception of m (3)Ψ1915, where about...... 80% of rRNA molecules were methylated. The modification status of a virulent clinical strain expressing the acquired methyltransferase RmtD was altered in two important respects: RmtD stoichiometrically modified m (7)G1405 conferring high resistance to the aminoglycoside tobramycin and, in doing so...

  17. Genotypic and phenotypic characteristics of aminoglycoside-resistant Mycobacterium tuberculosis isolates in Latvia.

    Science.gov (United States)

    Bauskenieks, Matiss; Pole, Ilva; Skenders, Girts; Jansone, Inta; Broka, Lonija; Nodieva, Anda; Ozere, Iveta; Kalvisa, Adrija; Ranka, Renate; Baumanis, Viesturs

    2015-03-01

    Mutations causing resistance to aminoglycosides, such as kanamycin (KAN), amikacin (AMK), and streptomycin, are not completely understood. In this study, polymorphisms of aminoglycoside resistance influencing genes such as rrs, eis, rpsL, and gidB in 41 drug-resistant and 17 pan-sensitive Mycobacterium tuberculosis clinical isolates in Latvia were analyzed. Mutation A1400G in rrs gene was detected in 92% isolates with high resistance level to KAN and diverse MIC level to AMK. Mutations in promoter region of eis were detected in 80% isolates with low-level MIC of KAN. The association of K43R mutation in rpsL gene, a mutation in the rrs gene at position 513, and various polymorphisms in gidB gene with distinct genetic lineages of M. tuberculosis was observed. The results of this study suggest that association of different controversial mutations of M. tuberculosis genes to the drug resistance phenotype should be done in respect to genetic lineages.

  18. Carriage of antibiotic-resistant enteric bacteria varies among sites in Galapagos reptiles.

    Science.gov (United States)

    Wheeler, Emily; Hong, Pei-Ying; Bedon, Lenin Cruz; Mackie, Roderick I

    2012-01-01

    Increased overlap between humans and wildlife populations has increased the risk for novel disease emergence. Detecting contacts with a high risk for transmission of pathogens requires the identification of dependable measures of microbial exchange. We evaluated antibiotic resistance as a molecular marker for the intensity of human-wildlife microbial connectivity in the Galápagos Islands. We isolated Escherichia coli and Salmonella enterica from the feces of land iguanas (Conolophus sp.), marine iguanas (Amblyrhynchus cristatus), giant tortoises (Geochelone nigra), and seawater, and tested these bacteria with the use of the disk diffusion method for resistance to 10 antibiotics. Antibiotic-resistant bacteria were found in reptile feces from two tourism sites (Isla Plaza Sur and La Galapaguera on Isla San Cristóbal) and from seawater close to a public use beach near Puerto Baquerizo Moreno on Isla San Cristóbal. No resistance was detected at two protected beaches on more isolated islands (El Miedo on Isla Santa Fe and Cape Douglas on Isla Fernandina) and at a coastal tourism site (La Lobería on Isla San Cristóbal). Eighteen E. coli isolates from three locations, all sites relatively proximate to a port town, were resistant to ampicillin, doxycycline, tetracycline, and trimethoprin/sulfamethoxazole. In contrast, only five S. enterica isolates showed a mild decrease in susceptibility to doxycycline and tetracycline from these same sites (i.e., an intermediate resistance phenotype), but no clinical resistance was detected in this bacterial species. These findings suggest that reptiles living in closer proximity to humans potentially have higher exposure to bacteria of human origin; however, it is not clear from this study to what extent this potential exposure translates to ongoing exchange of bacterial strains or genetic traits. Resistance patterns and bacterial exchange in this system warrant further investigation to understand better how human associations

  19. Carriage of antibiotic-resistant enteric bacteria varies among sites in Galapagos reptiles.

    Science.gov (United States)

    Wheeler, Emily; Hong, Pei-Ying; Bedon, Lenin Cruz; Mackie, Roderick I

    2012-01-01

    Increased overlap between humans and wildlife populations has increased the risk for novel disease emergence. Detecting contacts with a high risk for transmission of pathogens requires the identification of dependable measures of microbial exchange. We evaluated antibiotic resistance as a molecular marker for the intensity of human-wildlife microbial connectivity in the Galápagos Islands. We isolated Escherichia coli and Salmonella enterica from the feces of land iguanas (Conolophus sp.), marine iguanas (Amblyrhynchus cristatus), giant tortoises (Geochelone nigra), and seawater, and tested these bacteria with the use of the disk diffusion method for resistance to 10 antibiotics. Antibiotic-resistant bacteria were found in reptile feces from two tourism sites (Isla Plaza Sur and La Galapaguera on Isla San Cristóbal) and from seawater close to a public use beach near Puerto Baquerizo Moreno on Isla San Cristóbal. No resistance was detected at two protected beaches on more isolated islands (El Miedo on Isla Santa Fe and Cape Douglas on Isla Fernandina) and at a coastal tourism site (La Lobería on Isla San Cristóbal). Eighteen E. coli isolates from three locations, all sites relatively proximate to a port town, were resistant to ampicillin, doxycycline, tetracycline, and trimethoprin/sulfamethoxazole. In contrast, only five S. enterica isolates showed a mild decrease in susceptibility to doxycycline and tetracycline from these same sites (i.e., an intermediate resistance phenotype), but no clinical resistance was detected in this bacterial species. These findings suggest that reptiles living in closer proximity to humans potentially have higher exposure to bacteria of human origin; however, it is not clear from this study to what extent this potential exposure translates to ongoing exchange of bacterial strains or genetic traits. Resistance patterns and bacterial exchange in this system warrant further investigation to understand better how human associations

  20. Aquaculture can promote the presence and spread of antibiotic-resistant Enterococci in marine sediments.

    Directory of Open Access Journals (Sweden)

    Andrea Di Cesare

    Full Text Available Aquaculture is an expanding activity worldwide. However its rapid growth can affect the aquatic environment through release of large amounts of chemicals, including antibiotics. Moreover, the presence of organic matter and bacteria of different origin can favor gene transfer and recombination. Whereas the consequences of such activities on environmental microbiota are well explored, little is known of their effects on allochthonous and potentially pathogenic bacteria, such as enterococci. Sediments from three sampling stations (two inside and one outside collected in a fish farm in the Adriatic Sea were examined for enterococcal abundance and antibiotic resistance traits using the membrane filter technique and an improved quantitative PCR. Strains were tested for susceptibility to tetracycline, erythromycin, ampicillin and gentamicin; samples were directly screened for selected tetracycline [tet(M, tet(L, tet(O] and macrolide [erm(A, erm(B and mef] resistance genes by newly-developed multiplex PCRs. The abundance of benthic enterococci was higher inside than outside the farm. All isolates were susceptible to the four antimicrobials tested, although direct PCR evidenced tet(M and tet(L in sediment samples from all stations. Direct multiplex PCR of sediment samples cultured in rich broth supplemented with antibiotic (tetracycline, erythromycin, ampicillin or gentamicin highlighted changes in resistance gene profiles, with amplification of previously undetected tet(O, erm(B and mef genes and an increase in benthic enterococcal abundance after incubation in the presence of ampicillin and gentamicin. Despite being limited to a single farm, these data indicate that aquaculture may influence the abundance and spread of benthic enterococci and that farm sediments can be reservoirs of dormant antibiotic-resistant bacteria, including enterococci, which can rapidly revive in presence of new inputs of organic matter. This reservoir may constitute an

  1. Audiologic monitoring of multi-drug resistant tuberculosis patients on aminoglycoside treatment with long term follow-up

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    Sarkar Malay

    2007-11-01

    Full Text Available Abstract Background Multi-drug resistant tuberculosis has emerged as a significant problem with the resurfacing of tuberculosis and thus the need to use the second line drugs with the resultant increased incidence of adverse effects. We discuss the effect of second line aminoglycoside anti-tubercular drugs on the hearing status of MDR-TB patients. Methods Sixty four patients were put on second line aminoglycoside anti-TB drugs. These were divided into three groups: group I, 34 patients using amikacin, group II, 26 patients using kanamycin and group III, 4 patients using capreomycin. Results Of these, 18.75% of the patients developed sensorineural hearing loss involving higher frequencies while 6.25% had involvement of speech frequencies also. All patients were seen again approximately one year after aminoglycoside discontinuation and all hearing losses were permanent with no threshold improvement. Conclusion Aminoglycosides used in MDR-TB patients may result in irreversible hearing loss involving higher frequencies and can become a hearing handicap as speech frequencies are also involved in some of the patients thus underlining the need for regular audiologic evaluation in patients of MDR-TB during the treatment.

  2. Non-derivatization approach to high-performance liquid chromatography-fluorescence detection for aminoglycoside antibiotics based on a ligand displacement reaction.

    Science.gov (United States)

    Yang, M; Tomellini, S A

    2001-12-21

    An indirect fluorescence detection method has been developed for detecting the aminoglycoside antibiotics following chromatographic separation. This approach to detection is based on a displacement reaction between the aminoglycosides and a copper(II)-L-tryptophan (L-Trp) complex, Cu(L-Trp)2. The aminoglycosides, which contain multiple amino groups, have strong affinities for the Cu(II) ion and displace L-Trp from the Cu(L-Trp)2 complex. The resulting increase in L-Trp fluorescence, which is quenched when coordinated to Cu(II), is indicative of the presence of the aminoglycoside. Fluorescence titration data indicate that there is a stoichiometric ratio of 1:1 between the reaction of the aminoglycosides with Cu(L-Trp)2. This HPLC detection scheme is implemented postcolumn by mixing a buffered Cu(L-Trp)2 solution with the column eluent prior to detection. The aminoglycosides were separated with the use of a column packed with a polymeric strong cation-exchanger. Separation and detection variables were optimized and are discussed. The detection limits for the aminoglycosides tested ranged from 4.2 to 14.5 ng injected (S/N=3). A linear working curve was achieved for amikacin in the range of 29-586 ng for a six point linearity test. The developed separation and detection scheme was further tested by analyzing commercial pharmaceutical formulations of these antibiotics. PMID:11806546

  3. Chaperonin GroEL/GroES over-expression promotes multi-drug resistance in E. coli following exposure to aminoglycoside antibiotics

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    Lise eGoltermann

    2016-01-01

    Full Text Available Antibiotic resistance is an increasing challenge to modern healthcare. Aminoglycoside antiobiotics cause translation corruption and protein misfolding and aggregation in Escherichia coli. We previously showed that chaperonin GroEL/GroES depletion and overexpression sensitize and promote short-term tolerance, respectively, to this drug class. Here we show that chaperonin GroEL/GroES over-expression accelerates acquisition of aminoglycoside resistance and multi-drug resistance following sub-lethal aminoglycoside antibiotic exposure. Chaperonin buffering could provide a novel mechanism for antibiotic resistance and multi-drug resistance development.

  4. 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.

  5. Rapid and liquid-based selection of genetic switches using nucleoside kinase fused with aminoglycoside phosphotransferase.

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    Masahiro Tominaga

    Full Text Available The evolutionary design of genetic switches and circuits requires iterative rounds of positive (ON- and negative (OFF- selection. We previously reported a rapid OFF selection system based on the kinase activity of herpes simplex virus thymidine kinase (hsvTK on the artificial mutator nucleoside dP. By fusing hsvTK with the kanamycin resistance marker aminoglycoside-(3'-phosphotransferase (APH, we established a novel selector system for genetic switches. Due to the bactericidal nature of kanamycin and nucleoside-based lethal mutagenesis, both positive and negative selection could be completed within several hours. Using this new selector system, we isolated a series of homoserine lactone-inducible genetic switches with different expression efficiencies from libraries of the Vibrio fischeri lux promoter in two days, using only liquid handling.

  6. Study of the Interference between Plectranthus Species Essential Oils from Brazil and Aminoglycosides.

    Science.gov (United States)

    Galvão Rodrigues, Fabíola Fernandes; Costa, José Galberto Martins; Rodrigues, Fábio Fernandes Galvao; Campos, Adriana Rolim

    2013-01-01

    Plectranthus is one of the most representative genera of Lamiaceae family. In this study, the essential oils from Plectranthus amboinicus, Plectranthus ornatus, and Plectranthus barbatus were investigated for their chemical composition and antimicrobial and modulatory activities. The major components found were carvacrol (54.4%-P. amboinicus) and eugenol (22.9%-P. ornatus e 25.1%-P. barbatus). In vitro antimicrobial activity was conducted against Escherichia coli, Proteus vulgaris, Bacillus cereus, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus aureus (multiresistant) using microdilution method. The results of bioassay showed that all strains were sensitive to the oils, except P. aeruginosa that was resistant to P. amboinicus and P. ornatus. A synergistic effect of all essential oils combined with the aminoglycosides was demonstrated. These results show that P. amboinicus, P. ornatus, and P. barbatus inhibit the growth of pathogenic microorganism, and besides this they present antibiotic modifying activity, providing a new perspective against the problem of bacterial resistance to antibiotics. PMID:23662150

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

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    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. PMID:26255160

  8. Investigation on the Mechanism of Exacerbation of Myasthenia Gravis by Aminoglycoside Antibiotics in Mouse Model

    Institute of Scientific and Technical Information of China (English)

    LIU Changqin; HU Fang

    2005-01-01

    Summary: To investigate the underlying mechanism of the exacerbation of myasthenia gravis by aminoglycoside antibiotics. C57/BL6 mice were immunized with acetylcholine receptor (AChR), extracted from electric organ of Narcine timilei according to Xu Haopeng's methods, in complete Fruend's adjuvant (CFA) to establish experimental autoimmune myasthenia gravis (EAMG). EAMG mice were divided randomly into 5 groups: MG group, NS group and three antibiotics groups. The clinical symptom scores of mice were evaluated on d7 after the last immunization and d14 of antibiotics treatment. Repetitive nerve stimulation (RNS) was performed and the levels of anti-AChR antibody (AChR-Ab) were tested at the same time. The mean clinical symptom grades of gentamycin group (1.312, 2.067), amikacin group (1.111, 1.889) and etimicin group (1.263, 1.632) were significantly higher than those of MG group (1.000, 1.200) (P<0.05). The positive rates of RNS of three antibiotics groups were 69.23 %, 58.82 % and 63.16 % respectively, which were significantly higher than those of MG group and NS group (40.00 %, 40.00 %, P<0.05). The AChR-Ab level in serum and the expression of AChR on neuromuscular junction (NMJ) of mice in three antibiotics groups were also higher than those of MG group. Our results indicated that aminoglycoside antibiotics could aggravate the symptom of myasthenia gravis. The exacerbation of myasthenia gravis by these antibiotics probably involves competitively restraining the release of acetylcholine from presynaptic membrane, impairing the depolarization of postsynaptic membrane, depressing the irritability of myocyte membrane around the end-plate membrane and consequently leading to the blockade of neuromuscular junction.

  9. Factors impacting the aminoglycoside-induced UGA stop codon readthrough in selenoprotein translation.

    Science.gov (United States)

    Martitz, Janine; Hofmann, Peter Josef; Johannes, Jörg; Köhrle, Josef; Schomburg, Lutz; Renko, Kostja

    2016-09-01

    Aminoglycosides (AG) are oligosaccharide antibiotics that interfere with the small ribosomal subunit in aerobic, Gram-negative bacteria, causing pathogen-destructing error rates in their protein biosynthesis. Aminoglycosides also induce mRNA misinterpretation in eukaryotic cells, especially of the UGA (Opal)-stop codon, albeit to a lower extent. UGA recoding is essentially required for the incorporation of selenocysteine (Sec) into growing selenoproteins during translation. Selenocysteine incorporation requires the presence of a selenoprotein-specific stem-loop structure within the 3'-untranslated region of the mRNA, the so-called Sec-insertion sequence (SECIS) element. Interestingly, selenoprotein genes differ in their SECIS-element sequence and in their UGA base context. We hypothesized that the SECIS-element and the specific codon context synergize in controlling the effects of AG on stop codon readthrough. To this end, the SECIS-elements of glutathione peroxidase 1, glutathione peroxidase 4 and selenoprotein P transcripts were cloned into a reporter system and analyzed in combination with different UGA codon contexts. Our results indicate that a cytosine in position 4 (directly downstream of UGA) confers strongest effects on both the Se- and AG-dependent readthrough. Overall selenoprotein biosynthesis rate depends on the Se-status, AG concentration and the specific SECIS-element present in the transcript. These findings help to get a better understanding for the susceptibility of different transcripts towards AG-mediated interference with the biosynthesis of functional Se-containing selenoproteins, and highlight the importance of the Se-status for successful selenoprotein biosynthesis under antibiotic therapy. PMID:27157664

  10. Ribozyme-based aminoglycoside switches of gene expression engineered by genetic selection in S. cerevisiae.

    Science.gov (United States)

    Klauser, Benedikt; Atanasov, Janina; Siewert, Lena K; Hartig, Jörg S

    2015-05-15

    Systems for conditional gene expression are powerful tools in basic research as well as in biotechnology. For future applications, it is of great importance to engineer orthogonal genetic switches that function reliably in diverse contexts. RNA-based switches have the advantage that effector molecules interact immediately with regulatory modules inserted into the target RNAs, getting rid of the need of transcription factors usually mediating genetic control. Artificial riboswitches are characterized by their simplicity and small size accompanied by a high degree of modularity. We have recently reported a series of hammerhead ribozyme-based artificial riboswitches that allow for post-transcriptional regulation of gene expression via switching mRNA, tRNA, or rRNA functions. A more widespread application was so far hampered by moderate switching performances and a limited set of effector molecules available. Here, we report the re-engineering of hammerhead ribozymes in order to respond efficiently to aminoglycoside antibiotics. We first established an in vivo selection protocol in Saccharomyces cerevisiae that enabled us to search large sequence spaces for optimized switches. We then envisioned and characterized a novel strategy of attaching the aptamer to the ribozyme catalytic core, increasing the design options for rendering the ribozyme ligand-dependent. These innovations enabled the development of neomycin-dependent RNA modules that switch gene expression up to 25-fold. The presented aminoglycoside-responsive riboswitches belong to the best-performing RNA-based genetic regulators reported so far. The developed in vivo selection protocol should allow for sampling of large sequence spaces for engineering of further optimized riboswitches. PMID:24871672

  11. Natural products from the termite Nasutitermes corniger lowers aminoglycoside minimum inhibitory concentrations

    Directory of Open Access Journals (Sweden)

    Henrique D.M Coutinho

    2010-01-01

    Full Text Available Bacterial infectious agents present a risk to populations, as they are responsible for high morbidity and mortality. For combating these pathogens, our main line of defense is the use of antibiotics. However, indiscriminate use of these drugs develops resistant strains to these same drugs. The present study has tested the antibacterial and modifying antibiotic activity of natural products from Nasutitermes corniger (Termitidae (Motschulsky, a termite used in folk medicine in Northeast Brazil, by the microdilution and checkerboard methods, respectively. In this study, the aqueous extract from the nest of N. corniger (ANCE was prepared and tested with chlorpromazine (CPZ for its antimicrobial activity, using the microdilution method. CPZ and ANCE were used independently and also in combination with aminoglycosides, against a strain of Escherichia coli resistant to these antibiotics, to determine the participation of efflux systems in resistance mechanisms. The fractional inhibitory concentration (FIC index was calculated and evaluated for the occurrence of synergism, using the checkerboard method. The minimum inhibitory concentrations (MIC and minimum bactericidal concentrations (MBC values were ≥ 2048 µg/mL for both strains of E. coli assayed, indicating low antibacterial activity. However, synergism was observed with kanamycin when the decoction was used, but when chlorpromazine was used, synergism was observed with kanamycin, amikacin, and neomycin. This synergism with CPZ indicated the involvement of an efflux system in the resistance to these aminoglycosides. Therefore, it was suggested that the natural products from N. corniger could be used as a source of zoo-derived natural products with kanamycin-modifying activity, resulting in a new approach against bacterial resistance to antibiotics.

  12. In vitro susceptibility pattern of acinetobacter species to commonly used cephalosporins, quinolones, and aminoglycosides

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    Prashanth K

    2004-01-01

    Full Text Available PURPOSE: Acinetobacter spp. is an emerging important nosocomial pathogen. Clinical isolates of this genus are often resistant to many antibiotics. The in vitro susceptibility of Acinetobacter isolates obtained from patients were tested for currently used antibiotics. In addition, the study aimed at biotyping of Acinetobacter baumannii. METHODS: A total of 66 isolates were phenotypically characterised through a large panel of 25 carbon assimilation tests and susceptibility through disc diffusion method with 10 antimicrobial agents were tested. MICs were determined only for second line broad-spectrum drugs such as cefotaxime, ceftazidime, amikacin, ciprofloxacin, and ofloxacin using NCCLS guidelines. RESULTS: Multiple drug resistance (MDR was only witnessed in A. baumannii and not in other Acinetobacter species. Aminoglycosides such as amikacin, netilmicin were most active against the MDR isolates tested (60% susceptibility. Ceftazidime was more active than cefotaxime. MDR A. baumannii strains were susceptible only to amikacin, netilmicin and ceftadizime. Ciprofloxacin had poor activity irrespective of isolates belonging to different DNA groups tested (58% resistance overall, 79% among A. baumannii. Strains of Biotypes 6 and 19 of A. baumannii showed broader resistance than those of biotype 10 and others. CONCLUSIONS: Strains of A. baumannii from patients in our hospital, were generally more resistant to quinolones, -lactam antibiotics, first and second generation cephalosporins and partially resistant to third generation cephalosporins and aminoglycosides. The strains belonging to other DNA groups of Acinetobacter were comparatively less resistant than A.baumannii, except ciprofloxacin. This study suggests that, a combination therapy, using a third generation cephalosporin and amikacin, would be best choice for treating Acinetobacter infections.

  13. IncHI2 Plasmids Are Predominant in Antibiotic-Resistant Salmonella Isolates

    Science.gov (United States)

    Chen, Wenyao; Fang, Tingzi; Zhou, Xiujuan; Zhang, Daofeng; Shi, Xianming; Shi, Chunlei

    2016-01-01

    The wide usage of antibiotics contributes to the increase in the prevalence of antibiotic-resistant Salmonella. Plasmids play a critical role in horizontal transfer of antibiotic resistance markers in Salmonella. This study aimed to screen and characterize plasmid profiles responsible for antibiotic resistance in Salmonella and ultimately to clarify the molecular mechanism of transferable plasmid-mediated antibiotic resistance. A total of 226 Salmonella isolates were examined for antimicrobial susceptibility by a disk diffusion method. Thirty-two isolates (14.2%) were resistant to at least one antibiotic. The presence of plasmid-mediated quinolone resistance (PMQR) genes and β-lactamase genes were established by PCR amplification. PCR-based replicon typing revealed that these 32 isolates represented seven plasmid incompatibility groups (IncP, HI2, A/C, FIIs, FIA, FIB, and I1), and the IncHI2 (59.4%) was predominant. Antibiotic resistance markers located on plasmids were identified through plasmid curing. Fifteen phenotypic variants were obtained with the curing efficiency of 46.9% (15/32). The cured plasmids mainly belong to the HI2 incompatibility group. The elimination of IncHI2 plasmids correlated with the loss of β-lactamase genes (blaOXA-1 and blaTEM-1) and PMQR genes (qnrA and aac(6′)-Ib-cr). Both IncHI2 and IncI1 plasmids in a S. enterica serovar Indiana isolate SJTUF 10584 were lost by curing. The blaCMY -2-carrying plasmid pS10584 from SJTUF 10584 was fully sequenced. Sequence analysis revealed that it possessed a plasmid scaffold typical for IncI1 plasmids with the unique genetic arrangement of IS1294-ΔISEcp1-blaCMY -2-blc-sugE-ΔecnR inserted into the colicin gene cia. These data suggested that IncHI2 was the major plasmid lineage contributing to the dissemination of antibiotic resistance in Salmonella and the activity of multiple mobile genetic elements may contribute to antibiotic resistance evolution and dissemination between different plasmid

  14. Effect of biochar amendment on the control of soil sulfonamides, antibiotic-resistant bacteria, and gene enrichment in lettuce tissues.

    Science.gov (United States)

    Ye, Mao; Sun, Mingming; Feng, Yanfang; Wan, Jinzhong; Xie, Shanni; Tian, Da; Zhao, Yu; Wu, Jun; Hu, Feng; Li, Huixin; Jiang, Xin

    2016-05-15

    Considering the potential threat of vegetables growing in antibiotic-polluted soil with high abundance of antibiotic-resistant genes (ARGs) against human health through the food chain, it is thus urgent to develop novel control technology to ensure vegetable safety. In the present work, pot experiments were conducted in lettuce cultivation to assess the impedance effect of biochar amendment on soil sulfonamides (SAs), antibiotic-resistant bacteria (ARB), and ARG enrichment in lettuce tissues. After 100 days of cultivation, lettuce cultivation with biochar amendment exhibited the greatest soil SA dissipation as well as the significant improvement of lettuce growth indices, with residual soil SAs mainly existing as the tightly bound fraction. Moreover, the SA contents in roots and new/old leaves were reduced by one to two orders of magnitude compared to those without biochar amendment. In addition, isolate counts for SA-resistant bacterial endophytes in old leaves and sul gene abundances in roots and old leaves also decreased significantly after biochar application. However, neither SA resistant bacteria nor sul genes were detected in new leaves. It was the first study to demonstrate that biochar amendment can be a practical strategy to protect lettuce safety growing in SA-polluted soil with rich ARB and ARGs. PMID:26896719

  15. Advanced treatment of urban wastewater by UV radiation: Effect on antibiotics and antibiotic-resistant E. coli strains.

    Science.gov (United States)

    Rizzo, Luigi; Fiorentino, Antonino; Anselmo, Antonella

    2013-06-01

    Urban wastewater treatment plant (UWWTP) effluents are among the possible sources of antibiotics and antibiotic-resistant bacteria (ARB) spread into the environment. In this work, the effect of UV radiation on antibiotic-resistant Escherichia coli (E. coli) strains was compared with that of chlorination process. Under the investigated conditions, UV disinfection process resulted in a total inactivation after 60min of irradiation (1.25×10(4)μWscm(-2)) compared to 120min chlorine contact time (initial chlorine dose of 2mgL(-1)). Moreover, no change in E. coli strains' resistance to amoxicillin (AMX) (minimum inhibiting concentration (MIC)>256mgL(-1)) and sulfamethoxazole (SMZ) (MIC>1024mgL(-1)) could be observed after UV treatment, while the treatment affected resistance of the lower resistance strain to ciprofloxacin (CPX) (MIC decreased by 33% and 50% after 60 and 120min, respectively). Contrarily, chlorination process did not affect antibiotic resistance of the investigated E. coli strains. Finally, the effect of UV radiation on the mixture of three antibiotics was also investigated and photodegradation data fit quite well pseudo first order kinetic models with t1/2 values of 14, 20 and 25min for CPX, AMX and SMZ, respectively. According to these results, conventional disinfection processes may not be effective in the inactivation of ARB, and the simultaneous release of ARB and antibiotics at sub-lethal concentrations into UWWTP effluent may promote the development of resistance among bacteria in receiving water.

  16. Aminoglycoside 6′-N-Acetyltransferase Variants of the Ib Type with Altered Substrate Profile in Clinical Isolates of Enterobacter cloacae and Citrobacter freundii

    OpenAIRE

    Casin, Isabelle; Bordon, Florence; Bertin, Philippe; Coutrot, Anne; Podglajen, Isabelle; Brasseur, Robert; Collatz, Ekkehard

    1998-01-01

    Three clinical isolates, Enterobacter cloacae EC1562 and EC1563 and Citrobacter freundii CFr564, displayed an aminoglycoside resistance profile evocative of low-level 6′-N acetyltransferase type II [AAC(6′)-II] production, which conferred reduced susceptibility to gentamicin but not to amikacin or isepamicin. Aminoglycoside acetyltransferase assays suggested the synthesis in the three strains of an AAC(6′) which acetylated amikacin practically as well as it acetylated gentamicin in vitro. Bot...

  17. Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion

    OpenAIRE

    Bernacchi, Serena; Freisz, Séverine; Maechling, Clarisse; Spiess, Bernard; Marquet, Roland; Dumas, Philippe; Ennifar, Eric

    2007-01-01

    Owing to a striking, and most likely fortuitous, structural and sequence similarity with the bacterial 16 S ribosomal A site, the RNA kissing-loop complex formed by the HIV-1 genomic RNA dimerization initiation site (DIS) specifically binds 4,5-disubstituted 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics. We used chemical probing, molecular modeling, isothermal titration calorimetry (ITC) and UV melting to investigate aminoglycoside binding to the DIS loop–loop complex. We showed that ...

  18. Isolation and speciation of Enterococci from various clinical samples and their antimicrobial susceptibility pattern with special reference to high level Aminoglycoside resistance

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    :Saroj Golia, Nirmala AR, Asha S Kamath B

    2014-07-01

    Full Text Available Background and Objectives: Enterococci are important nosocomial agents and strains resistant to penicillin and other antibiotics occur frequently. Enterococci are intrinsically resistant to cephalosporins and offer low level resistance to aminoglycosides. In penicillin sensitive strains, synergism occurs with combination treatment with penicillin and aminoglycoside. Serious infections caused by them are treated with penicillin and aminoglycoside combination. But the synergistic effect is lost, when the strain develops high level aminoglycoside resistance. The choice of drug for infections due to such strains is vancomycin. The present study was carried out to isolate and speciate Enterococci from various clinical samples, to know the susceptibility pattern of the isolates, to determine the High Level Aminoglycoside Resistance (HLAR among Enterococcal isolates. Methods: A total of One hundred Enterococcal species isolated from various clinical samples were identified by various biochemical reactions. Antimicrobial susceptibility testing and HLAR were determined by Kirby- Bauer disc diffusion method. Results: Out of 100 Enterococcal isolates, 59 were E. faecalis, 38 were E. faecium, 3 were other Enterococcal species. Among these 53 isolates showed High Level Aminoglycoside Resistance. Conclusion: Present study shows the presence of drug resistance to most of commonly used antibiotics and HLAR is also more in E.faecium compared to E.fecalis.

  19. Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion.

    Science.gov (United States)

    Bernacchi, Serena; Freisz, Séverine; Maechling, Clarisse; Spiess, Bernard; Marquet, Roland; Dumas, Philippe; Ennifar, Eric

    2007-01-01

    Owing to a striking, and most likely fortuitous, structural and sequence similarity with the bacterial 16 S ribosomal A site, the RNA kissing-loop complex formed by the HIV-1 genomic RNA dimerization initiation site (DIS) specifically binds 4,5-disubstituted 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics. We used chemical probing, molecular modeling, isothermal titration calorimetry (ITC) and UV melting to investigate aminoglycoside binding to the DIS loop-loop complex. We showed that apramycin, an aminoglycoside containing a bicyclic moiety, also binds the DIS, but in a different way than 4,5-disubstituted 2-DOS aminoglycosides. The determination of thermodynamic parameters for various aminoglycosides revealed the role of the different rings in the drug-RNA interaction. Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (K(d) approximately 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (K(d) approximately 1.6 microM). In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop-loop interaction and also block the conversion from kissing-loop complex to extended duplex. Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA. PMID:17942426

  20. Ultrastructural analysis of aminoglycoside-induced hair cell death in the zebrafish lateral line reveals an early mitochondrial response.

    OpenAIRE

    Owens, Kelly,; Cunningham, Dale,; Macdonald, Glen; Rubel, Edwin,; Raible, David,; Pujol, Remy

    2007-01-01

    Loss of the mechanosensory hair cells in the auditory and vestibular organs leads to hearing and balance deficits. To investigate initial, in vivo events in aminoglycoside-induced hair cell damage, we examined hair cells from the lateral line of the zebrafish, Danio rerio. The mechanosensory lateral line is located externally on the animal and therefore allows direct manipulation and observation of hair cells. Labeling with vital dyes revealed a rapid response of hair cells to the aminoglycos...

  1. Prevalence of resistance to aminoglycosides and fluoroquinolones among Pseudomonas aeruginosa strains in a University Hospital in Northeastern Poland

    OpenAIRE

    Anna Diana Michalska; Pawel Tomasz Sacha; Dominika Ojdana; * Anna Wieczorek; Elzbieta Tryniszewska

    2014-01-01

    The present study was conducted to investigate the prevalence of genes encoding resistance to aminoglycosides and fluoroquinolones among twenty-five Pseudomonas aeruginosa isolated between 2002 and 2009. In PCR, following genes were detected: ant(2″)-Ia in 9 (36.0%), aac(6′)-Ib in 7 (28.0%), qnrB in 5 (20.0%), aph(3″)-Ib in 2 (8.0%) of isolates.

  2. Frequency of Aminoglycoside-Resistance Genes in Methicillin-Resistant Staphylococcus aureus (MRSA) Isolates from Hospitalized Patients

    Science.gov (United States)

    Mahdiyoun, Seyed Mohsen; Kazemian, Hossein; Ahanjan, Mohammad; Houri, Hamidreza; Goudarzi, Mehdi

    2016-01-01

    Background Staphylococcus aureus is one of the most important causative agents in community- and hospital-acquired infections. Aminoglycosides are powerful bactericidal drugs that are often used in combination with beta-lactams or glycopeptides to treat staphylococcal infections. Objectives The main objective of the present study was to determine the prevalence of aminoglycoside resistance among methicillin-resistant Staphylococcus aureus (MRSA) isolates in hospitalized patients in Sari and Tehran, Iran. Methods In this study, 174 MRSA strains isolated from different clinical samples, such as blood, sputum, tracheal exudates, bronchus, pleura, urine, wounds, and catheters, were collected from hospitalized patients in Tehran and Sari during 2014. Antibiotic susceptibility testing was performed against nine antibiotics with the Kirby-Bauer disk diffusion method according to CLSI guidelines. The MRSA strains were examined with oxacillin and cefoxitin disks. MRSA was then validated by detection of the mecA gene. PCR was used to evaluate the prevalence of the aminoglycoside-resistance genes aac (6’)-Ie/aph (2”), aph (3’)-IIIa, and ant (4’) among the MRSA isolates. Results The results of drug susceptibility testing showed that the highest rate of resistance was against erythromycin in Tehran (84.4%) and gentamicin (71.7%) in Sari. All isolates were sensitive to vancomycin, and all strains harbored the mecA gene. The aac (6’)-Ie/aph (2”), aph (3’)-IIIa, and ant (4’)-Ia genes were detected among 134 (77%), 119 (68.4%), and 122 (70.1%) of the isolates, respectively. Conclusions The present study showed a high prevalence of aminoglycoside-resistance genes among MRSA isolates in two cities in Iran.

  3. Appearance of amikacin and tobramycin resistance due to 4'-aminoglycoside nucleotidyltransferase [ANT(4')-II] in gram-negative pathogens.

    OpenAIRE

    Jacoby, G A; Blaser, M J; Santanam, P; Hächler, H; Kayser, F H; Hare, R S; Miller, G. H.

    1990-01-01

    Following the use of amikacin as the principal aminoglycoside at a Denver hospital, amikacin resistance appeared first in Pseudomonas aeruginosa and then in Escherichia coli, Klebsiella pneumoniae, and other enteric organisms from debilitated and compromised patients who had spent time in intensive care units and who had been treated with multiple antibiotics, usually including amikacin. In a P. aeruginosa isolate, resistance to amikacin and tobramycin was transferable by the IncP-2 plasmid p...

  4. Ablation of mixed lineage kinase 3 (Mlk3) does not inhibit ototoxicity induced by acoustic trauma or aminoglycoside exposure.

    Science.gov (United States)

    Polesskaya, Oksana; Cunningham, Lisa L; Francis, Shimon P; Luebke, Anne E; Zhu, Xiaoxia; Collins, David; Vasilyeva, Olga N; Sahler, Julie; Desmet, Emily A; Gelbard, Harris A; Maggirwar, Sanjay B; Walton, Joseph P; Frisina, Robert D; Dewhurst, Stephen

    2010-12-01

    Jun N-terminal kinase (JNK) is activated in cochlear hair cells following acoustic trauma or exposure to aminoglycoside antibiotics. Blockade of JNK activation using mixed lineage kinase (MLK) inhibitors prevents hearing loss and hair cell death following these stresses. Since current pharmacologic inhibitors of MLKs block multiple members of this kinase family, we examined the contribution of the major neuronal family member (MLK3) to stress-induced ototoxicity, usingMlk3(-/-) mice. Immunohistochemical staining revealed that MLK3 is expressed in cochlear hair cells of C57/BL6 mice (but not in Mlk3(-/-) animals). After exposure to acoustic trauma there was no significant difference in DPOAE and ABR values betweenMlk3(-/-) and wild-type mice at 48 h following exposure or 2 weeks later. Susceptibility of hair cells to aminoglycoside toxicity was tested by exposing explanted utricles to gentamicin. Gentamicin-induced hair cell death was equivalent in utricles from wild-type and Mlk3(-/-) mice. Blockade of JNK activation with the pharmacologic inhibitor SP600125 attenuated cell death in utricles from both wild-type and Mlk3(-/-) mice. These data show that MLK3 ablation does not protect against hair cell death following acoustic trauma or exposure to aminoglycoside antibiotics, suggesting that MLK3 is not the major upstream regulator of JNK-mediated hair cell death following these stresses. Rather, other MLK family members such as MLK1, which is also expressed in cochlea, may have a previously unappreciated role in noise- and aminoglycoside-induced ototoxicity.

  5. Activation of PI3K signaling prevents aminoglycoside-induced hair cell death in the murine cochlea

    Directory of Open Access Journals (Sweden)

    Azadeh Jadali

    2016-06-01

    Full Text Available Loss of sensory hair cells of the inner ear due to aminoglycoside exposure is a major cause of hearing loss. Using an immortalized multipotent otic progenitor (iMOP cell line, specific signaling pathways that promote otic cell survival were identified. Of the signaling pathways identified, the PI3K pathway emerged as a strong candidate for promoting hair cell survival. In aging animals, components for active PI3K signaling are present but decrease in hair cells. In this study, we determined whether activated PI3K signaling in hair cells promotes survival. To activate PI3K signaling in hair cells, we used a small molecule inhibitor of PTEN or genetically ablated PTEN using a conditional knockout animal. Hair cell survival was challenged by addition of gentamicin to cochlear cultures. Hair cells with activated PI3K signaling were more resistant to aminoglycoside-induced hair cell death. These results indicate that increased PI3K signaling in hair cells promote survival and the PI3K signaling pathway is a target for preventing aminoglycoside-induced hearing loss.

  6. The comparative effects of aminoglycoside antibiotics and muscle relaxants on electrical field stimulation response in rat bladder smooth muscle.

    Science.gov (United States)

    Min, Chang Ho; Min, Young Sil; Lee, Sang Joon; Sohn, Uy Dong

    2016-06-01

    It has been reported that several aminoglycoside antibiotics have a potential of prolonging the action of non-depolarizing muscle relaxants by drug interactions acting pre-synaptically to inhibit acetylcholine release, but antibiotics itself also have a strong effect on relaxing the smooth muscle. In this study, four antibiotics of aminoglycosides such as gentamicin, streptomycin, kanamycin and neomycin were compared with skeletal muscle relaxants baclofen, tubocurarine, pancuronium and succinylcholine, and a smooth muscle relaxant, papaverine. The muscle strips isolated from the rat bladder were stimulated with pulse trains of 40 V in amplitude and 10 s in duration, with pulse duration of 1 ms at the frequency of 1-8 Hz, at 1, 2, 4, 6, 8 Hz respectively. To test the effect of four antibiotics on bladder smooth muscle relaxation, each of them was treated cumulatively from 1 μM to 0.1 mM with an interval of 5 min. Among the four antibiotics, gentamicin and neomycin inhibited the EFS response. The skeletal muscle relaxants (baclofen, tubocurarine, pancuronium and succinylcholine) and inhibitory neurotransmitters (GABA and glycine) did not show any significant effect. However, papaverine, had a significant effect in the relaxation of the smooth muscle. It was suggested that the aminoglycoside antibiotics have inhibitory effect on the bladder smooth muscle. PMID:27260628

  7. Termite usage associated with antibiotic therapy: enhancement of aminoglycoside antibiotic activity by natural products of Nasutitermes corniger (Motschulsky 1855

    Directory of Open Access Journals (Sweden)

    Almeida-Filho Geraldo G

    2009-09-01

    Full Text Available Abstract Background Several species from Insecta are used as remedies. Among these species, the termite Nasutitermes corniger is commonly used in traditional medicine in Northeast Brazil. The present work tests the modifying antibiotic activity of Nasutitermes corniger, a termite used in folk medicine in Northeastern region of Brazil. Methods Chlorpromazine and decocts of N. corniger were collected from two different plant species used in the traditional medicine were tested for their antimicrobial activity against strains of Escherichia coli resistant to aminoglycosides. The growth of two bacterial strains of E. coli was tested using decocts and chlorpromazine alone or associeted with aminogycosides. Results The MIC and MBC values were ≥1024 μg/ml for both strains of E. coli assayed. A significant synergism was observed between both decocts and chlorpromazine when assyed with neomycin. This synergism with neomycin indicates the involvement of an efflux system in the resistance to this aminoglycoside. Conclusion Therefore it is suggested that natural products from N. corniger could be used as a source of zoo-derived natural products with modifying antibiotic activity to aminoglycosides, being a new weapon against the bacterial resistance to antibiotics.

  8. No evidence for transmission of antibiotic-resistant Escherichia coli strains from humans to wild western lowland gorillas in Lopé National Park, Gabon.

    Science.gov (United States)

    Benavides, Julio Andre; Godreuil, Sylvain; Bodenham, Rebecca; Ratiarison, Sandra; Devos, Céline; Petretto, Marie-Odile; Raymond, Michel; Escobar-Páramo, Patricia

    2012-06-01

    The intensification of human activities within the habitats of wild animals is increasing the risk of interspecies disease transmission. This risk is particularly important for great apes, given their close phylogenetic relationship with humans. Areas of high human density or intense research and ecotourism activities expose apes to a high risk of disease spillover from humans. Is this risk lower in areas of low human density? We determined the prevalence of Escherichia coli antibiotic-resistant isolates in a population of the critically endangered western lowland gorilla (Gorilla gorilla gorilla) and other wild mammals in Lopé National Park (LNP), Gabon, and we tested whether the observed pattern could be explained by bacterial transmission from humans and domestic animals into wildlife populations. Our results show a high prevalence of antibiotic-resistant bacterial isolates in humans and low levels in gorillas and other wildlife. The significant differences in the genetic background of the resistant bacteria isolated from humans and gorillas suggest that transmission is low or does not occur between these two species. These findings indicate that the presence of antibiotic-resistant strains in wildlife do not imply direct bacteria transmission from humans. Thus, in areas of low human density, human-wildlife E. coli transmission seems to be low. The presence of antibiotic-resistant isolates in gorillas may be better explained by other mechanisms for resistance acquisition, such as horizontal gene exchange among bacteria or naturally acquired resistance.

  9. Dispersal of antibiotic-resistant high-risk clones by hospital networks : changing the patient direction can make all the difference

    NARCIS (Netherlands)

    Donker, T.; Wallinga, J.; Grundmann, H.

    2014-01-01

    Background: Patients who seek treatment in hospitals can introduce high-risk clones of hospital-acquired, antibiotic-resistant pathogens from previous admissions. In this manner, different healthcare institutions become linked epidemiologically. All links combined form the national patient referral

  10. No evidence for transmission of antibiotic-resistant Escherichia coli strains from humans to wild western lowland gorillas in Lopé National Park, Gabon.

    Science.gov (United States)

    Benavides, Julio Andre; Godreuil, Sylvain; Bodenham, Rebecca; Ratiarison, Sandra; Devos, Céline; Petretto, Marie-Odile; Raymond, Michel; Escobar-Páramo, Patricia

    2012-06-01

    The intensification of human activities within the habitats of wild animals is increasing the risk of interspecies disease transmission. This risk is particularly important for great apes, given their close phylogenetic relationship with humans. Areas of high human density or intense research and ecotourism activities expose apes to a high risk of disease spillover from humans. Is this risk lower in areas of low human density? We determined the prevalence of Escherichia coli antibiotic-resistant isolates in a population of the critically endangered western lowland gorilla (Gorilla gorilla gorilla) and other wild mammals in Lopé National Park (LNP), Gabon, and we tested whether the observed pattern could be explained by bacterial transmission from humans and domestic animals into wildlife populations. Our results show a high prevalence of antibiotic-resistant bacterial isolates in humans and low levels in gorillas and other wildlife. The significant differences in the genetic background of the resistant bacteria isolated from humans and gorillas suggest that transmission is low or does not occur between these two species. These findings indicate that the presence of antibiotic-resistant strains in wildlife do not imply direct bacteria transmission from humans. Thus, in areas of low human density, human-wildlife E. coli transmission seems to be low. The presence of antibiotic-resistant isolates in gorillas may be better explained by other mechanisms for resistance acquisition, such as horizontal gene exchange among bacteria or naturally acquired resistance. PMID:22492436

  11. Prevalence of antibiotic-resistant E. coli in retail chicken: comparing conventional, organic, kosher, and raised without antibiotics [v2; ref status: indexed, http://f1000r.es/1pu

    Directory of Open Access Journals (Sweden)

    Jack M Millman

    2013-09-01

    Full Text Available Retail poultry products are known sources of antibiotic-resistant Escherichia coli, a major human health concern. Consumers have a range of choices for poultry, including conventional, organic, kosher, and raised without antibiotics (RWA – designations that are perceived to indicate differences in quality and safety. However, whether these categories vary in the frequency of contamination with antibiotic-resistant E. coli is unknown. We examined the occurrence of antibiotic-resistant E. coli on raw chicken marketed as conventional, organic, kosher and RWA. From April – June 2012, we purchased 213 samples of raw chicken from 15 locations in the New York City metropolitan area. We screened E. coli isolates from each sample for resistance to 12 common antibiotics. Although the organic and RWA labels restrict the use of antibiotics, the frequency of antibiotic-resistant E. coli tended to be only slightly lower for RWA, and organic chicken was statistically indistinguishable from conventional products that have no restrictions. Kosher chicken had the highest frequency of antibiotic-resistant E. coli, nearly twice that of conventional products, a result that belies the historical roots of kosher as a means to ensure food safety. These results indicate that production methods influence the frequency of antibiotic-resistant E. coli on poultry products available to consumers. Future research to identify the specific practices that cause the high frequency of antibiotic-resistant E. coli in kosher chicken could promote efforts to reduce consumer exposure to this potential pathogen.

  12. Dynamics of Mutator and Antibiotic-Resistant Populations in a Pharmacokinetic/Pharmacodynamic Model of Pseudomonas aeruginosa Biofilm Treatment

    DEFF Research Database (Denmark)

    Macià, María D.; Pérez, José L.; Molin, Søren;

    2011-01-01

    Biofilm growth, antibiotic resistance, and mutator phenotypes are key components of chronic respiratory infections by Pseudomonas aeruginosa in cystic fibrosis patients. We examined the dynamics of mutator and antibiotic-resistant populations in P. aeruginosa flow-cell biofilms, using fluorescently...... monitored by confocal laser scanning microscopy (CLSM), and the numbers of viable cells and resistant mutants (4- and 16-fold MICs) were determined. Despite optimized pharmacokinetic/pharmacodynamic (PK/PD) parameters, CIP treatment did not suppress resistance development in P. aeruginosa biofilms. One.......01 proportion, took over the whole biofilm after only 2 days of CIP treatment outnumbering PAO1 by 3 log at t4. Our results show that mutational mechanisms play a major role in biofilm antibiotic resistance and that theoretically optimized PK/PD parameters fail to suppress resistance development, suggesting...

  13. Cationic Amphiphiles Increase Activity of Aminoglycoside Antibiotic Tobramycin in the Presence of Airway Polyelectrolytes

    Energy Technology Data Exchange (ETDEWEB)

    Purdy Drew, Kirstin R.; Sanders, Lori K.; Culumber, Zachary W.; Zribi, Olena; Wong, Gerard C.L.; (UIUC)

    2009-06-17

    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.

  14. Cationic Amphiphiles Increase Activity of Aminoglycoside Antibiotic Tobramycin in the Presence of Airway Polyelectrolytes

    Energy Technology Data Exchange (ETDEWEB)

    Drew, K.R.Purdy; Sanders, L.K.; Culumber, Z.W.; Zribi, O.; Wong, G.C.L.

    2009-05-21

    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.

  15. 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. PMID:26245695

  16. Mutations in eukaryotic 18S ribosomal RNA affect translational fidelity and resistance to aminoglycoside antibiotics.

    Science.gov (United States)

    Chernoff, Y O; Vincent, A; Liebman, S W

    1994-02-15

    Mutations have been created in the Saccharomyces cerevisiae 18S rRNA gene that correspond to those known to be involved in the control of translational fidelity or antibiotic resistance in prokaryotes. Yeast strains, in which essentially all chromosomal rDNA repeats are deleted and all cellular rRNAs are encoded by plasmid, have been constructed that contain only mutant 18S rRNA. In Escherichia coli, a C-->U substitution at position 912 of the small subunit rRNA causes streptomycin resistance. Eukaryotes normally carry U at the corresponding position and are naturally resistant to streptomycin. We show that a U-->C transition (rdn-4) at this position of the yeast 18S rRNA gene decreases resistance to streptomycin. The rdn-4 mutation also increases resistance to paromomycin and G-418, and inhibits nonsense suppression induced by paromomycin. The same phenotypes, as well as a slow growth phenotype, are also associated with rdn-2, whose prokaryotic counterpart, 517 G-->A, manifests itself as a suppressor rather than an antisuppressor. Neither rdn-2- nor rdn-4-related phenotypes could be detected in the presence of the normal level of wild-type rDNA repeats. Our data demonstrate that eukaryotic rRNA is involved in the control of translational fidelity, and indicate that rRNA features important for interactions with aminoglycosides have been conserved throughout evolution.

  17. Introducing Aztreonam The First Monobactam Antibiotic, A Suitable Substitution for the Aminoglycosides

    Directory of Open Access Journals (Sweden)

    A. Jahanshahi M.Khajeh - Karamadeni S. Fazli Bazaz

    1992-07-01

    Full Text Available Aztreonam (Azactam for injection, squibb is the first member of a new and unique class of beta - lactam antibiotics designated by researchers at the Squibb Institute for Medical Research as monobactams (monocyclic bacterially produced beta - lactam antibiotics."nIn this research, for the first time, antimicrobial spectrum of aztrenoam was determined by Disk - Agar Diffusion (250 spp. and Macrodilution Broth Methods (150 Spp."nWe also compared this antibiotic with two routine aminoglycoside antibiotics (Amikacin, Gentamicin in Iran. The most active antibiotic in our study was aztreonam which had MIC50 & MIC90 of 4 & 32 ^g/ml specifically against Pseudomonas aeruginosa."nThis rate for the other aerobic gram - negative bacteria (E. coli, Kleb. pneumoniae, Proteus mirabilis, enterobacter spp., Shigella Spp. and Salmonella Spp. was less than 0.5 & 4 g/ml respectively."nAztreonam's MIC90 for kleb pneumoniae was 8/jg/mI our results were Correlated to the other studies"nAll aerobic gram - negative bacteria has been obtained from the Qhaem's Medical Center in Mashhad - IRAN."nThe results of Disk - Agar Diffusion Method determines that all bacteria were 100% susceptible against aztreonam except Pseudomonas aeruginosa with 83% susceptibility.

  18. Study of the Interference between Plectranthus Species Essential Oils from Brazil and Aminoglycosides

    Directory of Open Access Journals (Sweden)

    Fabíola Fernandes Galvão Rodrigues

    2013-01-01

    Full Text Available Plectranthus is one of the most representative genera of Lamiaceae family. In this study, the essential oils from Plectranthus amboinicus, Plectranthus ornatus, and Plectranthus barbatus were investigated for their chemical composition and antimicrobial and modulatory activities. The major components found were carvacrol (54.4%—P. amboinicus and eugenol (22.9%—P. ornatus e 25.1%—P. barbatus. In vitro antimicrobial activity was conducted against Escherichia coli, Proteus vulgaris, Bacillus cereus, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus aureus (multiresistant using microdilution method. The results of bioassay showed that all strains were sensitive to the oils, except P. aeruginosa that was resistant to P. amboinicus and P. ornatus. A synergistic effect of all essential oils combined with the aminoglycosides was demonstrated. These results show that P. amboinicus, P. ornatus, and P. barbatus inhibit the growth of pathogenic microorganism, and besides this they present antibiotic modifying activity, providing a new perspective against the problem of bacterial resistance to antibiotics.

  19. Cationic Amphiphiles Increase Activity of Aminoglycoside Antibiotic Tobramycin in the Presence of Airway Polyelectrolytes

    International Nuclear Information System (INIS)

    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

  20. Synergistic interaction of PMAP-36 and PRW4 with aminoglycoside antibiotics and their antibacterial mechanism.

    Science.gov (United States)

    Wang, Zeyun; Zhang, Licong; Wang, Jue; Wei, Dandan; Shi, Baoming; Shan, Anshan

    2014-12-01

    The antimicrobial peptide PMAP-36 is a highly cationic and amphipathic α-helical peptide. PRW4 is a truncated analog that replaces paired lysine residues with tryptophan along the N-terminal and deletes the C-terminal hydrophobic tail of PMAP-36. Studies on the two peptides have already been performed. However, whether there is a synergistic effect with antibiotics has not been investigated, and the study of the antibacterial mechanism of the peptides is inadequate. In this study, antibiotic-peptide combinations were tested against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, and the confocal laser scanning microscopy (LSCM) and DNA gel retardation were measured. The results indicated synergy between the peptides and gentamicin when tested against E. coli [fractional lethal concentration (FLC) peptides and gentamicin against S. aureus (0.5 peptides against E. coli and S. aureus (1 DNA binding suggest that PMAP-36 was able to translocate across the bacterial membranes and interact with intracellular DNA, but PRW4 presented no DNA-binding ability. These results indicate that the combination of PMAP-36 and PRW4 with aminoglycosides may provide useful information for clinical application, and the antibacterial mechanism of peptides likely does not solely involve cytoplasmic-membrane permeabilization.

  1. Cilia-Associated Genes Play Differing Roles in Aminoglycoside-Induced Hair Cell Death in Zebrafish

    Directory of Open Access Journals (Sweden)

    Tamara M. Stawicki

    2016-07-01

    Full Text Available Hair cells possess a single primary cilium, called the kinocilium, early in development. While the kinocilium is lost in auditory hair cells of most species it is maintained in vestibular hair cells. It has generally been believed that the primary role of the kinocilium and cilia-associated genes in hair cells is in the establishment of the polarity of actin-based stereocilia, the hair cell mechanotransduction apparatus. Through genetic screening and testing of candidate genes in zebrafish (Danio rerio we have found that mutations in multiple cilia genes implicated in intraflagellar transport (dync2h1, wdr35, ift88, and traf3ip, and the ciliary transition zone (cc2d2a, mks1, and cep290 lead to resistance to aminoglycoside-induced hair cell death. These genes appear to have differing roles in hair cells, as mutations in intraflagellar transport genes, but not transition zone genes, lead to defects in kinocilia formation and processes dependent upon hair cell mechanotransduction activity. These mutants highlight a novel role of cilia-associated genes in hair cells, and provide powerful tools for further study.

  2. Cilia-Associated Genes Play Differing Roles in Aminoglycoside-Induced Hair Cell Death in Zebrafish.

    Science.gov (United States)

    Stawicki, Tamara M; Hernandez, Liana; Esterberg, Robert; Linbo, Tor; Owens, Kelly N; Shah, Arish N; Thapa, Nihal; Roberts, Brock; Moens, Cecilia B; Rubel, Edwin W; Raible, David W

    2016-01-01

    Hair cells possess a single primary cilium, called the kinocilium, early in development. While the kinocilium is lost in auditory hair cells of most species it is maintained in vestibular hair cells. It has generally been believed that the primary role of the kinocilium and cilia-associated genes in hair cells is in the establishment of the polarity of actin-based stereocilia, the hair cell mechanotransduction apparatus. Through genetic screening and testing of candidate genes in zebrafish (Danio rerio) we have found that mutations in multiple cilia genes implicated in intraflagellar transport (dync2h1, wdr35, ift88, and traf3ip), and the ciliary transition zone (cc2d2a, mks1, and cep290) lead to resistance to aminoglycoside-induced hair cell death. These genes appear to have differing roles in hair cells, as mutations in intraflagellar transport genes, but not transition zone genes, lead to defects in kinocilia formation and processes dependent upon hair cell mechanotransduction activity. These mutants highlight a novel role of cilia-associated genes in hair cells, and provide powerful tools for further study.

  3. Increased prevalence of antibiotic-resistant E. coli in gulls sampled in Southcentral Alaska is associated with urban environments

    Science.gov (United States)

    Atterby, Clara; Ramey, Andrew M.; Hall, Gabriel Gustafsson; Järhult, Josef; Börjesson, Stefan; Bonnedahl, Jonas

    2016-01-01

    Background Antibiotic-resistant bacteria pose challenges to healthcare delivery systems globally; however, limited information is available regarding the prevalence and spread of such bacteria in the environment. The aim of this study was to compare the prevalence of antibiotic-resistant bacteria in large-bodied gulls (Larus spp.) at urban and remote locations in Southcentral Alaska to gain inference into the association between antibiotic resistance in wildlife and anthropogenically influenced habitats. Methods Escherichia coli was cultured (n=115 isolates) from fecal samples of gulls (n=160) collected from a remote location, Middleton Island, and a more urban setting on the Kenai Peninsula. Results Screening of E. coli from fecal samples collected from glaucous-winged gulls (Larus glaucescens) at Middleton Island revealed 8% of isolates were resistant to one or more antibiotics and 2% of the isolates were resistant to three or more antibiotics. In contrast, 55% of E. coli isolates derived from fecal samples collected from large-bodied gulls (i.e. glaucous, herring [Larus argentatus], and potentially hybrid gulls) on the Kenai Peninsula were resistant to one or more antibiotics and 22% were resistant to three or more antibiotics. In addition, total of 16% of the gull samples from locations on the Kenai Peninsula harbored extended-spectrum cephalosporin-resistant E. coli isolates (extended-spectrum beta-lactamases [ESBL] and plasmid-encoded AmpC [pAmpC]), in contrast to Middleton Island where no ESBL- or pAmpC-producing isolates were detected. Conclusion Our findings indicate that increased prevalence of antibiotic resistance is associated with urban environments in Southcentral Alaska and presumably influenced by anthropogenic impacts. Further investigation is warranted to assess how migratory birds may maintain and spread antimicrobial-resistant bacteria of relevance to human and animal health. PMID:27649798

  4. Increased prevalence of antibiotic-resistant E. coli in gulls sampled in Southcentral Alaska is associated with urban environments

    Directory of Open Access Journals (Sweden)

    Clara Atterby

    2016-09-01

    Full Text Available Background: Antibiotic-resistant bacteria pose challenges to healthcare delivery systems globally; however, limited information is available regarding the prevalence and spread of such bacteria in the environment. The aim of this study was to compare the prevalence of antibiotic-resistant bacteria in large-bodied gulls (Larus spp. at urban and remote locations in Southcentral Alaska to gain inference into the association between antibiotic resistance in wildlife and anthropogenically influenced habitats. Methods: Escherichia coli was cultured (n=115 isolates from fecal samples of gulls (n=160 collected from a remote location, Middleton Island, and a more urban setting on the Kenai Peninsula. Results: Screening of E. coli from fecal samples collected from glaucous-winged gulls (Larus glaucescens at Middleton Island revealed 8% of isolates were resistant to one or more antibiotics and 2% of the isolates were resistant to three or more antibiotics. In contrast, 55% of E. coli isolates derived from fecal samples collected from large-bodied gulls (i.e. glaucous, herring [Larus argentatus], and potentially hybrid gulls on the Kenai Peninsula were resistant to one or more antibiotics and 22% were resistant to three or more antibiotics. In addition, total of 16% of the gull samples from locations on the Kenai Peninsula harbored extended-spectrum cephalosporin-resistant E. coli isolates (extended-spectrum beta-lactamases [ESBL] and plasmid-encoded AmpC [pAmpC], in contrast to Middleton Island where no ESBL- or pAmpC-producing isolates were detected. Conclusion: Our findings indicate that increased prevalence of antibiotic resistance is associated with urban environments in Southcentral Alaska and presumably influenced by anthropogenic impacts. Further investigation is warranted to assess how migratory birds may maintain and spread antimicrobial-resistant bacteria of relevance to human and animal health.

  5. Antibiotic-resistant fecal bacteria, antibiotics, and mercury in surface waters of Oakland County, Michigan, 2005-2006

    Science.gov (United States)

    Fogarty, Lisa R.; Duris, Joseph W.; Crowley, Suzanne L.; Hardigan, Nicole

    2007-01-01

    Water samples collected from 20 stream sites in Oakland and Macomb Counties, Mich., were analyzed to learn more about the occurrence of cephalosporin-resistant Escherichia coli (E. coli) and vancomycin-resistant enterococci (VRE) and the co-occurrence of antibiotics and mercury in area streams. Fecal indicator bacteria concentrations exceeded the Michigan recreational water-quality standard of 300 E. coli colony forming units (CFU) per 100 milliliters of water in 19 of 35 stream-water samples collected in Oakland County. A gene commonly associated with enterococci from humans was detected in samples from Paint Creek at Rochester and Evans Ditch at Southfield, indicating that human fecal waste is a possible source of fecal contamination at these sites. E. coli resistant to the cephalosporin antibiotics (cefoxitin and/ or ceftriaxone) were found at all sites on at least one occasion. The highest percentages of E. coli isolates resistant to cefoxitin and ceftriaxone were 71 percent (Clinton River at Auburn Hills) and 19 percent (Sashabaw Creek near Drayton Plains), respectively. Cephalosporin-resistant E. coli was detected more frequently in samples from intensively urbanized or industrialized areas than in samples from less urbanized areas. VRE were not detected in any sample collected in this study. Multiple antibiotics (azithromycin, erythromycin, ofloxacin, sulfamethoxazole, and trimethoprim) were detected in water samples from the Clinton River at Auburn Hills, and tylosin (an antibiotic used in veterinary medicine and livestock production that belongs to the macrolide group, along with erythromycin) was detected in one water sample from Paint Creek at Rochester. Concentrations of total mercury were as high as 19.8 nanograms per liter (Evans Ditch at Southfield). There was no relation among percentage of antibiotic-resistant bacteria and measured concentrations of antibiotics or mercury in the water. Genetic elements capable of exchanging multiple antibiotic-resistance

  6. Food Enzymes

    Science.gov (United States)

    McBroom, Rachel; Oliver-Hoyo, Maria T.

    2007-01-01

    Many students view biology and chemistry as two unrelated, separate sciences; how these courses are generally taught in high schools may do little to change that impression. The study of enzymes provide a great opportunity for both biology and chemistry teachers to share with students the interdisciplinary nature of science. This article describes…

  7. Enzyme immunoassay

    DEFF Research Database (Denmark)

    Feldt-Rasmussen, B; Dinesen, B; Deckert, M

    1985-01-01

    An enzyme linked immunoadsorbent assay for urinary albumin using commercially available reagents is described. The assay range is 2.5-120 micrograms/l. When samples are analysed in two standard dilutions, the assayable albumin concentration range is 2.5-240 mg/l, covering the clinical range from...

  8. 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.

  9. Aminoglycoside ototoxicity in three murine strains and effects on NKCC1 of stria vascularis

    Institute of Scientific and Technical Information of China (English)

    CHU Han-qi; XIONG Hao; Zhou Xiao-qin; HAN Fang; WU Zhen-gong; ZHANG Ping; HUANG Xiao-wen; CUI Yong-hua

    2006-01-01

    Background After establishing a murine model of aminoglycoside antibiotic (AmAn) induced ototoxicity, the sensitivity of AmAn induced ototoxicity in three murine strains and the effect of kanamycin on the expression of Na-K-2C1 cotransporter-1 (NKCC 1) in stria vascularis were investigated.Methods C57BL/6J, CBA/CaJ, NKCC1+/- mice (24 of each strain) were randomly divided into four experimental groups: A: kanamycin alone; B: kanamycin plus 2,3-dihydroxybenzoate; C: 2,3-dihydroxybenzoate alone; and D: control group. Mice were injected with kanamycin or/and 2,3-dihydroxybenzoate twice daily for 14 days. Auditory brainstem response (ABR) was measured and morphology of cochlea delineated with succinate dehydrogenase staining. Expression of NKCC1 in stria vascularis was detected immunohistochemically.Results All three strains in groups A and B developed significant ABR threshold shifts (P<0.01), which were accompanied by outer hair cell loss. NKCC 1 expression in stria vascularis was the weakest in group A (A cf D,P<0.01) and the strongest in groups C and D (P<0.05). CBA/CaJ mice had the highest sensitivity to AmAn.Conclusions Administration of kanamycin established AmAn induced ototoxicity. Kanamycin inhibited the expression of NKCC1 in stria vascularis. 2, 3-dihydroxybenzoate attenuated AmAn induced ototoxicitypossibly by enhancing the expression of NKCC1. Age related hearing loss did not show additional sensitivity to AmAn induced ototoxicity in murine model.

  10. Highly stable, protein capped gold nanoparticles as effective drug delivery vehicles for amino-glycosidic antibiotics

    International Nuclear Information System (INIS)

    A method for the production of highly stable gold nanoparticles (Au NP) was optimized using sodium borohydride as reducing agent and bovine serum albumin as capping agent. The synthesized nanoparticles were characterized using UV–visible spectroscopy, transmission electron microscopy, X‐ray diffraction (XRD) and dynamic light scattering techniques. The formation of gold nanoparticles was confirmed from the appearance of pink colour and an absorption maximum at 532 nm. These protein capped nanoparticles exhibited excellent stability towards pH modification and electrolyte addition. The produced nanoparticles were found to be spherical in shape, nearly monodispersed and with an average particle size of 7.8 ± 1.7 nm. Crystalline nature of the nanoparticles in face centered cubic structure is confirmed from the selected‐area electron diffraction and XRD patterns. The nanoparticles were functionalized with various amino-glycosidic antibiotics for utilizing them as drug delivery vehicles. Using Fourier transform infrared spectroscopy, the possible functional groups of antibiotics bound to the nanoparticle surface have been examined. These drug loaded nanoparticle solutions were tested for their antibacterial activity against Gram-negative and Gram-positive bacterial strains, by well diffusion assay. The antibiotic conjugated Au NP exhibited enhanced antibacterial activity, compared to pure antibiotic at the same concentration. Being protein capped and highly stable, these gold nanoparticles can act as effective carriers for drugs and might have considerable applications in the field of infection prevention and therapeutics. - Highlights: ► Method for NaBH4 reduced and BSA capped gold nanoparticle was standardized. ► Nanoparticles were spherical and nearly monodispersed with a size of 7.8 nm. ► Nanoparticles are extremely stable towards pH modification and electrolyte addition. ► Antibiotic conjugated nanoparticles exhibited enhanced antibacterial activity

  11. Alkylating enzymes.

    Science.gov (United States)

    Wessjohann, Ludger A; Keim, Jeanette; Weigel, Benjamin; Dippe, Martin

    2013-04-01

    Chemospecific and regiospecific modifications of natural products by methyl, prenyl, or C-glycosyl moieties are a challenging and cumbersome task in organic synthesis. Because of the availability of an increasing number of stable and selective transferases and cofactor regeneration processes, enzyme-assisted strategies turn out to be promising alternatives to classical synthesis. Two categories of alkylating enzymes become increasingly relevant for applications: firstly prenyltransferases and terpene synthases (including terpene cyclases), which are used in the production of terpenoids such as artemisinin, or meroterpenoids like alkylated phenolics and indoles, and secondly methyltransferases, which modify flavonoids and alkaloids to yield products with a specific methylation pattern such as 7-O-methylaromadendrin and scopolamine.

  12. Engineering enzymes

    OpenAIRE

    Dutton, P. Leslie; Moser, Christopher C.

    2011-01-01

    Fundamental research into bioinorganic catalysis of the kind presented at this Faraday Discussion has the potential to turn inspiration drawn from impressive natural energy and chemical transformations into artificial catalyst constructions useful to mankind. Creating bio-inspired artificial constructions requires a level of understanding well beyond simple description of structures and mechanisms of natural enzymes. To be useful, such description must be augmented by a practical sense of str...

  13. SYNTHESIS AND CYTOTOXIC ACTIVITY OF NEW 5H-INDOLO[2,3-B]QUINOLINE O-AMINOGLYCOSIDES.

    Science.gov (United States)

    Badowska-Rosłonek, Katarzyna; Ciesielska, Agnieszka; Switalska, Marta; Piskozub, Małgorzata; Peczyńska-Czoch, Wanda; Wietrzyk, Joanna; Kaczmarek, Łukasz

    2016-01-01

    Novel 5H-indolo[2,3-b]quinoline O-aminoglycosides were synthesized in order to check the hypothesis that the construction of hybrids composed of the active 5H-indolo[2,3-b]quinoline chromophore and daunosaminyl or acosaminyl moiety may result in the cytotoxic activity of the obtained derivatives that is much higher than the one of the parent DIMIQ (5,11-dimethyl-5H-indolo[2,3-b]quinoline) and 6H-indoloquinoline analogs. Actually, 5H-indolo[2,3-b]indoloquinoline O-aminoglycosides showed the anti-proliferative activity in vitro against human lung adenocarcinoma A549, breast cancer MCF-7, melanoma Hs294T, promyelocytic leukemia HL-60, uterine sarcoma MES-SA and colon cancer LoVo cell lines, which was 10 times higher than that of the 6H-analogs and comparable to the one of the referential DIMIQ. Unexpectedly, it appeared that except for HL-60/MX2 (P-gp-independent and topoisomerase II-dependent resistance), other MDR tumor cell lines (LoVo/DX. P-gp-dependent, MRP-, LRP-dependent multidrug resistance) and MES-SA/DX5 (P-gp-dependent resistance to doxorubicin) are also resistant to the 5H-indolo[2,3-b]indoloquinoline O-aminoglycosides tested. This is surprising because 6H-analogs, in general, 10 times less active against non-MDR tumor cell lines, as well as the DIMIQ itself, are able to overcome drug resistance in all MDR cell lines examined. The cytotoxicity of the tested compounds against tumor cell lines and against normal cells (mice fibroblasts BALB/3T3) was comparable. PMID:27476287

  14. Natural antioxidant L-carnosine inhibits LPO intensification in structures of the auditory analyzer under conditions of chronic exposure to aminoglycoside antibiotics.

    Science.gov (United States)

    Zhuravskii, S G; Aleksandrova, L A; Sirot, V S; Ivanov, S A

    2004-10-01

    Intragastric administration of L-carnosine suspension to Wistar-Kyoto rats 3 days before and after 7-day course of intraperitoneal injections of ototoxic aminoglycoside antibiotic kanamycin compensated expenditures of tissue antioxidant systems and significantly eliminated kanamycin-induced intensification of MDA production in tissues of the membrane part of the cochlea and in the auditory cortex of the temporal lobe. L-NAME (competitive NO synthase inhibitor) also inhibited LPO, increased total antioxidant activity, and decreased ototoxicity of kanamycin, which confirms the contribution of NO into LPO intensification under conditions of aminoglycoside treatment. Inhibition of pathological intensification of LPO processes and increase in total antioxidant activity under conditions of induced acute aminoglycoside ototoxicity characterizes L-carnosine as a highly effective otoprotector. PMID:15665945

  15. Plasmid-Mediated High-Level Resistance to Aminoglycosides in Enterobacteriaceae Due to 16S rRNA Methylation

    OpenAIRE

    Galimand, Marc; Courvalin, Patrice; Lambert, Thierry

    2003-01-01

    A self-transferable plasmid of ca. 80 kb, pIP1204, conferred multiple-antibiotic resistance to Klebsiella pneumoniae BM4536, which was isolated from a urinary tract infection. Resistance to β-lactams was due to the blaTEM1 and blaCTX-M genes, resistance to trimethroprim was due to the dhfrXII gene, resistance to sulfonamides was due to the sul1 gene, resistance to streptomycin-spectinomycin was due to the ant3"9 gene, and resistance to nearly all remaining aminoglycosides was due to the aac3-...

  16. 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

  17. Antibiotic-Resistant Fecal Bacteria, Antibiotics, and Mercury in Surface Waters of Oakland County, Michigan, 2005-2006

    Science.gov (United States)

    Fogarty, Lisa R.; Duris, Joseph W.; Crowley, Suzanne L.; Hardigan, Nicole

    2007-01-01

    Water samples collected from 20 stream sites in Oakland and Macomb Counties, Mich., were analyzed to learn more about the occurrence of cephalosporin-resistant Escherichia coli (E. coli) and vancomycin-resistant enterococci (VRE) and the co-occurrence of antibiotics and mercury in area streams. Fecal indicator bacteria concentrations exceeded the Michigan recreational water-quality standard of 300 E. coli colony-forming units (CFU) per 100 milliliters of water in 19 of 35 stream-water samples collected in Oakland County. A gene commonly associated with enterococci from humans was detected in samples from Paint Creek at Rochester and Evans Ditch at Southfield, indicating that human fecal waste is a possible source of fecal contamination at these sites. E. coli resistant to the cephalosporin antibiotics (cefoxitin and/or ceftriaxone) were found at all sites on at least one occasion. The highest percentages of E. coli isolates resistant to cefoxitin and ceftriaxone were 71 percent (Clinton River at Auburn Hills) and 19 percent (Sashabaw Creek near Drayton Plains), respectively. Cephalosporin-resistant E. coli was detected more frequently in samples from intensively urbanized or industrialized areas than in samples from less urbanized areas. VRE were not detected in any sample collected in this study. Multiple antibiotics (azithromycin, erythromycin, ofloxacin, sulfamethoxazole, and trimethoprim) were detected in water samples from the Clinton River at Auburn Hills, and tylosin (an antibiotic used in veterinary medicine and livestock production that belongs to the macrolide group, along with erythromycin) was detected in one water sample from Paint Creek at Rochester. Concentrations of total mercury were as high as 19.8 nanograms per liter (Evans Ditch at Southfield). There was no relation among percentage of antibiotic-resistant bacteria and measured concentrations of antibiotics or mercury in the water. Genetic elements capable of exchanging multiple antibiotic-resistance

  18. Salmonella spp. and antibiotic-resistant strains in wild mammals and birds in north-western Italy from 2002 to 2010

    OpenAIRE

    Velca Botti; Francine Valérie Navillod; Lorenzo Domenis; Riccardo Orusa; Erika Pepe; Serena Robetto; Cristina Guidetti

    2013-01-01

    Salmonella is an important zoonotic pathogen of economic importance. In Europe, salmonellosis is the second food-borne infection, in Italy, Salmonella is still the major cause of food-borne outbreaks. In Europe, there are many Salmonella surveillance plans on farmed animals, while Salmonella survey of wild animals is occasionally performed. The aim of this study was to investigate the presence of Salmonella including the antibiotic-resistant strains in wild animals. Between 2002 and 2010, 2,7...

  19. Comparison of the incidence of pathogenic and antibiotic-resistant Escherichia coli strains in adult cattle and veal calf slaughterhouse effluents highlighted different risks for public health.

    Science.gov (United States)

    Um, Maryse Michèle; Barraud, Olivier; Kérourédan, Monique; Gaschet, Margaux; Stalder, Thibault; Oswald, Eric; Dagot, Christophe; Ploy, Marie-Cecile; Brugère, Hubert; Bibbal, Delphine

    2016-01-01

    The goal of this study was to investigate the involvement of bovine slaughterhouse effluents and biosolids in the risk of environmental dissemination of pathogenic and antibiotic-resistant Escherichia coli. Several samples were collected from one adult cattle and one veal calf slaughterhouse wastewater treatment plant (WWTP). The treatment process had no impact on the percentage of Shiga toxin-producing E. coli (STEC) and on the percentage of atypical enteropathogenic E. coli (aEPEC). A STEC O157:H7 was isolated from the thickened sludge of the adult cattle slaughterhouse. As thickened sludge is intended to be spread on agricultural lands, the detection of this pathogenic strain is a public health issue. The percentage of antibiotic-resistant E. coli was 5.0% and 87.5% in wastewater from the adult cattle and the veal calf slaughterhouse, respectively. These percentages were not significantly different after treatment. Integron-bearing E. coli isolates were only detected in the veal calf slaughterhouse WWTP with percentages above 50.0% for all sampling points whatever the step of the treatment process. Taken together, these findings highlighted the fact that different public health risks might be associated with adult cattle or veal calf slaughterhouses regarding the dissemination of pathogenic and antibiotic-resistant E. coli isolates into the environment.

  20. Comparison of the incidence of pathogenic and antibiotic-resistant Escherichia coli strains in adult cattle and veal calf slaughterhouse effluents highlighted different risks for public health.

    Science.gov (United States)

    Um, Maryse Michèle; Barraud, Olivier; Kérourédan, Monique; Gaschet, Margaux; Stalder, Thibault; Oswald, Eric; Dagot, Christophe; Ploy, Marie-Cecile; Brugère, Hubert; Bibbal, Delphine

    2016-01-01

    The goal of this study was to investigate the involvement of bovine slaughterhouse effluents and biosolids in the risk of environmental dissemination of pathogenic and antibiotic-resistant Escherichia coli. Several samples were collected from one adult cattle and one veal calf slaughterhouse wastewater treatment plant (WWTP). The treatment process had no impact on the percentage of Shiga toxin-producing E. coli (STEC) and on the percentage of atypical enteropathogenic E. coli (aEPEC). A STEC O157:H7 was isolated from the thickened sludge of the adult cattle slaughterhouse. As thickened sludge is intended to be spread on agricultural lands, the detection of this pathogenic strain is a public health issue. The percentage of antibiotic-resistant E. coli was 5.0% and 87.5% in wastewater from the adult cattle and the veal calf slaughterhouse, respectively. These percentages were not significantly different after treatment. Integron-bearing E. coli isolates were only detected in the veal calf slaughterhouse WWTP with percentages above 50.0% for all sampling points whatever the step of the treatment process. Taken together, these findings highlighted the fact that different public health risks might be associated with adult cattle or veal calf slaughterhouses regarding the dissemination of pathogenic and antibiotic-resistant E. coli isolates into the environment. PMID:26460853

  1. COMBINATIONAL ADMINISTRATION OF AMINOGLYCOSIDES AND LOOP DIURETICS AS AN EFFICIENT STRATEGY TO ESTABLISH DEAFNESS MODELS IN RATS

    Institute of Scientific and Technical Information of China (English)

    CONG Tao; LIU Riyuan; YUAN Shuolong; XU Liangwei; YANG Shiming

    2014-01-01

    It is known that aminoglycoside antibiotics can damage the vestibular and auditory sensory epithelia, and the loop diuretics can enhance the ototoxic effect of aminoglycosides. Previous studies on the synergistic effect of these two types of drugs have used mice, guinea pigs and cats, but not rats. The aim of this study was to determine this synergistic effects in rat cochleae. Rats received intravenous injections of different doses of furosemide and/or intramuscular injections of kanamycin sulfate. Au-ditory brainstem response (ABR), scanning electron microscopy (SEM) and immunocytochemistry were used to determine the effects of drug administration. In the group receiving combined administration of furosemide and kanamycin, the ABR thresh-old showed significant elevation 3 days after drug administration, greater than single drug administration. The hair cells showed various degrees of injury from the apical turn to the basal turn of the cochlea and from the outer hair cells to the inner hair cells. Neuron fibers of the hair cells showed significant loss 7 days after the drug administration, but the number of spiral ganglia did not decrease and supporting cells showed no signs of injury. Our study suggest that combined administration of fu-rosemide and kanamycin has an synergistic ototoxic effect, and can result in hair cell loss and hearing loss in rats.

  2. Cefditoren and ceftriaxone enhance complement-mediated immunity in the presence of specific antibodies against antibiotic-resistant pneumococcal strains.

    Directory of Open Access Journals (Sweden)

    Elisa Ramos-Sevillano

    Full Text Available BACKGROUND: Specific antibodies mediate humoral and cellular protection against invading pathogens such as Streptococcus pneumoniae by activating complement mediated immunity, promoting phagocytosis and stimulating bacterial clearance. The emergence of pneumococcal strains with high levels of antibiotic resistance is of great concern worldwide and a serious threat for public health. METHODOLOGY/PRINCIPAL FINDINGS: Flow cytometry was used to determine whether complement-mediated immunity against three antibiotic-resistant S. pneumoniae clinical isolates is enhanced in the presence of sub-inhibitory concentrations of cefditoren and ceftriaxone. The binding of acute phase proteins such as C-reactive protein and serum amyloid P component, and of complement component C1q, to pneumococci was enhanced in the presence of serum plus either of these antibiotics. Both antibiotics therefore trigger the activation of the classical complement pathway against S. pneumoniae. C3b deposition was also increased in the presence of specific anti-pneumococcal antibodies and sub-inhibitory concentrations of cefditoren and ceftriaxone confirming that the presence of these antibiotics enhances complement-mediated immunity to S. pneumoniae. CONCLUSIONS/SIGNIFICANCE: Using cefditoren and ceftriaxone to promote the binding of acute phase proteins and C1q to pneumococci, and to increase C3b deposition, when anti-pneumococcal antibodies are present, might help reduce the impact of antibiotic resistance in S. pneumoniae infections.

  3. Calcined Eggshell Waste for Mitigating Soil Antibiotic-Resistant Bacteria/Antibiotic Resistance Gene Dissemination and Accumulation in Bell Pepper.

    Science.gov (United States)

    Ye, Mao; Sun, Mingming; Feng, Yanfang; Li, Xu; Schwab, Arthur P; Wan, Jinzhong; Liu, Manqiang; Tian, Da; Liu, Kuan; Wu, Jun; Jiang, Xin

    2016-07-13

    The combined accumulation of antibiotics, heavy metals, antibiotic-resistant bacteria (ARB)/antibiotic resistance genes (ARGs) in vegetables has become a new threat to human health. This is the first study to investigate the feasibility of calcined eggshells modified by aluminum sulfate as novel agricultural wastes to impede mixed contaminants from transferring to bell pepper (Capsicum annuum L.). In this work, calcined eggshell amendment mitigated mixed pollutant accumulation in bell pepper significantly, enhanced the dissipation of soil tetracycline, sulfadiazine, roxithromycin, and chloramphenicol, decreased the water-soluble fractions of antibiotics, and declined the diversity of ARB/ARGs inside the vegetable. Moreover, quantitative polymerase chain reaction analysis detected that ARG levels in the bell pepper fruits significantly decreased to 10(-10) copies/16S copies, indicating limited risk of ARGs transferring along the food chain. Furthermore, the restoration of soil microbial biological function suggests that calcined eggshell is an environmentally friendly amendment to control the dissemination of soil ARB/ARGs in the soil-vegetable system. PMID:27333280

  4. The frequency of antibiotic-resistant bacteria in homes differing in their use of surface antibacterial agents.

    Science.gov (United States)

    Marshall, Bonnie M; Robleto, Eduardo; Dumont, Theresa; Levy, Stuart B

    2012-10-01

    Antibacterial agents are common in household cleaning and personal care products, but their long-range impacts on commensal and pathogenic household bacteria are largely unknown. In a one-time survey of 38 households from Boston, MA [19] and Cincinnati, OH [18], 13 kitchen and bathroom sites were sampled for total aerobic bacteria and screened for gram phenotype and susceptibility to six antibiotic drug families. The overall bacterial titers of both user (2 or more antibacterial cleaning or personal care products) and non-user (0 or 1 product) rooms were similar with sponges and sink drains consistently showing the highest overall titers and relatively high titers of antibiotic-resistant bacteria. The mean frequency of resistant bacteria ranged from ≤20 % to as high as 45 % and multi-drug resistance was common. However, no significant differences were noted between biocide users and non-users. The frequency of pathogen recovery was similar in both user and non-user groups. PMID:22752336

  5. Molecular design and genetic optimization of antimicrobial peptides containing unnatural amino acids against antibiotic-resistant bacterial infections.

    Science.gov (United States)

    He, Yongkang; He, Xiaofeng

    2016-09-01

    Antimicrobial peptides (AMPs) have been the focus of intense research towards the finding of a viable alternative to current small-molecule antibiotics, owing to their commonly observed and naturally occurring resistance against pathogens. However, natural peptides have many problems such as low bioavailability and high allergenicity that largely limit the clinical applications of AMPs. In the present study, an integrative protocol that combined chemoinformatics modeling, molecular dynamics simulations, and in vitro susceptibility test was described to design AMPs containing unnatural amino acids (AMP-UAAs). To fulfill this, a large panel of synthetic AMPs with determined activity was collected and used to perform quantitative structure-activity relationship (QSAR) modeling. The obtained QSAR predictors were then employed to direct genetic algorithm (GA)-based optimization of AMP-UAA population, to which a number of commercially available, structurally diverse unnatural amino acids were introduced during the optimization process. Subsequently, several designed AMP-UAAs were confirmed to have high antibacterial potency against two antibiotic-resistant strains, i.e. multidrug-resistant Pseudomonas aeruginosa (MDRPA) and methicillin-resistant Staphylococcus aureus (MRSA), with minimum inhibitory concentration (MIC) < 10 μg/ml. Structural dynamics characterizations revealed that the most potent AMP-UAA peptide is an amphipathic helix that can spontaneously embed into an artificial lipid bilayer and exhibits a strong destructuring tendency associated with the embedding process. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 746-756, 2016.

  6. Peptide IDR-1018: modulating the immune system and targeting bacterial biofilms to treat antibiotic-resistant bacterial infections.

    Science.gov (United States)

    Mansour, Sarah C; de la Fuente-Núñez, César; Hancock, Robert E W

    2015-05-01

    Host defense (antimicrobial) peptides, produced by all complex organisms, typically contain an abundance of positively charged and hydrophobic amino acid residues. A small synthetic peptide termed innate defense regulator (IDR-)1018 was derived by substantial modification of the bovine neutrophil host defense peptide bactenecin. Here, we review its intriguing properties that include anti-infective, anti-inflammatory, wound healing, and anti-biofilm activities. It was initially developed as an immune modulator with an ability to selectively enhance chemokine production and polarize cellular differentiation while suppressing/balancing the pro-inflammatory response. In this regard, it has demonstrated in vivo activity in murine models including enhancement of wound healing and an ability to protect against Staphylococcus aureus, multidrug resistant Mycobacterium tuberculosis, herpes virus, and inflammatory disorders, including cerebral malaria and neuronal damage in a pre-term birth model. More recently, IDR-1018 was shown, in a broad-spectrum fashion, to selectively target bacterial biofilms, which are adaptively resistant to many antibiotics and represent the most common growth state of bacteria in human infections. Furthermore, IDR-1018 demonstrated synergy with conventional antibiotics to both prevent biofilm formation and treat pre-existing biofilms. These data are consistent with a strong potential as an adjunctive therapy against antibiotic-resistant infections. PMID:25358509

  7. Potential use of Bacillus thuringiensis bacteriocins to control antibiotic-resistant bacteria associated with mastitis in dairy goats.

    Science.gov (United States)

    Gutiérrez-Chávez, A J; Martínez-Ortega, E A; Valencia-Posadas, M; León-Galván, M F; de la Fuente-Salcido, N M; Bideshi, D K; Barboza-Corona, J E

    2016-01-01

    Mastitis caused by microbial infections in dairy goats reduces milk yield, modifies milk composition, and potentially contributes to morbidity in herds and consumers of dairy products. Microorganisms associated with mastitis in dairy goats are commonly controlled with antibiotics, but it is known that continued use of these chemical agents promotes antibiotic resistance among bacterial populations. Recently, it has been shown that bacteriocins of Bacillus thuringiensis inhibit growth of food-borne pathogens and also bacteria associated with bovine mastitis. However, there is no report on their ability to inhibit microorganisms linked to mastitis in dairy goats. In this study, using 16S rDNA and ITS regions of rDNA, we identified nine bacterial isolates and an encapsulated yeast associated with mastitis in dairy goats. Enterococcus durans, Brevibacillus sp., and Staphylococcus epidermidis 2 were resistant to, respectively, 75, ~67, ~42, and ~42 % of the antibiotics screened. In addition, 60 % of the bacterial isolates were resistant to penicillin, ampicillin, vancomycin, and dicloxacillin. Importantly, 60 % of the isolates were inhibited by the bacteriocins, but S. epidermidis 1, Enterobacter sp., Escherichia vulneris, and Cryptococcus neoformans were not susceptible to these antimicrobial peptides. Using Brevibacillus sp. and Staphylococcus chromogenes as indicator bacteria, we show that peptides of ~10 kDa that correspond to the molecular mass of bacteriocins used in this study are responsible for the inhibitory activity. Our results demonstrate that multiple antibiotic-resistant bacteria associated with subclinical mastitis in dairy goats from Guanajuato, Mexico, are susceptible to bacteriocins produced by B. thuringiensis.

  8. Potential use of Bacillus thuringiensis bacteriocins to control antibiotic-resistant bacteria associated with mastitis in dairy goats.

    Science.gov (United States)

    Gutiérrez-Chávez, A J; Martínez-Ortega, E A; Valencia-Posadas, M; León-Galván, M F; de la Fuente-Salcido, N M; Bideshi, D K; Barboza-Corona, J E

    2016-01-01

    Mastitis caused by microbial infections in dairy goats reduces milk yield, modifies milk composition, and potentially contributes to morbidity in herds and consumers of dairy products. Microorganisms associated with mastitis in dairy goats are commonly controlled with antibiotics, but it is known that continued use of these chemical agents promotes antibiotic resistance among bacterial populations. Recently, it has been shown that bacteriocins of Bacillus thuringiensis inhibit growth of food-borne pathogens and also bacteria associated with bovine mastitis. However, there is no report on their ability to inhibit microorganisms linked to mastitis in dairy goats. In this study, using 16S rDNA and ITS regions of rDNA, we identified nine bacterial isolates and an encapsulated yeast associated with mastitis in dairy goats. Enterococcus durans, Brevibacillus sp., and Staphylococcus epidermidis 2 were resistant to, respectively, 75, ~67, ~42, and ~42 % of the antibiotics screened. In addition, 60 % of the bacterial isolates were resistant to penicillin, ampicillin, vancomycin, and dicloxacillin. Importantly, 60 % of the isolates were inhibited by the bacteriocins, but S. epidermidis 1, Enterobacter sp., Escherichia vulneris, and Cryptococcus neoformans were not susceptible to these antimicrobial peptides. Using Brevibacillus sp. and Staphylococcus chromogenes as indicator bacteria, we show that peptides of ~10 kDa that correspond to the molecular mass of bacteriocins used in this study are responsible for the inhibitory activity. Our results demonstrate that multiple antibiotic-resistant bacteria associated with subclinical mastitis in dairy goats from Guanajuato, Mexico, are susceptible to bacteriocins produced by B. thuringiensis. PMID:26022411

  9. A preliminary report on the susceptibility to aminoglycosides of Escherichia coli isolated from the community-acquired urinary tract infections in adults in south-east Poland

    Directory of Open Access Journals (Sweden)

    Fidecka-Skwarzynska Magdalena

    2015-03-01

    Full Text Available World-wide, urinary tract infections (UTIs are an important clinical problem. In such, the most frequently isolated uropathogen is Escherichia coli. In the treatment of uncomplicated UTIs, e.g. cystitis, the widely used antibiotics are nitrofurantoin, trimethoprim/sulfamethoxazole, fosfomycin trometamol or ciprofloxacin, while the treatment of pyelonephritis requires the usage of antibiotics with a broader spectrum of activity, such as cephalosporins of the 3rd and 4th generation, aminoglycosides or even carbapenems. The aim of this study was to assess the susceptibility to aminoglycosides (such as amikacin, gentamicin, netilmicin and tobramycin of E. coli isolated from UTIs in adult community patients living in Lubelszczyzna. We found that all of the 86 strains of E. coli encountered were susceptible to amikacin. Moreover, the prevalence of susceptibility to tobramycin, gentamicin or netilmicin among the tested strains was found to be 89,5%, 90,7% or 94,2%, respectively. The data obtained in the present study shows the high susceptibility to aminoglycosides of E. coli isolated from the community-acquired UTIS in adults. These data, together with that derived from current literature, indicate that aminoglycosides, when employed in combination therapy with other antibiotics, may still be very useful group of antibacterial agents in the treatment of UTI’s in Poland.

  10. HIGH-LEVEL AMINOGLYCOSIDE RESISTANCE ENTEROCOCCUS SPP IN A TERTIARY CARE HOSPITAL IN MEXICO

    Directory of Open Access Journals (Sweden)

    Silvia Giono Cerezo

    2005-01-01

    Full Text Available Enterococcus is one important cause hospital-acquired infections. High levels of resistance for aminoglycosides (HLAR as gentamicin (HLGR and streptomycin (HLSR in Enterococcus isolates in a tertiary clinical care in Mexico City were studied. Identified using Microscan® system. Resistance to ampicillin, streptomycin, gentamicin and vancomycin according to NCCLS. HLGR and HLSR were confirmed using disks. 91 strains were isolated and identified from clinical samples from January 1998 to January 1999. Two species were identified. 83 (91.2 % E. faecalis and 8/91 (8.8 % were E. faecium. E. faecalis in urine samples were 67/91 (73.6%. Neither showed vancomycin or ampicillin resistance; 1/8 E. faecium was ampicillin resistant. 30/83 (36% E. faecalis and 3/8 E. faecium were gentamicin resistant; while 39/83 (47.0% E. faecalis and 4/8(50% E. faecium were streptomycin resistant. 14/83 (16% E. faecalis, 3/8 E. faecium showed sensitive pattern for gentamicin and streptomycin. None strains were -lactamases producer. E. faecalis 12/83 (14.4% were HLGR and 28/83 (33.7% were HLSR. E. faecium. 2/8 were HLGR and 2/8 were HLSR. HLAR 33/83 (39.7% were E. faecalis and 3/8(37.5% were E. faecium isolated from urine. E. faecalis was more frequent than E. faecium and show that HLAR in Enterococci is high and could be a serious problem if spread as nosocomial infection. RESUMEN: Enterococcus es una causa importante de infección intrahospitalaria. Se determinaron los niveles altos de resistencia para aminoglucósidos(HLAR, gentamicina (HLGR y estreptomicina (HLSR en Enterococcus aislados de diversos casos clínicos en un hospital de tercer nivel en México, D.F. La identificación se realizó usando el sistema de Microscan® y la resistencia a ampicilina, estreptomicina, gentamicina, vancomicina, HLGR y HLSR de acuerdo a la NCCLS. 91 cepas fueron aisladas de muestras clínicas de Enero de 1998 a Enero 1999, se identificaron dos especies. 83 (91.2% E. faecalis y 8/91 (8

  11. Diversity and dynamics of antibiotic-resistant bacteria in cheese as determined by PCR denaturing gradient gel electrophoresis.

    Science.gov (United States)

    Flórez, Ana Belén; Mayo, Baltasar

    2015-12-01

    This work reports the composition and succession of tetracycline- and erythromycin-resistant bacterial communities in a model cheese, monitored by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Bacterial 16S rRNA genes were examined using this technique to detect structural changes in the cheese microbiota over manufacturing and ripening. Total bacterial genomic DNA, used as a template, was extracted from cultivable bacteria grown without and with tetracycline or erythromycin (both at 25 μg ml(-1)) on a non-selective medium used for enumeration of total and viable cells (Plate Count agar with Milk; PCA-M), and from those grown on selective and/or differential agar media used for counting various bacterial groups; i.e., lactic acid bacteria (de Man, Rogosa and Sharpe agar; MRSA), micrococci and staphylococci (Baird-Parker agar; BPA), and enterobacteria (Violet Red Bile Glucose agar; VRBGA). Large numbers of tetracycline- and erythromycin-resistant bacteria were detected in cheese samples at all stages of ripening. Counts of antibiotic-resistant bacteria varied widely depending on the microbial group and the point of sampling. In general, resistant bacteria were 0.5-1.0 Log10 units fewer in number than the corresponding susceptible bacteria. The PCR-DGGE profiles obtained with DNA isolated from the plates for total bacteria and the different bacterial groups suggested Escherichia coli, Lactococcus lactis, Enterococcus faecalis and Staphylococcus spp. as the microbial types resistant to both antibiotics tested. This study shows the suitability of the PCR-DGGE technique for rapidly identifying and tracking antibiotic resistant populations in cheese and, by extension, in other foods.

  12. Microbiological and biochemical studies on certain antibiotic-resistant bacteria isolated from certain clinical specimens

    International Nuclear Information System (INIS)

    . Determination of B-lactamase and AmpC-B-lactamase enzyme production were carried out by two methods. Cefotaxime, ceftazidime, cefoperazone, cefoxitin and aztreonam were used with and without amoxycillin clavulanic acid to detect the presence of extended-spectrum B-lactamase (ESBL) harbouring isolates by using double-disk diffusion synergy test (DDST). Combined disk method was used also to detect the presence of ESBL harbouring isolates by using cefoperazone (CFP) and cefoperazone sulbactam (SCF) among the tested strains. Agar dilution method was used to determine minimum inhibitory concentration (MIC) of ampicillin sulbactam, cefoperazone, gentamycin and levofloxacin alone and in combination (Ampicillin sulbactam with both of gentamycin and levofloxacin) and (cefoperazone with both of gentamycin and levofloxacin). Fractional inhibitory concentration (FIC) for the combined antibiotics were calculated according to checkerboard method and synergistic effect were determined. Some resistant isolates were subjected to molecular studies including plasmid profile (Kleb.52 Morg.60 and Ps.72 ) by using a high pure plasmid isolation kit and protein pattern of Ps.72 before and after irradiation in the presence of different antibiotics alone (cefoperazone, gentamycin and ampicillin sulbactam) or in combined (cefoperazone with gentamycin and ampicillin sulbactam with gentamycin)The result of the present investigation showed that, 9 multi-drug resistant isolates were identified as; 2 isolates Escherichia coli, 2 isolates Pseudomonas aeruginosa, 1 isolate Citrobacter freundii, 1 isolate Morganella morganii all were isolated from urine samples

  13. Study on aminoglycoside resistance and drug resistance gene of ESBLs-producing Escherichia coil%产超广谱β-内酰胺酶大肠埃希菌对氨基糖甙类药物耐药性及耐药基因研究

    Institute of Scientific and Technical Information of China (English)

    郑为平; 史伟峰

    2009-01-01

    Objective To investigate the characteristics of aminoglycoside resistance of extend-ed-spectrum β-lactamases(ESBLs)-producing Escherichia coli(E, cold and expression of aminoglyco-side-modifying enzyme genes. Methods The minimal inhibitory concentrations(MICs) of gentamicin,amikacin, kanamycin, tobramycin, netilmicin and neomycin for 37 strains of ESBLs-producing E. Coli were detected by agar dilution. In addition, six aminoglycoside-modifying enzyme genes were amplified by polymersae chain reaction(PCR) and verified by DNA sequencing. Results MIC and MIC90 of gentamicin, amikacin, kanamycin, tobramycin and netilmicin for 37 strains of ESBLs-producing E. Co-Il all excelled 256 μg/mL, the resistance rates of the above antibiotics were 78.4%, 45.9%, 72.9%,83.8%and 64.90%, respectively. However, neomycin still had powerful antibacterial activity. In ad-dition, five modifying enzyme genes, including aac(3)-Ⅱ , aac(6′)-Ⅰ b, aac(6′)-Ⅱ , ant(2″)-Ⅰ and ant(3″)- Ⅰ genes, were found in 37 isoaltes except aac(3)- Ⅰ , and their positive rates were 56.8%,27.0 %, 2.7 %, 5.4 % and 13. 5 %, respectively. Conclusion The aminoglycoside resistance of ES-BLs-producing E. Coil may be associated with the expression of aminoglycoside-modifying enzyme genes.%目的 研究产超广谱β-内酰胺酶(ESBLs)大肠埃希菌对氨基糖甙类药物的耐药特性及耐药基因表达.方法 用琼脂稀释法检测庆大霉素、阿米卡星、卡那霉素、妥布霉素、奈替米星和新霉素6种药物对37株产ESBLs大肠埃希菌的最低抑菌浓度.用PCR法检测5种氨基糖甙类药物修饰酶基因,并使用DNA测序加以证实.结果 庆大霉素、阿米卡星、卡那霉素、妥布霉素和奈替米星对37株大肠埃希菌MIC50、MIC90.均大于256 mg/L,其耐药率分别为78.4%、45.9%、72.9%、83.8%和64.9%,而新霉素仍具有较高的抗菌活性.从37株菌中检出5种修饰酶基因,aac(3)-Ⅱ、aac(6′)-Ⅰ b、aac(6′)-Ⅱ、ant(2

  14. 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

    Decoding of genetic information occurs upon interaction of an mRNA codon-tRNA anticodon complex with the small subunit of the ribosome. The ribosomal decoding region is associated with highly conserved sequences near the 3' end of 16 S rRNA. The decoding process is perturbed by the aminoglycoside...... of universally conserved nucleotides at 1406 to 1408 and 1494 to 1495 in the decoding region of plasmid-encoded bacterial 16 S rRNA. Phenotypic changes range from the benign effect of U1406-->A or A1408-->G substitutions, to the highly deleterious 1406G and 1495 mutations that assemble into 30 S subunits...... but are defective in forming functional ribosomes. Changes in the local conformation of the decoding region caused by these mutations were identified by chemical probing of isolated 30 S subunits. Ribosomes containing 16 S rRNA with mutations at positions 1408, 1407+1494, or 1495 had reduced affinity...

  15. Development of aminoglycoside and β-lactamase resistance in intestinal microbiota of swine treated with lincomycin, chlorotetracycline and amoxicillin

    Directory of Open Access Journals (Sweden)

    Jian eSun

    2014-11-01

    Full Text Available Lincomycin, chlortetracycline, and amoxicillin are commonly used antimicrobials for growth promotion and infectious disease prophylaxis in swine production. In this study, we investigated the shifts and resistance development among intestinal microbiota in pregnant sows before and after lincomycin, chlortetracycline, and amoxicillin treatment by using phylogenetic analysis, bacterial enumeration, and PCR. After the antimicrobial treatment, shifts in microbial community, an increased proportion of resistant bacteria, and genes related to antimicrobial resistance as compared to the day before antimicrobial administration (day 0 were observed. Importantly, a positive correlation between antimicrobial resistance gene expression in different categories, especially those encoding aminoglycoside and β-lactamase and antimicrobial resistance, was observed. These findings demonstrate an important role of antimicrobial usage in animals in the development of antimicrobial resistance, and support the notion that prudent use of antimicrobials in swine is needed to reduce the risk of the emergence of multi-drug resistant (MDR zoonotic pathogens.

  16. Characterization of carbapenemases, extended spectrum β-lactamases, quinolone resistance and aminoglycoside resistance determinants in carbapenem-non-susceptible Escherichia coli from a teaching hospital in Chongqing, Southwest China.

    Science.gov (United States)

    Zhang, Chuanming; Xu, Xiuyu; Pu, Shuli; Huang, Shifeng; Sun, Jide; Yang, Shuangshuang; Zhang, Liping

    2014-10-01

    Carbapenem-resistant Escherichiacoli isolates harboring carbapenemases or combining an extended-spectrum β-lactamase (ESBL) enzyme with loss of porins present an increasingly urgent clinical danger. Combined resistance to aminoglycosides and fluoroquinolones in carbapeneme non-susceptible (CNS) isolates will inevitably create problems. In the current study, we characterized the carbapenemases and ESBLs, and the prevalence of quinolone resistance determinants and aminoglycoside resistance determinants in carbapenem-non-susceptible (CNS) E.coli isolates from a teaching hospital in Chongqing, Southwest China in 2012. Thirty non-duplicated CNS E.coli isolates were screened via antimicrobial susceptibility testing, and the drug resistance profiles of the 30 strains were analyzed. Carbapenemase genes blaKPC-2, ESBL genes including blaCTX-M-3, blaCTX-M-14, blaCTX-M-55 and blaTEM, ARD genes including aac(6')-Ib, armA and rmtB, and QRD genes including qnrA, qnrB, qnrC, qnrD, qnrS and aac(6')-Ib-cr were identified and clonal relatedness was investigated by pulsed-field gel electrophoresis. Of the 30 isolates, 2 (6.7%) harbored carbapenemase gene blaKPC-2; 29 (96.7%) carried ESBLs; 20 (66.7%) were QRD positive; and 11 (36.7%) were ARD positive. Between the two blaKPC-2 positive strains, one contained ESBL, QRD and ARD genes, while the other expressed ESBL genes but was negative for both QRD and ARD genes. Of the 29 ESBLs positive isolates, 2 (6.9%) were carbapenemase positive, 19 (65.5%) were QRD positive, and 11 (37.9%) were ARD positive. PFGE revealed genetic diversity among the 30 isolates, indicating that the high prevalence of CNS E. coli isolates was not caused by clonal dissemination. Production of ESBLs was associated with the carbapenem resistance and QRD genes were highly prevalent among the CNS E. coli isolates. Multiple resistant genes were co-expressed in the same isolates. This is the first report of a multidrug resistant carbapenem-non-susceptible E.coli co

  17. Involvement of aph(3′)-IIa in the formation of mosaic aminoglycoside resistance genes in natural environments

    Science.gov (United States)

    Woegerbauer, Markus; Kuffner, Melanie; Domingues, Sara; Nielsen, Kaare M.

    2015-01-01

    Intragenic recombination leading to mosaic gene formation is known to alter resistance profiles for particular genes and bacterial species. Few studies have examined to what extent aminoglycoside resistance genes undergo intragenic recombination. We screened the GenBank database for mosaic gene formation in homologs of the aph(3′)-IIa (nptII) gene. APH(3′)-IIa inactivates important aminoglycoside antibiotics. The gene is widely used as a selectable marker in biotechnology and enters the environment via laboratory discharges and the release of transgenic organisms. Such releases may provide opportunities for recombination in competent environmental bacteria. The retrieved GenBank sequences were grouped in three datasets comprising river water samples, duck pathogens and full-length variants from various bacterial genomes and plasmids. Analysis for recombination in these datasets was performed with the Recombination Detection Program (RDP4), and the Genetic Algorithm for Recombination Detection (GARD). From a total of 89 homologous sequences, 83% showed 99–100% sequence identity with aph(3′)-IIa originally described as part of transposon Tn5. Fifty one were unique sequence variants eligible for recombination analysis. Only a single recombination event was identified with high confidence and indicated the involvement of aph(3′)-IIa in the formation of a mosaic gene located on a plasmid of environmental origin in the multi-resistant isolate Pseudomonas aeruginosa PA96. The available data suggest that aph(3′)-IIa is not an archetypical mosaic gene as the divergence between the described sequence variants and the number of detectable recombination events is low. This is in contrast to the numerous mosaic alleles reported for certain penicillin or tetracycline resistance determinants. PMID:26042098

  18. Involvement of aph(3‘-IIa in the formation of mosaic aminoglycoside resistance genes in natural environments

    Directory of Open Access Journals (Sweden)

    Markus eWoegerbauer

    2015-05-01

    Full Text Available Intragenic recombination leading to mosaic gene formation is known to alter resistance profiles for particular genes and bacterial species. Few studies have examined to what extent aminoglycoside resistance genes undergo intragenic recombination.We screened the GenBank database for mosaic gene formation in homologs of the aph(3’-IIa (nptII gene. APH(3’-IIa inactivates important aminoglycoside antibiotics. The gene is widely used as a selectable marker in biotechnology and enters the environment via laboratory discharges and the release of transgenic organisms. Such releases may provide opportunities for recombination in competent environmental bacteria.The retrieved GenBank sequences were grouped in 3 datasets comprising river water samples, duck pathogens and full-length variants from various bacterial genomes and plasmids. Analysis for recombination in these datasets was performed with the Recombination Detection Program, RDP4, and the Genetic Algorithm for Recombination Detection, GARD.From a total of 89 homologous sequences, 83% showed 99% - 100% sequence identity with aph(3’-IIa originally described as part of transposon Tn5. Fifty one were unique sequence variants eligible for recombination analysis. Only a single recombination event was identified with high confidence and indicated the involvement of aph(3’-IIa in the formation of a mosaic gene located on a plasmid of environmental origin in the multi-resistant isolate Pseudomonas aeruginosa PA96. The available data suggest that aph(3’-IIa is not an archetypical mosaic gene as the divergence between the described sequence variants and the number of detectable recombination events is low. This is in contrast to the numerous mosaic alleles reported for certain penicillin or tetracycline resistance determinants.

  19. Adeno-associated virus-mediated Bcl-xL prevents aminoglycoside-induced hearing loss in mice

    Institute of Scientific and Technical Information of China (English)

    LIU Yu-he; KE Xiao-mei; QIN Yong; GU Zhi-ping; XIAO Shui-fang

    2007-01-01

    Background Recent studies showed that aminoglycosides destroyed the cochlear cells and induced ototoxicity by producing reactive oxygen species, including free radicals in the mitochondria, damaging the membrane of mitochondria and resulting in apoptotic cell death. Bcl-xL is a well characterized anti-apoptotic member of the Bcl-2 family. The aim of this study was to determine the potential cochlear protective effect of Bcl-xL as a therapeutic agent in the murine model of aminoglycoside ototoxicity.Methods Serotype 2 of adeno-associated virus (AAV2) as a vector encoding the mouse Bcl-xL gene was injected into mice cochleae prior to injection of kanamycin. Bcl-xL expression in vitro and in vivo was examined with Western blotting and immunohistochemistry separately. Cochlear dissection and auditory steady state responses were checked to evaluate the cochlear structure and function.Results The animals in the AAV2-Bcl-xL/kanamycin group displayed better auditory steady state responses hearing thresholds and cochlear structure than those in the artificial perilymph/kanamycin or AAV2-enhanced humanized green fluorescent protein/kanamycin control group at all tested frequencies. The auditory steady state responses hearing thresholds and cochlear structure in the inoculated side were better than that in the contralateral side.Conclusions AAV2-Bcl-xL afforded significant preservation of the cochlear hair cells against ototoxic insults and protected the cochlear function. AAV2-mediated Bcl-xL might be an approach with respect to potential therapeutic application in the cochlear degeneration.

  20. The MisR Response Regulator Is Necessary for Intrinsic Cationic Antimicrobial Peptide and Aminoglycoside Resistance in Neisseria gonorrhoeae.

    Science.gov (United States)

    Kandler, Justin L; Holley, Concerta L; Reimche, Jennifer L; Dhulipala, Vijaya; Balthazar, Jacqueline T; Muszyński, Artur; Carlson, Russell W; Shafer, William M

    2016-08-01

    During infection, the sexually transmitted pathogen Neisseria gonorrhoeae (the gonococcus) encounters numerous host-derived antimicrobials, including cationic antimicrobial peptides (CAMPs) produced by epithelial and phagocytic cells. CAMPs have both direct and indirect killing mechanisms and help link the innate and adaptive immune responses during infection. Gonococcal CAMP resistance is likely important for avoidance of host nonoxidative killing systems expressed by polymorphonuclear granulocytes (e.g., neutrophils) and intracellular survival. Previously studied gonococcal CAMP resistance mechanisms include modification of lipid A with phosphoethanolamine by LptA and export of CAMPs by the MtrCDE efflux pump. In the related pathogen Neisseria meningitidis, a two-component regulatory system (2CRS) termed MisR-MisS has been shown to contribute to the capacity of the meningococcus to resist CAMP killing. We report that the gonococcal MisR response regulator but not the MisS sensor kinase is involved in constitutive and inducible CAMP resistance and is also required for intrinsic low-level resistance to aminoglycosides. The 4- to 8-fold increased susceptibility of misR-deficient gonococci to CAMPs and aminoglycosides was independent of phosphoethanolamine decoration of lipid A and the levels of the MtrCDE efflux pump and seemed to correlate with a general increase in membrane permeability. Transcriptional profiling and biochemical studies confirmed that expression of lptA and mtrCDE was not impacted by the loss of MisR. However, several genes encoding proteins involved in membrane integrity and redox control gave evidence of being MisR regulated. We propose that MisR modulates the levels of gonococcal susceptibility to antimicrobials by influencing the expression of genes involved in determining membrane integrity. PMID:27216061

  1. Persistence of antibiotic-resistant and -sensitive Proteus mirabilis strains in the digestive tract of the housefly (Musca domestica) and green bottle flies (Calliphoridae).

    Science.gov (United States)

    Wei, Ting; Miyanaga, Kazuhiko; Tanji, Yasunori

    2014-10-01

    Synanthropic flies have been implicated in the rapid dissemination of antibiotic-resistant bacteria and resistance determinants in the biosphere. These flies stably harbor a considerable number of bacteria that exhibit resistance to various antibiotics, but the mechanisms underlying this phenomenon remain unclear. In this study, we investigated the persistence of antibiotic-resistant bacteria in the digestive tract of houseflies and green bottle flies, using Proteus mirabilis as a model microorganism. One resistant strain carried the blaTEM and aphA1 genes, and another carried a plasmid containing qnrD gene. Quantitative PCR and 454 pyrosequencing were used to monitor the relative abundance of the Proteus strains, as well as potential changes in the overall structure of the whole bacterial community incurred by the artificial induction of Proteus cultures. Both antibiotic-resistant and -sensitive P. mirabilis strains persisted in the fly digestive tract for at least 3 days, and there was no significant difference in the relative abundance of resistant and sensitive strains despite the lower growth rate of resistant strains when cultured in vitro. Therefore, conditions in the fly digestive tract may allow resistant strains to survive the competition with sensitive strains in the absence of antibiotic selective pressure. The composition of the fly-associated bacterial community changed over time, but the contribution of the artificially introduced P. mirabilis strains to these changes was not clear. In order to explain these changes, it will be necessary to obtain more information about bacterial interspecies antagonism in the fly digestive tract.

  2. 铜绿假单胞菌耐氨基糖苷类相关耐药基因的检测%Tests on the correlated resistant genes of Pseudomonas aerugionsa to Aminoglycosides

    Institute of Scientific and Technical Information of China (English)

    屈艳; 张崇; 张春晓

    2012-01-01

    Objective To study the existing situation of aminoglycoside modifying enzyme( AMEs) gene and methyla-tion gene of aminoglycoside antibiotics resistant Pseudomonas aerugumsa. Methods One hundred and seventy strains of amin-oglycoside antibiotics resistant Pseudomonas aerugionsa were separated,meanwhile,5 main kinds of AMEs gene and 2 methyla-tion genes were detected by polymerase chain reaction(PCR). Results The AMEs gene carrying tate of Pseudomonas acru-gionsa was high(94.71%) ,and the detection rates of its modification genes were aac(3)-Ⅱ 77.6% ,aac(6')-Ⅰ41-2% ,ant(3")-Ⅰ40.0% ,aph(3')-Ⅵ21.8% ,aac(3)-Ⅰ0.0% ,resectively The isolates rate that simultaneously carrying two or more genetypes was 63.53% ,and the detection rates of melhylation genes were rmtD 1.8%,rmtA 0.0% Conclnsion The Pseudomonas acru-gionsa that carrying the AMEs gene has a high level,and the genelype is multiple;bul the carrying rate of methylation is very low.%目的 了解对氨基糖苷类抗生素耐药的铜绿假单胞菌氨基糖苷类修饰酶(AMEs)基因和甲酰化酶基因的存在情况.方法 用聚合酶链反应(PCR)测定临床分离的170株对氨基糖苷类抗生素耐药的铜绿假单胞菌5种主要的AMEs基因和2种甲酰化酶基因.结果 铜绿假单胞菌的AMEs基因携带率较高(94.71%),其修饰酶基因检出率分别为aac(3)-Ⅱ77.6%、aac(6′)-Ⅰ 41.2%、ant(3″)-Ⅰ40.0%、aph(3′)-Ⅵ21.8%、aac(3)-Ⅰ 0.0%,同时携带2种或2种以上AMEs基因的菌株占63.53%,甲酰化酶基因检出率分别为rmtD 1.8%、rmtA 0.0%.结论 铜绿假单胞菌中AMEs基因携带率较高,同一菌株携带多个不同的AMEs基因现象较多;甲酰化酶基因携带率极低.

  3. Enzyme detection by microfluidics

    DEFF Research Database (Denmark)

    2013-01-01

    Microfluidic-implemented methods of detecting an enzyme, in particular a DNA-modifying enzyme, are provided, as well as methods for detecting a cell, or a microorganism expressing said enzyme. The enzyme is detected by providing a nucleic acid substrate, which is specifically targeted...... by that enzyme...

  4. Evaluation of Antibacterial Activity of Aminoglycosides and Modulating the Essential Oil of Cymbopogon citratus (DC. Stapf

    Directory of Open Access Journals (Sweden)

    Saulo R. TINTINO

    2014-05-01

    Full Text Available  Several works demonstrated the importance of the study of natural products as an alternative source for new antimicrobial drugs or for modulators for these ones. In this point, the aim of this was to investigate the antibacterial activity and the possible interactions between the essential oil of Cymbopogon citratus alone and in association with aminoglycosides against standard and clinically isolated strains of multidrug-resistant bacteria such as S. aureus, E. coli and P. aeruginosa by microdilution method. The results indicated a synergism between the antibiotics and the essential oil with a subinhibitory concentration (MIC/8, reducing the minimal inhibitory concentration (MIC sixteen times against the multidrug-resistant strains of S. aureus 358, E. coli 27 and P. aeruginosa 143, but none modulatory activity was observed against P. aeruginosa 78 and P. aeruginosa 91 strains. By our results, can be concluded that the essential oil of Cymbopogon citratus can be an interesting source of natural products with antibacterial and/or modulatory antibiotic activitieAVALIAÇÃO DA ATIVIDADE ANTIBACTERIANA E MODULADORA DE AMINOGLICOSÍDEOS DO ÓLEO ESSENCIAL DE Cymbopogon citratus (DC. STAPFVários trabalhos vêm demonstrando a importância do estudo de produtos naturais como fonte alternativa para novos antimicrobianos ou que venham potencializar os já existentes. Neste contexto este trabalho teve como objetivo investigar a atividade antibacteriana e as possíveis interações entre o óleo essencial de Cymbopogon citratus combinados a aminoglicosídeos frente a linhagens padrões e multirresistentes de S. aureus, E. coli e de P. aeruginosa provenientes de isolados clínicos. Um ensaio de microdiluição foi realizado para verificar a atividade antibacteriana e as possíveis interacções entre o produto natural e os antibióticos, utilizando uma concentração sub-inibitória. Através dos resultados foi constatado a interferência sinérgica dos

  5. Antibiotic, cytotoxic and enzyme inhibitory activity of crude extracts from Brazilian marine invertebrates Atividade antibiótica, citotóxica e de inibição enzimática de extratos brutos de invertebrados marinhos do Brasil

    OpenAIRE

    Mirna H.R. Seleghim; Simone P. Lira; Miriam H. Kossuga; Tatiana Batista; Roberto G. S. Berlinck; Eduardo Hajdu; Guilherme Muricy; Rosana M. da Rocha; Gislene G. F. do Nascimento; Marcio Silva; Eli F. Pimenta; Thiemann, Otávio H.; Glaucius Oliva; Bruno C. Cavalcanti; Claudia Pessoa

    2007-01-01

    Herein we present the results of a screening with 349 crude extracts of Brazilian marine sponges, ascidians, bryozoans and octocorals, against 16 strains of susceptible and antibiotic-resistant bacteria, one yeast (Candida albicans), Mycobacterium tuberculosis H37Rv, three cancer cell lines MCF-7 (breast), B16 (murine melanoma ) and HCT8 (colon), and Leishmania tarentolae adenine phosphoribosyl transferase (L-APRT) enzyme. Less than 15% of marine sponge crude extracts displayed antibacterial ...

  6. Phenotypic and genome-wide analysis of an antibiotic-resistant small colony variant (SCV of Pseudomonas aeruginosa.

    Directory of Open Access Journals (Sweden)

    Qing Wei

    Full Text Available BACKGROUND: Small colony variants (SCVs are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch. METHODOLOGY/PRINCIPAL FINDINGS: One SCV (termed PAO-SCV was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10(-5 on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS. Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM, the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAO-SCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexAB-oprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels. CONCLUSIONS: By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the

  7. Toward an Alternative Therapeutic Approach for Skin Infections: Antagonistic Activity of Lactobacilli Against Antibiotic-Resistant Staphylococcus aureus and Pseudomonas aeruginosa.

    Science.gov (United States)

    Hafez, Mohamed M; Maghrabi, Ibrahim A; Zaki, Noha M

    2013-09-01

    The wide spread of antimicrobial resistance has urged the need of alternative therapeutic approach. In this context, probiotic lactobacilli have been reported for the prevention and treatment of many gastrointestinal and urogenital infections. However, very little is known about their antagonistic activity against skin pathogens. Accordingly, the present study aimed to investigate the potential of lactobacilli to interfere with pathogenesis features of two antibiotic-resistant skin pathogens, namely methicillin-resistant Staphylococcus aureus and multiple-resistant Pseudomonas aeruginosa. A total of 49 lactobacilli were recovered, identified and tested for their antagonistic activities against the aforementioned pathogens. Of these, eight isolates were capable of blocking the adherence of pathogens to mammalian cells independent of the skin pathogen tested or model adopted. Moreover, three Lactobacillus isolates (LRA4, LC2 and LR5) effectively prevented the pathogen internalization into epithelial cells in addition to potentiating phagocyte-mediated pathogen killing. Interestingly, the lactobacilli LC2, LF9 and LRA4 markedly inhibited the growth of P. aeruginosa and S. aureus isolates in coculture experiments. Besides, the lactobacilli LRA4, LC2, LR5 and LF9 have counteracted pathogen cytotoxicity. Taken together, the present study revealed some inhibitory activities of lactobacilli against two antibiotic-resistant skin pathogens. Moreover, it revealed two lactobacilli, namely LC2 and LRA4, with antagonistic capacity against different virulence determinants of skin pathogens. These lactobacilli are considered promising probiotic candidates that may represent an alternative therapeutic approach for skin infections.

  8. Chemoprophylactic efficacy against experimental endocarditis caused by beta-lactamase-producing, aminoglycoside-resistant enterococci is associated with prolonged serum inhibitory activity.

    OpenAIRE

    Bayer, A S; Tu, J

    1990-01-01

    We studied the prevention of experimental aortic endocarditis caused by a beta-lactamase-producing, aminoglycoside-resistant strain of Enterococcus faecalis (HH22) in 146 catheterized rabbits. Both vancomycin and ampicillin-sulbactam readily killed this resistant enterococcus strain in vitro. At a challenge inoculum of approximately 10(9) CFU, vancomycin (40 mg/kg intravenously [i.v.]), ampicillin (40 mg/kg i.v.), or a combination of ampicillin plus a beta-lactamase inhibitor, sulbactam (20 m...

  9. 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

    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......RNA nucleotide at C1404. EfmM uses the methyl group donor S-adenosyl-L-methionine to catalyze formation of m(5)C1404 on the 30S ribosomal subunit, whereas naked 16S rRNA and the 70S ribosome are not substrates. Addition of the 5-methyl to C1404 sterically hinders aminoglycoside binding. Crystallographic......Aminoglycosides are ribosome-targeting antibiotics and a major drug group of choice in the treatment of serious enterococcal infections. Here we show that aminoglycoside resistance in Enterococcus faecium strain CIP 54-32 is conferred by the chromosomal gene efmM, encoding the E. faecium...

  10. Vancomycin and High Level Aminoglycoside Resistance in Enterococcus spp. in a Tertiary Health Care Centre: A Therapeutic Concern

    Directory of Open Access Journals (Sweden)

    Seema Mittal

    2016-01-01

    Full Text Available Aims. This study was aimed at knowing the prevalence of vancomycin and high level aminoglycoside resistance in enterococcal strains among clinical samples. Study Design. It was an investigational study. Place and Duration of Study. It was conducted on 100 Enterococcus isolates, in the Department of Microbiology, Pt. BDS PGIMS, Rohtak, over a period of six months from July to December 2014. Methodology. Clinical specimens including urine, pus, blood, semen, vaginal swab, and throat swab were processed and Enterococcus isolates were identified by standard protocols. Antibiotic sensitivity testing of enterococci was performed using Kirby-Bauer disc diffusion method. Results. High level gentamicin resistance (HLGR was more common in urine samples (41.5% followed by blood (36% samples. High level streptomycin resistance (HLSR was more common in pus samples (52.6% followed by blood samples (36%. Resistance to vancomycin was maximum in blood isolates. Conclusion. Enterococci resistant to multiple antimicrobial agents have been recognized. Thus, it is crucial for laboratories to provide accurate antimicrobial resistance patterns for enterococci so that effective therapy and infection control measures can be initiated.

  11. 氨基糖苷类耐药的肠杆菌科细菌16SrRNA甲基化酶基因研究%Research on 16S rRNA methylase in aminoglycoside-resistant Enterobacteriaceae

    Institute of Scientific and Technical Information of China (English)

    吴琼; 韩立中; 孙景勇; 倪语星; 陈敏

    2014-01-01

    目的:探讨临床分离的对氨基糖苷类耐药的肠杆菌科细菌产16S rRNA甲基化酶状况,分析其分子流行趋势及其耐药性形成和传播的机制。方法采用纸片扩散法筛选庆大霉素和/或阿米卡星耐药的肠杆菌科细菌;采用聚合酶链反应(PCR)扩增16S rRNA甲基化酶基因、氨基糖苷修饰酶基因、β-内酰胺酶基因;采用质粒接合试验验证16S rRNA甲基化酶的转移性;应用脉冲场凝胶电泳(PFGE)对16S rRNA甲基化酶基因阳性菌株进行分型。结果201株对庆大霉素和/或阿米卡星耐药的肠杆菌科细菌中共检出38株16S rRNA甲基化酶阳性株(armA基因16株,rmtB基因22株)。其中30株可通过接合试验将耐药质粒转移至受体菌。blaCTX-M-14、blaTEM-1和 blaSHV-12可连同armA或rmtB分别转移到11、20和7个接合子中。肺炎克雷伯菌、大肠埃希菌和阴沟肠杆菌分别被PFGE分为4、21和1个型别。结论本研究分离的肠杆菌科细菌16S rRNA甲基化酶以armA和rmtB为主要流行型别,且后者分离率较高。该甲基化酶可导致氨基糖苷类高水平耐药,而且酶编码基因位于质粒上,具有转移性,β-内酰胺酶基因和氟喹诺酮耐药决定因子可随之一同转移。%Objective To investigate the molecular epidemiological characterization and the drug resistance and prevalence mechanism of 16S rRNA methylase in aminoglycoside-resistant Enterobacteriaceae isolated clinically. Methods Gentamicin-and or amikacin-resistant Enterobacteriaceae were screened by disc diffusion method.16S rRNA methylase genes,aminoglycoside modification enzyme genes and beta-lactamase genes were amplified by polymerase chain reaction(PCR).The conjugal transfer of aminoglycoside-resistant determination was performed.Pulsed-field gel electrophoresis (PFGE)was carried out to analyze genotyping.Results A total of 16 armA gene and 22 rmtB gene 16S rRNA methylase positive isolates were

  12. Enzyme inhibition by iminosugars

    DEFF Research Database (Denmark)

    López, Óscar; Qing, Feng-Ling; Pedersen, Christian Marcus;

    2013-01-01

    Imino- and azasugar glycosidase inhibitors display pH dependant inhibition reflecting that both the inhibitor and the enzyme active site have groups that change protonation state with pH. With the enzyme having two acidic groups and the inhibitor one basic group, enzyme-inhibitor complexes...

  13. Special characteristics of fluorescence and resonance Rayleigh scattering for cadmium telluride nanocrystal aqueous solution and its interactions with aminoglycoside antibiotics

    Institute of Scientific and Technical Information of China (English)

    LI TaiShan; LIU ShaoPu; LIU ZhongFang; HU XiaoLi; ZHANG LiPing

    2009-01-01

    CdTe nanocrystals (CdTe NCs) were achieved by reaction of CdCl2 with KHTe solution and were capped with sodium mercaptoacetate. The product was detected by transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), energy dispersive spectroscopy (EDS), fluorescence spectra, ultraviolet-visible spectra and X-ray diffraction (XRD). The CdTe NCs are of cubic structure and the average size is about 5 nm. The fluorescence quantum yield of CdTe NCs aqueous solution increased from 37% to 97% after 20 d under room light. The maximum λem of fluorescence changed from 543 nm to 510 nm and the blue shift was 33 nm. CdTe NCs aqueous solution can be steady for at least 10 months at 4℃ in a refrigerator. The resonance Rayleigh scattering (RRS) of CdTe NCs in the aqueous solution was investigated. The maximum scattering peak was located at about 554 nm. The interactions of CdTe NCs with amikacin sulfate (AS) and micronomicin sulfate (MS) were in-vestigated respectively. The effects of AS and MS on fluorescence and RRS of CdTe NCs were analyzed. It was found that AS and MS quenched the photoluminescence of CdTe NCs and enhanced RRS of CdTe NCs. Under optimum conditions, there are linear relationships between quenching intensity (F0-F), intensity of RRS (1-10) and concentration of AS and MS. The detection limits (3σ) of AS and MS are re-spectively 3.4 ng.mL-1 and 2.6 ng.mL-1 by the fluorescence quenching method, and 15.2 ng.mL-1 and 14.0 ng.mL-1 by the RRS method. The methods have high sensitivity, thus CdTe NCs may be used as fluorescence probes and RRS probes for the detection of aminoglycoside antibiotics.

  14. RpoS plays a central role in the SOS induction by sub-lethal aminoglycoside concentrations in Vibrio cholerae.

    Directory of Open Access Journals (Sweden)

    Zeynep Baharoglu

    Full Text Available 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.

  15. Comparative Proteomic Analysis of Aminoglycosides Resistant and Susceptible Mycobacterium tuberculosis Clinical Isolates for Exploring Potential Drug Targets.

    Directory of Open Access Journals (Sweden)

    Divakar Sharma

    Full Text Available Aminoglycosides, amikacin (AK and kanamycin (KM are second line anti-tuberculosis drugs used to treat tuberculosis (TB and resistance to them affects the treatment. Membrane and membrane associated proteins have an anticipated role in biological processes and pathogenesis and are potential targets for the development of new diagnostics/vaccine/therapeutics. In this study we compared membrane and membrane associated proteins of AK and KM resistant and susceptible Mycobacterium tuberculosis isolates by 2DE coupled with MALDI-TOF/TOF-MS and bioinformatic tools. Twelve proteins were found to have increased intensities (PDQuest Advanced Software in resistant isolates and were identified as ATP synthase subunit alpha (Rv1308, Trigger factor (Rv2462c, Dihydrolipoyl dehydrogenase (Rv0462, Elongation factor Tu (Rv0685, Transcriptional regulator MoxR1(Rv1479, Universal stress protein (Rv2005c, 35kDa hypothetical protein (Rv2744c, Proteasome subunit alpha (Rv2109c, Putative short-chain type dehydrogenase/reductase (Rv0148, Bacterioferritin (Rv1876, Ferritin (Rv3841 and Alpha-crystallin/HspX (Rv2031c. Among these Rv2005c, Rv2744c and Rv0148 are proteins with unknown functions. Docking showed that both drugs bind to the conserved domain (Usp, PspA and SDR domain of these hypothetical proteins and GPS-PUP predicted potential pupylation sites within them. Increased intensities of these proteins and proteasome subunit alpha might not only be neutralized/modulated the drug molecules but also involved in protein turnover to overcome the AK and KM resistance. Besides that Rv1876, Rv3841 and Rv0685 were found to be associated with iron regulation signifying the role of iron in resistance. Further research is needed to explore how these potential protein targets contribute to resistance of AK and KM.

  16. Comparative Proteomic Analysis of Aminoglycosides Resistant and Susceptible Mycobacterium tuberculosis Clinical Isolates for Exploring Potential Drug Targets

    Science.gov (United States)

    Sharma, Divakar; Kumar, Bhavnesh; Lata, Manju; Joshi, Beenu; Venkatesan, Krishnamurthy; Shukla, Sangeeta; Bisht, Deepa

    2015-01-01

    Aminoglycosides, amikacin (AK) and kanamycin (KM) are second line anti-tuberculosis drugs used to treat tuberculosis (TB) and resistance to them affects the treatment. Membrane and membrane associated proteins have an anticipated role in biological processes and pathogenesis and are potential targets for the development of new diagnostics/vaccine/therapeutics. In this study we compared membrane and membrane associated proteins of AK and KM resistant and susceptible Mycobacterium tuberculosis isolates by 2DE coupled with MALDI-TOF/TOF-MS and bioinformatic tools. Twelve proteins were found to have increased intensities (PDQuest Advanced Software) in resistant isolates and were identified as ATP synthase subunit alpha (Rv1308), Trigger factor (Rv2462c), Dihydrolipoyl dehydrogenase (Rv0462), Elongation factor Tu (Rv0685), Transcriptional regulator MoxR1(Rv1479), Universal stress protein (Rv2005c), 35kDa hypothetical protein (Rv2744c), Proteasome subunit alpha (Rv2109c), Putative short-chain type dehydrogenase/reductase (Rv0148), Bacterioferritin (Rv1876), Ferritin (Rv3841) and Alpha-crystallin/HspX (Rv2031c). Among these Rv2005c, Rv2744c and Rv0148 are proteins with unknown functions. Docking showed that both drugs bind to the conserved domain (Usp, PspA and SDR domain) of these hypothetical proteins and GPS-PUP predicted potential pupylation sites within them. Increased intensities of these proteins and proteasome subunit alpha might not only be neutralized/modulated the drug molecules but also involved in protein turnover to overcome the AK and KM resistance. Besides that Rv1876, Rv3841 and Rv0685 were found to be associated with iron regulation signifying the role of iron in resistance. Further research is needed to explore how these potential protein targets contribute to resistance of AK and KM. PMID:26436944

  17. Comparative Proteomic Analysis of Aminoglycosides Resistant and Susceptible Mycobacterium tuberculosis Clinical Isolates for Exploring Potential Drug Targets.

    Science.gov (United States)

    Sharma, Divakar; Kumar, Bhavnesh; Lata, Manju; Joshi, Beenu; Venkatesan, Krishnamurthy; Shukla, Sangeeta; Bisht, Deepa

    2015-01-01

    Aminoglycosides, amikacin (AK) and kanamycin (KM) are second line anti-tuberculosis drugs used to treat tuberculosis (TB) and resistance to them affects the treatment. Membrane and membrane associated proteins have an anticipated role in biological processes and pathogenesis and are potential targets for the development of new diagnostics/vaccine/therapeutics. In this study we compared membrane and membrane associated proteins of AK and KM resistant and susceptible Mycobacterium tuberculosis isolates by 2DE coupled with MALDI-TOF/TOF-MS and bioinformatic tools. Twelve proteins were found to have increased intensities (PDQuest Advanced Software) in resistant isolates and were identified as ATP synthase subunit alpha (Rv1308), Trigger factor (Rv2462c), Dihydrolipoyl dehydrogenase (Rv0462), Elongation factor Tu (Rv0685), Transcriptional regulator MoxR1(Rv1479), Universal stress protein (Rv2005c), 35kDa hypothetical protein (Rv2744c), Proteasome subunit alpha (Rv2109c), Putative short-chain type dehydrogenase/reductase (Rv0148), Bacterioferritin (Rv1876), Ferritin (Rv3841) and Alpha-crystallin/HspX (Rv2031c). Among these Rv2005c, Rv2744c and Rv0148 are proteins with unknown functions. Docking showed that both drugs bind to the conserved domain (Usp, PspA and SDR domain) of these hypothetical proteins and GPS-PUP predicted potential pupylation sites within them. Increased intensities of these proteins and proteasome subunit alpha might not only be neutralized/modulated the drug molecules but also involved in protein turnover to overcome the AK and KM resistance. Besides that Rv1876, Rv3841 and Rv0685 were found to be associated with iron regulation signifying the role of iron in resistance. Further research is needed to explore how these potential protein targets contribute to resistance of AK and KM.

  18. Kinetics of kill of bacterial conjunctivitis isolates with moxifloxacin, a fluoroquinolone, compared with the aminoglycosides tobramycin and gentamicin

    Directory of Open Access Journals (Sweden)

    Rudolph S Wagner

    2010-01-01

    Full Text Available Rudolph S Wagner1, David B Granet2, Steven J Lichtenstein3, Tiffany Jamison4, Joseph J Dajcs4, Robert D Gross5, Paul Cockrum41New Jersey Medical School, Newark, NJ, USA; 2Ratner Children’s Eye Center, University of California – San Diego, La Jolla, CA, USA; 3University of Illinois College of Medicine at Peoria, Peoria, Illinois, USA; 4Alcon Research, Ltd, Fort Worth, TX, USA; 5Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX, USAPurpose: To compare the kinetics and speed of kill of Streptococcus pneumoniae and Haemophilus influenzae on exposure to three topical ophthalmic antibiotic solutions.Materials and methods: Bacterial conjunctivitis isolates of S. pneumoniae and H. influenzae were exposed to 1:1000 dilutions of moxifloxacin 0.5%, tobramycin 0.3%, gentamicin 0.3%, and water (control. At 15, 30, 60, 120, and 180 minutes after exposure, aliquots were collected, cells were cultured, and viable cell counts were determined using standard microbiological methods.Results: Moxifloxacin achieved 99.9% kill (3-log reduction at approximately 2 hours for S. pneumoniae and at 15 minutes for H. influenzae. Tobramycin and gentamicin did not achieve 3-log reduction of S. pneumoniae during the 180-minute study period. An increase in bacterial growth was noted for these isolates. Gentamicin took more than 120 minutes to achieve the 3-log reduction of H. influenzae and tobramycin did not reach the 3-log reduction of this pathogen during the 180-minute study period.Conclusion: Moxifloxacin killed S. pneumoniae and H. influenzae in vitro faster than tobramycin and gentamicin, suggesting its potential clinical benefit as a first-line treatment for bacterial conjunctivitis to minimize patient symptoms and to limit the contagiousness of the disease.Keywords: kinetics of kill, bacterial conjunctivitis, in vitro, Streptococcus pneumoniae, Haemophilus influenzae, fluoroquinolones, aminoglycosides

  19. Combinations of β-lactam or aminoglycoside antibiotics with plectasin are synergistic against methicillin-sensitive and methicillin-resistant Staphylococcus aureus.

    Science.gov (United States)

    Hu, Yanmin; Liu, Alexander; Vaudrey, James; Vaiciunaite, Brigita; Moigboi, Christiana; McTavish, Sharla M; Kearns, Angela; Coates, Anthony

    2015-01-01

    Bacterial infections remain the leading killer worldwide which is worsened by the continuous emergence of antibiotic resistance. In particular, methicillin-sensitive (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) are prevalent and the latter can be difficult to treat. The traditional strategy of novel therapeutic drug development inevitably leads to emergence of resistant strains, rendering the new drugs ineffective. Therefore, rejuvenating the therapeutic potentials of existing antibiotics offers an attractive novel strategy. Plectasin, a defensin antimicrobial peptide, potentiates the activities of other antibiotics such as β-lactams, aminoglycosides and glycopeptides against MSSA and MRSA. We performed in vitro and in vivo investigations to test against genetically diverse clinical isolates of MSSA (n = 101) and MRSA (n = 115). Minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. The effects of combining plectasin with β-lactams, aminoglycosides and glycopeptides were examined using the chequerboard method and time kill curves. A murine neutropenic thigh model and a murine peritoneal infection model were used to test the effect of combination in vivo. Determined by factional inhibitory concentration index (FICI), plectasin in combination with aminoglycosides (gentamicin, neomycin or amikacin) displayed synergistic effects in 76-78% of MSSA and MRSA. A similar synergistic response was observed when plectasin was combined with β-lactams (penicillin, amoxicillin or flucloxacillin) in 87-89% of MSSA and MRSA. Interestingly, no such interaction was observed when plectasin was paired with vancomycin. Time kill analysis also demonstrated significant synergistic activities when plectasin was combined with amoxicillin, gentamicin or neomycin. In the murine models, plectasin at doses as low as 8 mg/kg augmented the activities of amoxicillin and gentamicin in successful treatment of MSSA and MRSA infections. We

  20. Combinations of β-lactam or aminoglycoside antibiotics with plectasin are synergistic against methicillin-sensitive and methicillin-resistant Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Yanmin Hu

    Full Text Available Bacterial infections remain the leading killer worldwide which is worsened by the continuous emergence of antibiotic resistance. In particular, methicillin-sensitive (MSSA and methicillin-resistant Staphylococcus aureus (MRSA are prevalent and the latter can be difficult to treat. The traditional strategy of novel therapeutic drug development inevitably leads to emergence of resistant strains, rendering the new drugs ineffective. Therefore, rejuvenating the therapeutic potentials of existing antibiotics offers an attractive novel strategy. Plectasin, a defensin antimicrobial peptide, potentiates the activities of other antibiotics such as β-lactams, aminoglycosides and glycopeptides against MSSA and MRSA. We performed in vitro and in vivo investigations to test against genetically diverse clinical isolates of MSSA (n = 101 and MRSA (n = 115. Minimum inhibitory concentrations (MIC were determined by the broth microdilution method. The effects of combining plectasin with β-lactams, aminoglycosides and glycopeptides were examined using the chequerboard method and time kill curves. A murine neutropenic thigh model and a murine peritoneal infection model were used to test the effect of combination in vivo. Determined by factional inhibitory concentration index (FICI, plectasin in combination with aminoglycosides (gentamicin, neomycin or amikacin displayed synergistic effects in 76-78% of MSSA and MRSA. A similar synergistic response was observed when plectasin was combined with β-lactams (penicillin, amoxicillin or flucloxacillin in 87-89% of MSSA and MRSA. Interestingly, no such interaction was observed when plectasin was paired with vancomycin. Time kill analysis also demonstrated significant synergistic activities when plectasin was combined with amoxicillin, gentamicin or neomycin. In the murine models, plectasin at doses as low as 8 mg/kg augmented the activities of amoxicillin and gentamicin in successful treatment of MSSA and MRSA

  1. Ex vivo treatment with a novel synthetic aminoglycoside NB54 in primary fibroblasts from Rett syndrome patients suppresses MECP2 nonsense mutations.

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    Manuela Vecsler

    Full Text Available BACKGROUND: Nonsense mutations in the X-linked methyl CpG-binding protein 2 (MECP2 comprise a significant proportion of causative MECP2 mutations in Rett syndrome (RTT. Naturally occurring aminoglycosides, such as gentamicin, have been shown to enable partial suppression of nonsense mutations related to several human genetic disorders, however, their clinical applicability has been compromised by parallel findings of severe toxic effects. Recently developed synthetic NB aminoglycosides have demonstrated significantly improved effects compared to gentamicin evident in substantially higher suppression and reduced acute toxicity in vitro. RESULTS: We performed comparative study of suppression effects of the novel NB54 and gentamicin on three MECP2 nonsense mutations (R294X, R270X and R168X common in RTT, using ex vivo treatment of primary fibroblasts from RTT patients harboring these mutations and testing for the C-terminal containing full-length MeCP2. We observed that NB54 induces dose-dependent suppression of MECP2 nonsense mutations more efficiently than gentamicin, which was evident at concentrations as low as 50 µg/ml. NB54 read-through activity was mutation specific, with maximal full-length MeCP2 recovery in R168X (38%, R270X (27% and R294X (18%. In addition, the recovered MeCP2 was translocated to the cell nucleus and moreover led to parallel increase in one of the most important MeCP2 downstream effectors, the brain derived neurotrophic factor (BDNF. CONCLUSION: Our findings suggest that NB54 may induce restoration of the potentially functional MeCP2 in primary RTT fibroblasts and encourage further studies of NB54 and other rationally designed aminoglycoside derivatives as potential therapeutic agents for nonsense MECP2 mutations in RTT.

  2. 大肠埃希菌氨基糖苷类药物获得性耐药机制探讨%Investigation of acquired resistant genes to aminoglycosides of Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    黄东标; 周茂亮; 李嫦珍; 陈江平; 胡晓燕

    2011-01-01

    目的:调查耐药大肠埃希菌分离株中氨基糖昔类修饰酶基因和16S rRNA甲基化酶基因的情况.方法:收集浙江省磐安县人民医院2009年6月-2010年6月临床分离的耐药大肠埃希菌共20株,采用聚合酶链反应(PCR)方法分析6种AMEs基因和2种16S rRNA甲基化酶基因.结果:20株耐药大肠埃希菌共检出3种氨基糖苷类修饰酶基因aac(6')-Ib4株、ant(3")-I1株和aadA5 10株,1种16S rRNA甲基化酶基因rmtB 2株.4株sac(6')-IbPCR阳性产物测序比对后确认1株为aac(6')-Ib型和3株为aac(6')-Ib-cr型.结论:本文在浙江省中部地区首次查出氨基糖苷类修饰酶aac(6')-Ib-cr f和16S rRNA甲基化酶rmtB型.产氨基糖苷类修饰酶基因、16S rRNA甲基化酶基因与氨基糖苷类药物耐药性相关.%Objective :To investigate the distribution of aminoglycoside modifying enzyme genes(AMEs) and 16S rRNA methylase genes in drug - resistant of Escherichia coli. Methods: From June 2009 to June 2010, 20 strains of drug - resistant E. coli were collected from Pan'an Hospital. Then, 6 kinds of AMEs (aac(3) - Ⅰ , aac(3) - Ⅱ , aac(6') - Ⅰ b, ant(3") - Ⅰ , aadA5, aph(3') - Ⅰ ) and 2 kinds of 16S rRNA methylase genes (armA, rmtB) were analyzed by PCR. Results: In 20 strains of E. coli, 10 strains, 4 strains, 2 strains, and 1 strain were detected to carry aadA5, aac(6') - Ⅰ b, rmtB, and ant(3") - Ⅰ respectively. After verificated by DNA sequencing, 4 PCR positive products of aac(6') - Ⅰ b were confirmed as 1 strain of aac (6') - Ⅰ b and 3 strains of aac(6') - Ⅰ b-cr. Conclusion: It's the first report that AMEs aac(6') - Ⅰ b-cr and 16S rRNA methylase gene rmtB were detected in central region of Zhejiang. AMEs and 16S rRNA methylase genes play a role in resistance to aminoglycosides.

  3. Enzymes for improved biomass conversion

    Energy Technology Data Exchange (ETDEWEB)

    Brunecky, Roman; Himmel, Michael E.

    2016-02-02

    Disclosed herein are enzymes and combinations of the enzymes useful for the hydrolysis of cellulose and the conversion of biomass. Methods of degrading cellulose and biomass using enzymes and cocktails of enzymes are also disclosed.

  4. A nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistance genes in Enterococcus species

    Directory of Open Access Journals (Sweden)

    Ravichandran Manickam

    2007-12-01

    Full Text Available Abstract Background Enterococci have emerged as a significant cause of nosocomial infections in many parts of the world over the last decade. The most common enterococci strains present in clinical isolates are E. faecalis and E. faecium which have acquired resistant to either gentamicin or vancomycin. The conventional culture test takes 2–5 days to yield complete information of the organism and its antibiotic sensitivity pattern. Hence our present study was focused on developing a nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistant enterococci (V-BiA-RE. This assay simultaneously detects 8 genes namely 16S rRNA of Enterococcus genus, ddl of E. faecalis and E. faecium, aacA-aphD that encodes high level gentamicin resistance (HLGR, multilevel vancomycin resistant genotypes such as vanA, vanB, vanC and vanD and one internal control gene. Results Unique and specific primer pairs were designed to amplify the 8 genes. The specificity of the primers was confirmed by DNA sequencing of the nanoplex PCR products and BLAST analysis. The sensitivity and specificity of V-BiA-RE nanoplex PCR assay was evaluated against the conventional culture method. The analytical sensitivity of the assay was found to be 1 ng at the DNA level while the analytical specificity was evaluated with 43 reference enterococci and non-enterococcal strains and was found to be 100%. The diagnostic accuracy was determined using 159 clinical specimens, which showed that 97% of the clinical isolates belonged to E. faecalis, of which 26% showed the HLGR genotype, but none were vancomycin resistant. The presence of an internal control in the V-BiA-RE nanoplex PCR assay helped us to rule out false negative cases. Conclusion The nanoplex PCR assay is robust and can give results within 4 hours about the 8 genes that are essential for the identification of the most common Enterococcus spp. and their antibiotic sensitivity pattern. The PCR assay

  5. Special characteristics of fluorescence and resonance Rayleigh scattering for cadmium telluride nanocrystal aqueous solution and its interactions with aminoglycoside antibiotics

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    CdTe nanocrystals(CdTe NCs) were achieved by reaction of CdCl2 with KHTe solution and were capped with sodium mercaptoacetate.The product was detected by transmission electron microscopy(TEM),high-resolution transmission electron microscopy(HRTEM),energy dispersive spectroscopy(EDS),fluorescence spectra,ultraviolet-visible spectra and X-ray diffraction(XRD).The CdTe NCs are of cubic structure and the average size is about 5 nm.The fluorescence quantum yield of CdTe NCs aqueous solution increased from 37% to 97% after 20 d under room light.The maximum λem of fluorescence changed from 543 nm to 510 nm and the blue shift was 33 nm.CdTe NCs aqueous solution can be steady for at least 10 months at 4℃ in a refrigerator.The resonance Rayleigh scattering(RRS) of CdTe NCs in the aqueous solution was investigated.The maximum scattering peak was located at about 554 nm.The interactions of CdTe NCs with amikacin sulfate(AS) and micronomicin sulfate(MS) were investigated respectively.The effects of AS and MS on fluorescence and RRS of CdTe NCs were analyzed.It was found that AS and MS quenched the photoluminescence of CdTe NCs and enhanced RRS of CdTe NCs.Under optimum conditions,there are linear relationships between quenching intensity(F0-F),intensity of RRS(I-I0) and concentration of AS and MS.The detection limits(3б) of AS and MS are respectively 3.4 ng·mL-1 and 2.6 ng·mL-1 by the fluorescence quenching method,and 15.2 ng·mL-1 and 14.0 ng·mL-1 by the RRS method.The methods have high sensitivity,thus CdTe NCs may be used as fluorescence probes and RRS probes for the detection of aminoglycoside antibiotics.

  6. Magnetically responsive enzyme powders

    Energy Technology Data Exchange (ETDEWEB)

    Pospiskova, Kristyna, E-mail: kristyna.pospiskova@upol.cz [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Safarik, Ivo, E-mail: ivosaf@yahoo.com [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Department of Nanobiotechnology, Institute of Nanobiology and Structural Biology of GCRC, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic)

    2015-04-15

    Powdered enzymes were transformed into their insoluble magnetic derivatives retaining their catalytic activity. Enzyme powders (e.g., trypsin and lipase) were suspended in various liquid media not allowing their solubilization (e.g., saturated ammonium sulfate and highly concentrated polyethylene glycol solutions, ethanol, methanol, 2-propanol) and subsequently cross-linked with glutaraldehyde. Magnetic modification was successfully performed at low temperature in a freezer (−20 °C) using magnetic iron oxides nano- and microparticles prepared by microwave-assisted synthesis from ferrous sulfate. Magnetized cross-linked enzyme powders were stable at least for two months in water suspension without leakage of fixed magnetic particles. Operational stability of magnetically responsive enzymes during eight repeated reaction cycles was generally without loss of enzyme activity. Separation of magnetically modified cross-linked powdered enzymes from reaction mixtures was significantly simplified due to their magnetic properties. - Highlights: • Cross-linked enzyme powders were prepared in various liquid media. • Insoluble enzymes were magnetized using iron oxides particles. • Magnetic iron oxides particles were prepared by microwave-assisted synthesis. • Magnetic modification was performed under low (freezing) temperature. • Cross-linked powdered trypsin and lipase can be used repeatedly for reaction.

  7. Magnetically responsive enzyme powders

    International Nuclear Information System (INIS)

    Powdered enzymes were transformed into their insoluble magnetic derivatives retaining their catalytic activity. Enzyme powders (e.g., trypsin and lipase) were suspended in various liquid media not allowing their solubilization (e.g., saturated ammonium sulfate and highly concentrated polyethylene glycol solutions, ethanol, methanol, 2-propanol) and subsequently cross-linked with glutaraldehyde. Magnetic modification was successfully performed at low temperature in a freezer (−20 °C) using magnetic iron oxides nano- and microparticles prepared by microwave-assisted synthesis from ferrous sulfate. Magnetized cross-linked enzyme powders were stable at least for two months in water suspension without leakage of fixed magnetic particles. Operational stability of magnetically responsive enzymes during eight repeated reaction cycles was generally without loss of enzyme activity. Separation of magnetically modified cross-linked powdered enzymes from reaction mixtures was significantly simplified due to their magnetic properties. - Highlights: • Cross-linked enzyme powders were prepared in various liquid media. • Insoluble enzymes were magnetized using iron oxides particles. • Magnetic iron oxides particles were prepared by microwave-assisted synthesis. • Magnetic modification was performed under low (freezing) temperature. • Cross-linked powdered trypsin and lipase can be used repeatedly for reaction

  8. HYDRATION AND ENZYME ACTIVITY

    OpenAIRE

    Poole, P.

    1984-01-01

    Hydration induced conformation and dynamic changes are followed using a variety of experimental techniques applied to hen egg white lysozyme. These changes are completed just before the onset of enzyme activity, which occurs before all polar groups are hydrated, and before monolayer coverage is attained. We suggest that these hydration induced changes are necessary for the return of enzyme activity.

  9. Artificial Enzymes, "Chemzymes"

    DEFF Research Database (Denmark)

    Bjerre, Jeannette; Rousseau, Cyril Andre Raphaël; Pedersen, Lavinia Georgeta M;

    2008-01-01

    "Chemzymes", based on cyclodextrins and other molecules. Only the chemzymes that have shown enzyme-like activity that has been quantified by different methods will be mentioned. This review will summarize the work done in the field of artificial glycosidases, oxidases, epoxidases, and esterases, as well...... as chemzymes that catalyze conjugate additions, cycloadditions, and self-replicating processes. The focus will be mainly on cyclodextrin-based chemzymes since they have shown to be good candidate structures to base an enzyme model skeleton on. In addition hereto, other molecules that encompass binding......Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models...

  10. Impact of Some Ecological Factors on Fecal Contamination of Drinking Water by Diarrheagenic Antibiotic-Resistant Escherichia coli in Zagazig City, Egypt

    Directory of Open Access Journals (Sweden)

    Ahmed Elsadek Fakhr

    2016-01-01

    Full Text Available Fecal contamination of drinking water is a major health problem which accounts for many cases of diarrhea mainly in infants and foreigners. This contamination is a complex interaction of many parameters. Antibiotic resistance among bacterial isolates complicates the problem. The study was done to identify fecal contamination of drinking water by Diarrheagenic Antibiotic-Resistant Escherichia coli in Zagazig city and to trace reasons for such contamination, three hundred potable water samples were investigated for E. coli existence. Locations of E. coli positive samples were investigated in relation to population density, water source, and type of water pipe. Sixteen E. coli strains were isolated. Antibiotic sensitivity was done and enterotoxigenic, enteropathogenic, and enterohaemorrhagic virulence genes were investigated by PCR. Probability of fecal contamination correlated with higher population density, with increased distance from Zagazig water plant, and with asbestos cement water pipes. Resistance to at least one antimicrobial drug was found in all isolates. Virulence genes were detected in a rate of 26.27%, 13.13%, 20%, 6.67%, and 33.33% for LT, ST, stx1, stx2, and eae genes, respectively. This relatively high frequency of fecal contamination points towards the high risk of developing diarrhea by antibiotic resistant DEC in low socioeconomic communities particularly with old fashion distribution systems.

  11. CRISPR/Cas9-Mediated Re-Sensitization of Antibiotic-Resistant Escherichia coli Harboring Extended-Spectrum β-Lactamases.

    Science.gov (United States)

    Kim, Jun-Seob; Cho, Da-Hyeong; Park, Myeongseo; Chung, Woo-Jae; Shin, Dongwoo; Ko, Kwan Soo; Kweon, Dae-Hyuk

    2016-02-01

    Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) system, a genome editing technology, was shown to be versatile in treating several antibiotic-resistant bacteria. In the present study, we applied the CRISPR/ Cas9 technology to kill extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli. ESBL bacteria are mostly multidrug resistant (MDR), and have plasmid-mediated antibiotic resistance genes that can be easily transferred to other members of the bacterial community by horizontal gene transfer. To restore sensitivity to antibiotics in these bacteria, we searched for a CRISPR/Cas9 target sequence that was conserved among >1,000 ESBL mutants. There was only one target sequence for each TEM- and SHV-type ESBL, with each of these sequences found in ~200 ESBL strains of each type. Furthermore, we showed that these target sequences can be exploited to re-sensitize MDR cells in which resistance is mediated by genes that are not the target of the CRISPR/Cas9 system, but by genes that are present on the same plasmid as target genes. We believe our Re-Sensitization to Antibiotics from Resistance (ReSAFR) technology, which enhances the practical value of the CRISPR/Cas9 system, will be an effective method of treatment against plasmid-carrying MDR bacteria.

  12. Membrane Assisted Enzyme Fractionation

    DEFF Research Database (Denmark)

    Yuan, Linfeng

    . In this thesis, separations using crossflow elecro-membrane filtration (EMF) of amino acids, bovine serum albumin (BSA) and industrial enzymes from Novozymes were performed. The main objective of this study was to investigate the technological feasibility of EMF in the application of industrial enzyme...... fractionation, such as removal of a side activity from the main enzyme activity. As a proof-of-concept, amino acids were used as model solution to test the feasibility of EMF in the application of amphoteric molecule separation. A single amino acid was used to illustrate the effect of an electric field...... on the separation performance were very small in the investigated range. The mass transport of each enzyme can be well explained by the Extended-Nernst-Planck equation. Better separation was observed at lower feed concentration, higher solution pH in the investigated range and with a polysulfone (PS) MF membrane...

  13. Overproduction of ligninolytic enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Elisashvili, Vladimir; Kachlishvili, Eva; Torok, Tamas

    2014-06-17

    Methods, compositions, and systems for overproducing ligninolytic enzymes from the basidiomycetous fungus are described herein. As described, the method can include incubating a fungal strain of Cerrena unicolor IBB 303 in a fermentation system having growth medium which includes lignocellulosic material and then cultivating the fungal strain in the fermentation system under conditions wherein the fungus expresses the ligninolytic enzymes. In some cases, the lignocellulosic material is mandarin peel, ethanol production residue, walnut pericarp, wheat bran, wheat straw, or banana peel.

  14. Enzyme with rhamnogalacturonase activity.

    OpenAIRE

    Kofod, L.V.; Andersen, L N; Dalboge, H; Kauppinen, M.S.; Christgau, S; Heldt-Hansen, H.P.; Christophersen, C.; Nielsen, P.M.; Voragen, A. G. J.; Schols, H.A.

    1998-01-01

    An enzyme exhibiting rhamnogalacturonase activity, capable of cleaving a rhamnogalacturonan backbone in such a manner that galacturonic acids are left as the non-reducing ends, and which exhibits activity on hairy regions from a soy bean material and/or on saponified hairy regions from a sugar beet material. The enzyme has the amino acid sequence of SEQ ID NO:2 and is encoded by the DNA sequence of SEQ ID NO:1

  15. RNA-modifying enzymes.

    Science.gov (United States)

    Ferré-D'Amaré, Adrian R

    2003-02-01

    A bewildering number of post-transcriptional modifications are introduced into cellular RNAs by enzymes that are often conserved among archaea, bacteria and eukaryotes. The modifications range from those with well-understood functions, such as tRNA aminoacylation, to widespread but more mysterious ones, such as pseudouridylation. Recent structure determinations have included two types of RNA nucleobase modifying enzyme: pseudouridine synthases and tRNA guanine transglycosylases.

  16. Mitochondrial DNA A1555G mutation screening using a testing kit method and its significance in preventing aminoglycoside-related hearing loss

    Institute of Scientific and Technical Information of China (English)

    LIU Xin; YANG Weiyan; HAN Dongyi; JIN Zhengce; GUAN Minxin; DAI Pu; HUANG Deliang; YUAN Huijun; LI Weiming; YU Fei; ZHANG Xin; KANG Dongyang; CAO Juyang

    2006-01-01

    To report a new screening method for mitochondrial DNA 1555A→G mutation and the results of genotype analysis in 19 maternal inherited deafness pedigrees. Method Five hundred and forty-six non-syndromic neuro-sensory hearing loss patients were tested for 1555A→G mutation using a new compact testing kit, which allows clear distinction between wild type and 1555 A→G mutated mtDNAs. Results Nineteen subjects among the 546 patients (3.48%) were found to carry mtDNA A1555G mutation. The results were confirmed by sequencing in an ABI 3100 Avant sequencer. Conclusions Maternal inherited deafness families are a frequently seen in outpatient group. The detection ofmtDNA 1555 A→G mutation with a low cost, ready to use detection kit is needed and suitable in China for large scale screening and preventive testing before usage of aminoglycoside antibiotics.

  17. Partial characterization of an endemic strain of a methicillin- and aminoglycoside-resistant Staphylococcus aureus (MARSA) homogeneously resistant to beta-lactam antibiotics.

    Science.gov (United States)

    Jacob, J; Meers, P D

    1992-06-01

    Selected strains of methicillin- and aminoglycoside-resistant Staphylococcus aureus (MARSA) were subjected to a preliminary examination. They were representative of a larger group collected in a routine clinical microbiology laboratory over a period of 2 years. MARSA was endemic in the associated hospital. The characteristics investigated were antimicrobial resistance, the production of beta-lactamase, free and bound coagulase, protein A, DNA-ase, urease, lipase and pigment. The MARSA strains were generally indistinguishable, other than in their antimicrobial resistances. The resistance to methicillin was completely homogeneous. Except with imipenem, growth extended to the edge of discs containing methicillin and the other beta-lactam antibiotics tested when the strains were cultured at 37 degrees C on media without added salt. Homogeneous resistance may confer an epidemiological advantage on strains of this phenotype. PMID:1353087

  18. The impact of methicillin- and aminoglycoside-resistant Staphylococcus aureus on the pattern of hospital-acquired infection in an acute hospital.

    Science.gov (United States)

    Meers, P D; Leong, K Y

    1990-10-01

    Infections due to methicillin- and aminoglycoside-resistant Staphylococcus aureus (MARSA) appeared in a new teaching hospital shortly after it opened. The effect this had on the pattern of hospital-acquired infections in the four years that followed is described. No control measures were applied and MARSA became endemic. New infections appeared at a rate of about four for each 1000 patients discharged. It established itself at different levels of incidence in various specialist units, patients under intensive care being most severely affected. MARSA was implicated in half of all hospital-acquired infections due to S. aureus but it was not more pathogenic than its more sensitive counterpart. It had little impact on the life of the hospital. PMID:1979573

  19. Detection of high-level aminoglycoside resistant pattern of Enterococci isolated from urine samples at a tertiary care hospital in Bengaluru

    Directory of Open Access Journals (Sweden)

    Smeeta Huidrom

    2016-01-01

    Full Text Available Aims: Enterococcus species are major nosocomial pathogens and they most commonly cause urinary tract infections (UTIs, exhibiting vancomycin and high-level aminoglycoside resistance (HLAR with increasing frequency, resulting in high mortality of patients with serious enterococcal infections. Detection of resistance is thus of paramount importance. The present study aims to detect and determine the HLAR pattern of Enterococci isolated from urine samples of patients diagnosed with UTI at our hospital. Materials and Methods: This study was carried out at a tertiary care hospital in Bengaluru for a period of 1 year from January 2013 to December 2013. A total of 105 enterococcal strains were isolated from urine samples and speciated as per the scheme of Facklam and Collins. Antibiotic susceptibility was determined for various drugs by Kirby–Bauer disc diffusion method. The results were interpreted as per the Clinical and Laboratory Standards Institute (CLSI guidelines. Results: Ninety-three of the 105 (88.6% isolates showed high-level resistance to gentamicin and/or streptomycin. Combined resistance to both the aminoglycosides, high level gentamicin and streptomycin (HLAR, was seen only in Enterococcus faecalis 20/105 (19.04%. Of the two isolates of Enterococcus faecium, 1 (50% was seen to be resistant to high level gentamicin. The HLAR E. faecalis and E. faecium isolates also showed concordant resistance to multiple antibiotics including vancomycin. Conclusion: This study highlights the need to screen for HLAR in patients suffering from enterococcal infections. Routine screening for HLAR is important to limit the spread of resistance and to have a surveillance program.

  20. Decellularized human amniotic membrane: more is needed for an efficient dressing for protection of burns against antibiotic-resistant bacteria isolated from burn patients.

    Science.gov (United States)

    Gholipourmalekabadi, M; Bandehpour, M; Mozafari, M; Hashemi, A; Ghanbarian, H; Sameni, M; Salimi, M; Gholami, M; Samadikuchaksaraei, A

    2015-11-01

    Human amniotic membranes (HAMs) have attracted the attention of burn surgeons for decades due to favorable properties such as their antibacterial activity and promising support of cell proliferation. On the other hand, as a major implication in the health of burn patients, the prevalence of bacteria resistant to multiple antibiotics is increasing due to overuse of antibiotics. The aim of this study was to investigate whether HAMs (both fresh and acellular) are an effective antibacterial agent against antibiotic-resistant bacteria isolated from burn patients. Therefore, a HAM was decellularized and tested for its antibacterial activity. Decellularization of the tissue was confirmed by hematoxylin and eosin (H&E) and 4,6-diamidino-2-phenylindole (DAPI) staining. In addition, the cyto-biocompatibility of the acellular HAM was proven by the cell viability test (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, MTT) and scanning electron microscopy (SEM). The resistant bacteria were isolated from burns, identified, and tested for their susceptibility to antibiotics using both the antibiogram and polymerase chain reaction (PCR) techniques. Among the isolated bacteria, three blaIMP gene-positive Pseudomonas aeruginosa strains were chosen for their high resistance to the tested antibiotics. The antibacterial activity of the HAM was also tested for Klebsiella pneumoniae (American Type Culture Collection (ATCC) 700603) as a resistant ATCC bacterium; Staphylococcus aureus (mecA positive); and three standard strains of ATCC bacteria including Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27833), and S. aureus (ATCC 25923). Antibacterial assay revealed that only the latter three bacteria were susceptible to the HAM. All the data obtained from this study suggest that an alternative strategy is required to complement HAM grafting in order to fully protect burns from nosocomial infections.

  1. Red cell enzymes.

    Science.gov (United States)

    Paniker, N V

    1975-03-01

    As compared to other cells of the body, the mammalian red cell has one of the simplest structural organizations. As a result, this cell has been extensively used in studies involving the structure, function, and integrity of cell membranes as well as cytoplasmic events. Additionally, the metabolic activities of the red blood cell are also relatively simple. During the past quarter century or so, an ocean of knowledge has been gathered on various aspects of red cell metabolism and function. The fields of enzymes, hemoglobin, membrane, and metabolic products comprise the major portion of this knowledge. These advances have made valuable contributions to biochemistry and medicine. Despite these favorable aspects of this simple, anucleated cell, it must be conceded that our knowledge about the red cell is far from complete. We are still in the dark concerning the mechanism involved in several aspects of its membrane, hemoglobin, enzymes, and a large number of other constituents. For example, a large number of enzymes with known catalytic activity but with unknown function have eluded investigators despite active pursuit. This review will be a consolidation of our present knowledge of human red cell enzymes, with particular reference to their usefulness in the diagnosis and therapy of disease. Owing to the multitude of publications by prominent investigators on each of the approximately 50 enzymes discussed in this review, it was impossible to cite a majority of them.

  2. Random-walk enzymes

    Science.gov (United States)

    Mak, Chi H.; Pham, Phuong; Afif, Samir A.; Goodman, Myron F.

    2015-09-01

    Enzymes that rely on random walk to search for substrate targets in a heterogeneously dispersed medium can leave behind complex spatial profiles of their catalyzed conversions. The catalytic signatures of these random-walk enzymes are the result of two coupled stochastic processes: scanning and catalysis. Here we develop analytical models to understand the conversion profiles produced by these enzymes, comparing an intrusive model, in which scanning and catalysis are tightly coupled, against a loosely coupled passive model. Diagrammatic theory and path-integral solutions of these models revealed clearly distinct predictions. Comparison to experimental data from catalyzed deaminations deposited on single-stranded DNA by the enzyme activation-induced deoxycytidine deaminase (AID) demonstrates that catalysis and diffusion are strongly intertwined, where the chemical conversions give rise to new stochastic trajectories that were absent if the substrate DNA was homogeneous. The C →U deamination profiles in both analytical predictions and experiments exhibit a strong contextual dependence, where the conversion rate of each target site is strongly contingent on the identities of other surrounding targets, with the intrusive model showing an excellent fit to the data. These methods can be applied to deduce sequence-dependent catalytic signatures of other DNA modification enzymes, with potential applications to cancer, gene regulation, and epigenetics.

  3. Random-walk enzymes.

    Science.gov (United States)

    Mak, Chi H; Pham, Phuong; Afif, Samir A; Goodman, Myron F

    2015-09-01

    Enzymes that rely on random walk to search for substrate targets in a heterogeneously dispersed medium can leave behind complex spatial profiles of their catalyzed conversions. The catalytic signatures of these random-walk enzymes are the result of two coupled stochastic processes: scanning and catalysis. Here we develop analytical models to understand the conversion profiles produced by these enzymes, comparing an intrusive model, in which scanning and catalysis are tightly coupled, against a loosely coupled passive model. Diagrammatic theory and path-integral solutions of these models revealed clearly distinct predictions. Comparison to experimental data from catalyzed deaminations deposited on single-stranded DNA by the enzyme activation-induced deoxycytidine deaminase (AID) demonstrates that catalysis and diffusion are strongly intertwined, where the chemical conversions give rise to new stochastic trajectories that were absent if the substrate DNA was homogeneous. The C→U deamination profiles in both analytical predictions and experiments exhibit a strong contextual dependence, where the conversion rate of each target site is strongly contingent on the identities of other surrounding targets, with the intrusive model showing an excellent fit to the data. These methods can be applied to deduce sequence-dependent catalytic signatures of other DNA modification enzymes, with potential applications to cancer, gene regulation, and epigenetics.

  4. Angiotensin-converting enzyme

    DEFF Research Database (Denmark)

    Sørensen, P G; Rømer, F K; Cortes, D

    1984-01-01

    In order to evaluate bleomycin-associated lung damage in humans, lung function parameters and serum levels of the endothelial-bound angiotensin-converting enzyme (ACE) were determined by serial measurements in 11 patients who were treated for testicular cancer. None developed clinical or radiolog......In order to evaluate bleomycin-associated lung damage in humans, lung function parameters and serum levels of the endothelial-bound angiotensin-converting enzyme (ACE) were determined by serial measurements in 11 patients who were treated for testicular cancer. None developed clinical...

  5. The surface science of enzymes

    DEFF Research Database (Denmark)

    Rod, Thomas Holm; Nørskov, Jens Kehlet

    2002-01-01

    ? To solve these problems we must understand in some detail how enzymes interact with reactants from its surroundings. These interactions take place at the surface of the enzyme and the question of enzyme function can be viewed as the surface science of enzymes. In this article we discuss how to describe......One of the largest challenges to science in the coming years is to find the relation between enzyme structure and function. Can we predict which reactions an enzyme catalyzes from knowledge of its structure-or from its amino acid sequence? Can we use that knowledge to modify enzyme function...

  6. ISFET based enzyme sensors

    NARCIS (Netherlands)

    Schoot, van der Bart H.; Bergveld, Piet

    1987-01-01

    This paper reviews the results that have been reported on ISFET based enzyme sensors. The most important improvement that results from the application of ISFETs instead of glass membrane electrodes is in the method of fabrication. Problems with regard to the pH dependence of the response and the dyn

  7. Computational enzyme design

    Science.gov (United States)

    Bolon, Daniel N.

    2002-08-01

    The long-term objective of computational enzyme design is the ability to generate efficient protein catalysts for any chemical reaction. This thesis develops and experimentally validates a general computational approach for the design of enzymes with novel function. In order to include catalytic mechanism in protein design, a high-energy state (HES) rotamer (side chain representation) was constructed. In this rotamer, substrate atoms are in a HES. In addition, at least one amino acid side chain is positioned to interact favorably with substrate atoms in their HES and facilitate the reaction. Including an amino acid side chain in the HES rotamer automatically positions substrate relative to a protein scaffold and allows protein design algorithms to search for sequences capable of interacting favorably with the substrate. Because chemical similarity exists between the transition state and the high-energy state, optimizing the protein sequence to interact favorably with the HES rotamer should lead to transition state stabilization. In addition, the HES rotamer model focuses the subsequent computational active site design on a relevant phase space where an amino acid is capable of interacting in a catalytically active geometry with substrate. Using a HES rotamer model of the histidine mediated nucleophilic hydrolysis of p-nitrophenyl acetate, the catalytically inert 108 residue E. coli thioredoxin as a scaffold, and the ORBIT protein design software to compute sequences, an active site scan identified two promising active site designs. Experimentally, both candidate ?protozymes? demonstrated catalytic activity significantly above background. In addition, the rate enhancement of one of these ?protozymes? was the same order of magnitude as the first catalytic antibodies. Because polar groups are frequently buried at enzyme-substrate interfaces, improved modeling of buried polar interactions may benefit enzyme design. By studying native protein structures, rules have been

  8. The Moderately Efficient Enzyme: Futile Encounters and Enzyme Floppiness.

    Science.gov (United States)

    Bar-Even, Arren; Milo, Ron; Noor, Elad; Tawfik, Dan S

    2015-08-18

    The pioneering model of Henri, Michaelis, and Menten was based on the fast equilibrium assumption: the substrate binds its enzyme reversibly, and substrate dissociation is much faster than product formation. Here, we examine this assumption from a somewhat different point of view, asking what fraction of enzyme-substrate complexes are futile, i.e., result in dissociation rather than product formation. In Knowles' notion of a "perfect" enzyme, all encounters of the enzyme with its substrate result in conversion to product. Thus, the perfect enzyme's catalytic efficiency, kcat/KM, is constrained by only the diffusion on-rate, and the fraction of futile encounters (defined as φ) approaches zero. The available data on >1000 different enzymes suggest that for ≥90% of enzymes φ > 0.99 and for the "average enzyme" φ ≥ 0.9999; namely, <1 of 10(4) encounters is productive. Thus, the "fast equilibrium" assumption holds for the vast majority of enzymes. We discuss possible molecular origins for the dominance of futile encounters, including the coexistence of multiple sub-states of an enzyme's active site (enzyme floppiness) and/or its substrate. Floppiness relates to the inherent flexibility of proteins, but also to conflicting demands, or trade-offs, between rate acceleration (the rate-determining chemical step) and catalytic turnover, or between turnover rate and accuracy. The study of futile encounters and active-site floppiness may contribute to a better understanding of enzyme catalysis, enzyme evolution, and improved enzyme design.

  9. Prevalence of 16S rRNA methylase, modifying enzyme, and extended-spectrum beta-lactamase genes among Acinetobacter baumannii isolates.

    Science.gov (United States)

    Liu, Zhenru; Ling, Baodong; Zhou, Liming

    2015-08-01

    Multidrug-resistant Acinetobacter baumannii has become a worldwide problem, and methylation of 16S rRNA has recently emerged as a new mechanism of resistance to aminoglycosides, which is mediated by a newly recognized group of 16S rRNA methylases. 16S rRNA methylase confers a high-level resistance to all 4,6-substituted deoxystreptamine aminoglycosides that are currently used in clinical practice. Some of the A. baumannii isolates have been found to coproduce extended-spectrum beta-lactamases (ESBLs), contributing to their multidrug resistance. The aim of this study was to detect the determinants of the 16S rRNA methylase genes armA, rmtA, rmtB, rmtC, rmtD, rmtE, and npmA, the modifying enzyme genes aac(6')-Ib, ant(3″)-Ia, aph(3')-I, and the extended-spectrum beta-lactamase genes bla(TEM), bla(SHV), and bla(CTX-M-3) among A. baumannii isolates in northeastern Sichuan, China. Minimum inhibitory concentrations (MICs) of 21 different antimicrobial agents against the A. baumannii isolates were determined. The clinical isolates showed a high level of resistance (MIC≧256 μg/ml) to aminoglycosides, which ranged from 50·1 to 83·8%. The resistances to meropenem and imipenem, two of the beta-lactam antibiotics and the most active antibiotics against A. baumannii, were 9·1 and 8·2%, respectively. Among 60 amikacin-resistant isolates, only the 16S rRNA methylase gene armA was found to be prevalent (66·7%), but the other 16S rRNA methylase genes rmtA, rmtB, rmtC, rmtD, rmtE, and npmA were not detected. The prevalences of the modifying enzyme genes aac (6')-Ib, ant (3″)-Ia, and aph (3')-I were 51·7, 81·7, and 58·3%, respectively, which are different from a previous study in which the occurrences of these genes were 3, 64, and 72%, respectively. Among the 40 isolates that were armA-positive, the prevalences of bla(TEM), bla(SHV), and bla(CTX-M-3) genes were detected for the first time in China, and their occurrences were 45, 65, and 52·5%, respectively. In all, A

  10. The effects of N-acylhomoserine lactones, β-lactam antibiotics and adenosine on biofilm formation in the multi-β-lactam antibiotic-resistant bacterium Acidovorax sp. strain MR-S7.

    Science.gov (United States)

    Kusada, Hiroyuki; Hanada, Satoshi; Kamagata, Yoichi; Kimura, Nobutada

    2014-07-01

    Bacteria in the natural ecosystem frequently live as adherent communities called biofilms. Some chemical compounds are known to affect biofilm formation. We investigated the effect of exogenous small molecules, N-acylhomoserine lactones (AHLs), β-lactam antibiotics, and adenosine, on biofilm formation in the β-lactam antibiotic-resistant bacterium Acidovorax sp. strain MR-S7. Biofilm formation was induced by the addition of various types of AHL isomers and β-lactam antibiotics, whereas the addition of adenosine strongly interfered with the biofilm formation. A gene (macP) encoding adenosine deaminase (that converts adenosine to inosine controlling intracellular adenosine concentration) was successfully cloned from MR-S7 genome and heterologously expressed in Escherichia coli. The purified MacP protein clearly catalyzed the deamination of adenosine to produce inosine. A transcriptional analysis revealed that biofilm-inducing molecules, an AHL and a β-lactam antibiotic, strongly induced not only biofilm formation but also adenosine deaminase gene expression, suggesting that an elaborate gene regulation network for biofilm formation is present in the β-lactam antibiotic-resistant bacterium studied here.

  11. Long-time follow-up study of localized gastric mucosa-associated lymphoid tissue (MALT) lymphoma and the clinical features of antibiotic-resistant cases of gastric MALT lymphoma

    International Nuclear Information System (INIS)

    To clarify the clinical features of gastric mucosa-associated lymphoid tissue (MALT) lymphoma (GML) with persistent lymphoma after eradication therapy of Helicobacter pylori (H. pylori), and the outcome of long-time follow-up study after treatment against GML, seventy-six patients with localized GML were studied. The median follow-up period was 44.4 months. Thirty-eight of 49 patients (77.6%) with H. pylori-positive GML had been cured of GML by antibiotic therapy alone. On the other hand, none of 13 patients with H. pylori-negative GML had been cured by antibiotic therapy (77.6% vs 0%, p<0.001). ''H. pylori-negative'' is one of the clinical features of antibiotic-resistant cases with GML. There was no significant difference in sex, age, stage, endoscopic finding, depth, and affected region between the two groups of cured and persistent GML with H. pylori infection. Twenty-two of 29 patients (75.6%) with antibiotic-resistant or H. pylori-negative cases of GML had been cured by 30 Gy radiation therapy. Low-dose radiation was thought to be a useful therapeutic procedure as a second line treatment'' of localized GML. (author)

  12. Enzyme linked immunoassay with stabilized polymer saccharide enzyme conjugates

    Science.gov (United States)

    Callstrom, M.R.; Bednarski, M.D.; Gruber, P.R.

    1997-11-25

    An improvement in enzyme linked immunoassays is disclosed wherein the enzyme is in the form of a water soluble polymer saccharide conjugate which is stable in hostile environments. The conjugate comprises the enzyme which is linked to the polymer at multiple points through saccharide linker groups. 19 figs.

  13. Treating Wastewater With Immobilized Enzymes

    Science.gov (United States)

    Jolly, Clifford D.

    1991-01-01

    Experiments show enzymes are immobilized on supporting materials to make biocatalyst beds for treatment of wastewater. With suitable combination of enzymes, concentrations of various inorganic and organic contaminants, including ammonia and urea, reduced significantly.

  14. [Aztreonam as monotherapy in urinary tract infections with a systemic repercussion in patients with a relative contraindication for the use of aminoglycosides].

    Science.gov (United States)

    Pedrajas, J M; Pieltain, R; Mesa, N; Ramón, P; García, C; Pontes, J C; Fernández-Cruz, A

    1993-04-01

    A comparative study has been performed to evaluate the clinical efficacy and safety of aztreonam in the treatment of urinary tract infections with systemic affectation, in a group of patients who showed a relative contraindication to be treated with aminoglycosides. The group studied was formed by 30 patients (21 females and 9 males). Mean age of said group was 78 years and percentage of patients over 65 years was 93%. Moreover, 53.3% of patients showed nephropathy, 30% diabetes mellitus and 16.6% hearing disorders. Responsible germ of the infection was identified through blood and urine culture in 24 patients (80%) being the most frequent isolated E. Coli (60%). The cure rate was of 76.6% and improvement rate was 13.3%. Three deaths happened on the studied group (10%). In a patient superinfection due to Enterococcus was detected. There were no significative adverse effects (hypersensibility reactions, hematological disorders, nephrotoxicity or hepatotoxicity). Aztreonam could be an efficacious alternative in the treatment of urinary infection with systemic affectation, caused by gram-negative germs, showing low toxicity. PMID:8497718

  15. High-level aminoglycoside resistance in Enterococcus faecalis and Enterococcus faecium causing invasive infection: Twelve-year surveillance in the Minami Ibaraki Area.

    Science.gov (United States)

    Osuka, Hanako; Nakajima, Jun; Oishi, Tsuyoshi; Funayama, Yasunori; Ebihara, Tsugio; Ishikawa, Hiroichi; Saito, Kazuto; Koganemaru, Hiroshi; Hitomi, Shigemi

    2016-01-01

    We examined prevalence of high-level aminoglycoside resistance (HLAR) in Enterococcus faecalis and Enterococcus faecium causing invasive infection in the Minami Ibaraki Area. Ten strains of both species each, recovered from the blood or the cerebrospinal fluid between 2003 and 2014, were randomly selected every year. High-level resistance to gentamicin (HLR-GM) and streptomycin (HLR-SM) was detected in 34% (41 of 120 strains) and 18% (21) of E. faecalis and 9% (11) and 39% (48) of E. faecium, respectively. In comparisons of the proportions among three four-year periods, HLR-SM among E. faecium was significantly lower in the 2011-2014 period. All strains with HLR-GM were positive for the aac(6')-Ie-aph(2″)-Ia gene. The ant(6')-Ia gene was detected in all with HLR-SM except for one E. faecalis strain. The present study showed that prevalence of HLR-GM among E. faecalis and E. faecium causing invasive infection in this area was nearly equivalent to that described in previous studies in Japan and that proportions of strains with HLAR did not vary during the study period except for that of HLR-SM among E. faecium.

  16. A Site-Specific Integrative Plasmid Found in Pseudomonas aeruginosa Clinical Isolate HS87 along with A Plasmid Carrying an Aminoglycoside-Resistant Gene.

    Directory of Open Access Journals (Sweden)

    Dexi Bi

    Full Text Available Plasmids play critical roles in bacterial fitness and evolution of Pseudomonas aeruginosa. Here two plasmids found in a drug-resistant P. aeruginosa clinical isolate HS87 were completely sequenced. The pHS87b plasmid (11.2 kb carries phage-related genes and function-unknown genes. Notably, pHS87b encodes an integrase and has an adjacent tRNAThr-associated attachment site. A corresponding integrated form of pHS87b at the tRNAThr locus was identified on the chromosome of P. aeruginosa, showing that pHS87b is able to site-specifically integrate into the 3'-end of the tRNAThr gene. The pHS87a plasmid (26.8 kb displays a plastic structure containing a putative replication module, stability factors and a variable region. The RepA of pHS87a shows significant similarity to the replication proteins of pPT23A-family plasmids. pHS87a carries a transposon Tn6049, a truncated insertion sequence ΔIS1071 and a Tn402-like class 1 integron which contains an aacA4 cassette that may confer aminoglycoside resistance. Thus, pHS87b is a site-specific integrative plasmid whereas pHS87a is a plastic antibiotic resistance plasmid. The two native plasmids may promote the fitness and evolution of P. aeruginosa.

  17. A Site-Specific Integrative Plasmid Found in Pseudomonas aeruginosa Clinical Isolate HS87 along with A Plasmid Carrying an Aminoglycoside-Resistant Gene.

    Science.gov (United States)

    Bi, Dexi; Xie, Yingzhou; Tai, Cui; Jiang, Xiaofei; Zhang, Jie; Harrison, Ewan M; Jia, Shiru; Deng, Zixin; Rajakumar, Kumar; Ou, Hong-Yu

    2016-01-01

    Plasmids play critical roles in bacterial fitness and evolution of Pseudomonas aeruginosa. Here two plasmids found in a drug-resistant P. aeruginosa clinical isolate HS87 were completely sequenced. The pHS87b plasmid (11.2 kb) carries phage-related genes and function-unknown genes. Notably, pHS87b encodes an integrase and has an adjacent tRNAThr-associated attachment site. A corresponding integrated form of pHS87b at the tRNAThr locus was identified on the chromosome of P. aeruginosa, showing that pHS87b is able to site-specifically integrate into the 3'-end of the tRNAThr gene. The pHS87a plasmid (26.8 kb) displays a plastic structure containing a putative replication module, stability factors and a variable region. The RepA of pHS87a shows significant similarity to the replication proteins of pPT23A-family plasmids. pHS87a carries a transposon Tn6049, a truncated insertion sequence ΔIS1071 and a Tn402-like class 1 integron which contains an aacA4 cassette that may confer aminoglycoside resistance. Thus, pHS87b is a site-specific integrative plasmid whereas pHS87a is a plastic antibiotic resistance plasmid. The two native plasmids may promote the fitness and evolution of P. aeruginosa. PMID:26841043

  18. The Catalytic Function of Enzymes.

    Science.gov (United States)

    Splittgerber, Allan G.

    1985-01-01

    Discusses: structure of the enzyme molecule; active site; reaction mechanism; transition state; factors affecting enzyme reaction rates, concentration of enzyme; concentration of substrate; product concentration; temperature effects and pH effects; factors causing a lowering of activation energy; proximity and orientation effects; substrate strain…

  19. 噬菌体治疗耐药性幽门螺杆菌的研究进展%Advances of phage therapy in antibiotic-resistant Helicobacter pylori infection

    Institute of Scientific and Technical Information of China (English)

    熊婧; 白杨

    2011-01-01

    幽门螺杆菌感染是胃癌重要致病因子.目前,幽门螺杆菌对抗生素耐药情况日趋普遍.噬菌体治疗作为一种生物疗法在治疗幽门螺杆菌方面有极大的潜力.本文就噬菌体治疗耐药性幽门螺杆菌的现状及趋势进行综述.%Helicobacter pylori (H. pylori) infection is one of the most important risk factors leading to gastric cancer.To date, it has been more and more popular that H. pylori is resistant to antibiotics. Phage therapy has a great advantages in control of H. pylori infection. In this article, we will review the advance of phage therapy in antibiotic-resistant H.pylori infection.

  20. Measuring the Enzyme Activity of Arabidopsis Deubiquitylating Enzymes.

    Science.gov (United States)

    Kalinowska, Kamila; Nagel, Marie-Kristin; Isono, Erika

    2016-01-01

    Deubiquitylating enzymes, or DUBs, are important regulators of ubiquitin homeostasis and substrate stability, though the molecular mechanisms of most of the DUBs in plants are not yet understood. As different ubiquitin chain types are implicated in different biological pathways, it is important to analyze the enzyme characteristic for studying a DUB. Quantitative analysis of DUB activity is also important to determine enzyme kinetics and the influence of DUB binding proteins on the enzyme activity. Here, we show methods to analyze DUB activity using immunodetection, Coomassie Brilliant Blue staining, and fluorescence measurement that can be useful for understanding the basic characteristic of DUBs.

  1. [Development and application of indirect competitive enzyme immunoassay for detection of neomycin in milk].

    Science.gov (United States)

    Burkin, M A; Gal'vidis, I A

    2011-01-01

    As a result of immunization of rabbits with neomycin B (N M) conjugated to periodate-oxidized transferrin, polyclonal antibodies were generated and used to develop an indirect competitive enzyme-linked immunosorbent assay (ELISA) of NM. Several heterologous conjugates, namely, glutaraldehyde (GA)-polymerized NM, gelatin-ribostamycin (sp), and gelatin-NM (ga) were used as coating antigens in different ELISA variants for quantification of NM in milk. These variants were characterized by different dynamic ranges and detection limits of 1.0, 0.1, and 0.01 ng/ml, respectively. Maximum residue level (MRL) of this antibiotic in milk accepted in the EU can be detected without any special pretreatment at a 100-fold sample dilution in the least sensitive assay variant. The mean recovery rate from NM-spiked milk containing 1.5-10% fat was 111.7% and ranged from 84 to 125.2%. We found that 57 of 106 tested milk samples contained NM at concentrations higher than 100 ng/ml. In ten percent of cases (11/1 06), the residual level of this antibiotic was greater than 500 ng/ml. In one case, the M RL was exceeded (1690 ng/ml). The assay developed in this study is specific shows no cross-reactivity with other veterinary aminoglycosides, has a good sensitivity reserve, and can serve as an effective tool to monitor the NM content in milk stuff.

  2. Negative cooperativity in regulatory enzymes.

    Science.gov (United States)

    Levitzki, A; Koshland, D E

    1969-04-01

    Negative cooperativity has been observed in CTP synthetase, an allosteric enzyme which contains a regulatory site. Thus, the same enzyme exhibits negative cooperativity for GTP (an effector) and glutamine (a substrate) and positive cooperativity for ATP and UTP (both substrates). In the process of the delineation of these phenomena, diagnostic procedures for negative cooperativity were developed. Application of these procedures to other enzymes indicates that negative cooperativity is a characteristic of many of them. These findings add strong support for the sequential model of subunit interactions which postulates that ligand-induced conformational changes are responsible for regulatory and cooperative phenomena in enzymes. PMID:5256410

  3. Enzyme therapeutics for systemic detoxification.

    Science.gov (United States)

    Liu, Yang; Li, Jie; Lu, Yunfeng

    2015-08-01

    Life relies on numerous biochemical processes working synergistically and correctly. Certain substances disrupt these processes, inducing living organism into an abnormal state termed intoxication. Managing intoxication usually requires interventions, which is referred as detoxification. Decades of development on detoxification reveals the potential of enzymes as ideal therapeutics and antidotes, because their high substrate specificity and catalytic efficiency are essential for clearing intoxicating substances without adverse effects. However, intrinsic shortcomings of enzymes including low stability and high immunogenicity are major hurdles, which could be overcome by delivering enzymes with specially designed nanocarriers. Extensive investigations on protein delivery indicate three types of enzyme-nanocarrier architectures that show more promise than others for systemic detoxification, including liposome-wrapped enzymes, polymer-enzyme conjugates, and polymer-encapsulated enzymes. This review highlights recent advances in these nano-architectures and discusses their applications in systemic detoxifications. Therapeutic potential of various enzymes as well as associated challenges in achieving effective delivery of therapeutic enzymes will also be discussed.

  4. Synthesis of kanamycin A derivatives by regioselective masking drug resistant enzymes targeting hydroxyl groups%选择性封闭卡那霉素A耐药酶羟基衍生物的合成

    Institute of Scientific and Technical Information of China (English)

    陈颖; 孟祥豹; 陈桂辉; 潘攀; 李中军

    2008-01-01

    The 3'-OH, 4'-OH and 2"-OH of kanamycin A were modified in search of new aminoglycosides to overcome resistant enzymes, ANTs and APHs. The key intermediate was a dibenzylidene-protected derivative of kanamycin A. The aimed sites were masked by benzyl, methyl and allyl groups. Multi-step reactions gave the desired aminoglycoside derivatives but showed less antibiotic activity than kanamycin A.%为寻找能抵抗耐药酶作用的氨基糖苷类抗生素,本文对卡那霉素A的3'-OH,4'-OH和2"-OH位点进行了修饰.合成路线中的关键中间体为引入双苄叉保护基的卡那霉素A衍生物.目标羟基通过苄基化,甲基化和烯丙基化的方法进行修饰.最后通过多步反应脱保护,得到目标产物.活性结果显示,目标产物对敏感菌和耐药菌未显示出良好的抗菌活性.

  5. Study on risk factors for nosocomial infections caused by high-level aminoglycoside-resistant Enterococcus and aminoglycoside resistance-related genes%耐氨基苷类高水平肠球菌医院感染的危险因素及氨基糖苷类耐药相关基因研究

    Institute of Scientific and Technical Information of China (English)

    范建中; 周田美; 董晓勤; 王贤军

    2012-01-01

    目的 了解耐氨基糖苷类高水平肠球菌(HLAR)的耐药性和医院感染的危险因素,研究HLAR氨基糖苷类耐药相关基因类型分布.方法 采用全自动微生物鉴定仪VITEK-AMS对857株肠球菌属进行鉴定及抗菌药物敏感性检测;PCR法检测HLAR氨基糖苷类耐药相关基因,并对PCR结果进行测序分析.结果 肠球菌属中HLAR占50.4%,利奈唑胺、万古霉素和替考拉宁对HLAR的抗菌作用最好,但有3株屎肠球菌对万古霉素和替考拉宁耐药,粪肠球菌对氯霉素和四环素的耐药率高于屎肠球菌,而屎肠球菌对其他常用抗菌药物的耐药率明显高于粪肠球菌,粪肠球菌和屎肠球菌的耐药谱明显不同,aac(6')-Ie-aph(2〃)-Ia基因为耐庆大霉素高水平肠球菌(HLGR)的主要耐药基因,占HLGR的88.0%,严重的基础疾病、侵入性操作和头孢三代抗菌药物和激素的应用是肠球菌属医院感染的常见危险因素.结论 HLAR已成为医院感染的重要耐药菌,HLGR产生的主要机制是aac(6')-Ie-aph(2〃)-Ia基因介导对庆大霉素高水平耐药,控制常见医院感染危险因素,合理使用抗菌药物,可减少HLAR医院感染的发生.%OBJECTIVE To explore the antibiotic resistance and risk factors for nosocomial infections caused by high-level aminoglycoside-resistant (HLAR) Enterococcus, and investigate the genotypes related to high-level aminoglycoside resistance. METHODS A total of 857 strains of Enterococcus were identified and analyzed for their antimicrobial susceptibility by VITEK-AMS. The aminoglycoside resistance-related genes were detected by PCR. The sequencing analysis of PCR products was performed. RESULTS A total of 50. 4% of Enterococcus isolates were HLAR Enterococcus. Linezolid, vancomycin and teicoplanin were mostly effective against HLAR Enterococcus, but there were three isolates resistant to vancomycin and teicoplanin. The resistance rates to chloramphenicol and tetracycline of E. Faecium were

  6. Computational enzyme design: transitioning from catalytic proteins to enzymes.

    Science.gov (United States)

    Mak, Wai Shun; Siegel, Justin B

    2014-08-01

    The widespread interest in enzymes stem from their ability to catalyze chemical reactions under mild and ecologically friendly conditions with unparalleled catalytic proficiencies. While thousands of naturally occurring enzymes have been identified and characterized, there are still numerous important applications for which there are no biological catalysts capable of performing the desired chemical transformation. In order to engineer enzymes for which there is no natural starting point, efforts using a combination of quantum chemistry and force-field based protein molecular modeling have led to the design of novel proteins capable of catalyzing chemical reactions not catalyzed by naturally occurring enzymes. Here we discuss the current status and potential avenues to pursue as the field of computational enzyme design moves forward.

  7. 两个携带线粒体12S rRNA 1494C>T突变的耳聋家系的遗传学特征%Characterization of two Chinese families with aminoglycoside-induced and nonsyndromic hearing loss both carrying a mitochondrial 12S rRNA 1494C>T mutation

    Institute of Scientific and Technical Information of China (English)

    龚莎莎; 管敏鑫; 陈波蓓; 彭光华; 郑静; 张婷; 郑斌娇; 方芳; 张初琴; 吕建新

    2012-01-01

    Objective To evaluate the effect of mitochondrial DNA(mtDNA) secondary mutations,haplotypes,GJB2 gene mutations on phenotype of 1494C > T mutation,and to study the molecular pathogenic mechanism of maternally transmitted aminoglycoside-induced and nonsyndromic hearing loss.Methods Two Chinese Han pedigrees of maternally transmitted aminoglycoside induced and nonsyndromic hearing loss were collected.The two probands and their family members underwent clinical,genetic and molecular evaluations including audiological examinations and mutational analysis of mitochondrial genome and GJB2 gene.Results Clinical evaluation revealed wide range of severity,age-at-onset and audiometric configuration of hearing impairment in matrilineal relatives in both families,for which the penetrance of hearing loss was respectively 42.9 % and 28.6% when aminoglycoside-induced deafness was included.When the effect of aminoglycosides was excluded,the penetrances of hearing loss were 14.3% and 14.3%.Sequence analysis of mitochondrial genomes identified a known 12S rRNA 1494C>T mutation,in addition with distinct sets of mtDNA polymorphisms belonging to Eastern Asian haplogroups C4a1a and B4b1c,respectively.Conclusion Mitochondrial 12S rRNA 1494C>T mutation probably underlie the deafness in both families.Lack of significant mutation in the GJB2 gene ruled out involvement of GJB2 in the phenotypic expression.However,aminoglycosides and other nuclear modifier genes may still modify the phenotype of the 1494C>T mutation in these families.The B4b1c is a newly identified haplogroup in aminoglycoside-induced and nonsyndromic hearing loss family carrying the 1494C>T mutation.The 1494C>T mutation seems to have occurred sporadically through evolution.

  8. Enzyme immunoassay for human ferritin

    International Nuclear Information System (INIS)

    We described an enzyme immunoassay with use of β-D-galactosidase for quantitation of ferritin in human serum. The minimum detectable ferritin concentration is 0.25 μg/L of serum, which is comparable to results obtained by radioimmunoassay. The correlation coefficient between values determined by enzyme immunoassay and radioimmunoassay was 0.95

  9. Phage lytic enzymes: a history

    Institute of Scientific and Technical Information of China (English)

    David; Trudil

    2015-01-01

    There are many recent studies regarding the efficacy of bacteriophage-related lytic enzymes: the enzymes of ‘bacteria-eaters’ or viruses that infect bacteria. By degrading the cell wall of the targeted bacteria, these lytic enzymes have been shown to efficiently lyse Gram-positive bacteria without affecting normal flora and non-related bacteria. Recent studies have suggested approaches for lysing Gram-negative bacteria as well(Briersa Y, et al., 2014). These enzymes include: phage-lysozyme, endolysin, lysozyme, lysin, phage lysin, phage lytic enzymes, phageassociated enzymes, enzybiotics, muralysin, muramidase, virolysin and designations such as Ply, PAE and others. Bacteriophages are viruses that kill bacteria, do not contribute to antimicrobial resistance, are easy to develop, inexpensive to manufacture and safe for humans, animals and the environment. The current focus on lytic enzymes has been on their use as anti-infectives in humans and more recently in agricultural research models. The initial translational application of lytic enzymes, however, was not associated with treating or preventing a specifi c disease but rather as an extraction method to be incorporated in a rapid bacterial detection assay(Bernstein D, 1997).The current review traces the translational history of phage lytic enzymes–from their initial discovery in 1986 for the rapid detection of group A streptococcus in clinical specimens to evolving applications in the detection and prevention of disease in humans and in agriculture.

  10. Enzyme immobilization on graft copolymers

    NARCIS (Netherlands)

    Mohy Eldin, M.S.

    1999-01-01

    Immobilised enzymes can be reused, easily separated from the reaction medium, and are more stable in most of the cases. Despite of these advantages, there are still some problems facing the usage of the immobilised enzyme in industry. One of those problems is diffusion-limitation of both the reactan

  11. Engineering Cellulase Enzymes for Bioenergy

    Science.gov (United States)

    Atreya, Meera Elizabeth

    Sustainable energy sources, such as biofuels, offer increasingly important alternatives to fossil fuels that contribute less to global climate change. The energy contained within cellulosic biofuels derives from sunlight energy stored in the form of carbon-carbon bonds comprising sugars such as glucose. Second-generation biofuels are produced from lignocellulosic biomass feedstocks, including agricultural waste products and non-food crops like Miscanthus, that contain lignin and the polysaccharides hemicellulose and cellulose. Cellulose is the most abundant biological material on Earth; it is a polymer of glucose and a structural component of plant cell walls. Accessing the sugar is challenging, as the crystalline structure of cellulose resists degradation; biochemical and thermochemical means can be used to depolymerize cellulose. Cellulase enzymes catalyze the biochemical depolymerization of cellulose into glucose. Glucose can be used as a carbon source for growth of a biofuel-producing microorganism. When it converts glucose to a hydrocarbon fuel, this microbe completes the biofuels process of transforming sunlight energy into accessible, chemical energy capable of replacing non-renewable transportation fuels. Due to strong intermolecular interactions between polymer chains, cellulose is significantly more challenging to depolymerize than starch, a more accessible polymer of glucose utilized in first-generation biofuels processes (often derived from corn). While most mammals cannot digest cellulose (dietary fiber), certain fungi and bacteria produce cellulase enzymes capable of hydrolyzing it. These organisms secrete a wide variety of glycoside hydrolase and other classes of enzymes that work in concert. Because cellulase enzymes are slow-acting and expensive to produce, my aim has been to improve the properties of these enzymes as a means to make a cellulosic biofuels process possible that is more efficient and, consequently, more economical than current

  12. Moonlighting enzymes in parasitic protozoa.

    Science.gov (United States)

    Collingridge, Peter W; Brown, Robert W B; Ginger, Michael L

    2010-08-01

    Enzymes moonlight in a non-enzymatic capacity in a diverse variety of cellular processes. The discovery of these non-enzymatic functions is generally unexpected, and moonlighting enzymes are known in both prokaryotes and eukaryotes. Importantly, this unexpected multi-functionality indicates that caution might be needed on some occasions in interpreting phenotypes that result from the deletion or gene-silencing of some enzymes, including some of the best known enzymes from classic intermediary metabolism. Here, we provide an overview of enzyme moonlighting in parasitic protists. Unequivocal and putative examples of moonlighting are discussed, together with the possibility that the unusual biological characteristics of some parasites either limit opportunities for moonlighting to arise or perhaps contribute to the evolution of novel proteins with clear metabolic ancestry.

  13. An enzyme with rhamnogalacturonase activity.

    OpenAIRE

    Kovod, L.V.; Dalboge, H; Andersen, L N; Kauppinen, M.; Christgan, S.; Heldt-Hansen, H.P.; Christophersen, C.; Nielsen, P.M.; Voragen, A. G. J.; Schols, H.A.

    1994-01-01

    An enzyme exhibiting rhamnogalacturonase activity, which enzyme: a) is encoded by the DNA sequence shown in SEQ ID No. 1 or a sequence homologous thereto encoding a polypeptide with RGase activity, b) has the amino acid sequence shown in SEQ ID No. 2 or an analogue thereof, c) is reactive with an antibody raised against the enzyme encoded by the DNA sequence shown in SEQ ID No. 1, d) has a pH optimum above pH 5, and/or e) has a relative activity of at least 30t a pH in the range of 5.5-6.5. T...

  14. 黄芩苷消除鲍曼不动杆菌耐药质粒的实验研究%Experimental Study of Baicalin Curing Antibiotic-Resistant Plasmid in Acinetobacter Baumanii

    Institute of Scientific and Technical Information of China (English)

    汪东海; 陈敏; 姜志强; 王萍; 陈学锋; 徐菊玲

    2012-01-01

    OBJECTIVE To investigate if baicalin was able to cure antibiotic-resistant plasmid in Acinetobacter baumanii. and affect minimum inhibitory concentrations (MIC) of antibiotics to bacteria. METHODS The growth curve of A. baumanii treated with baicalin was detected. 16 strains of A. baumanii, gentamicin(GEN) and ciprofloxacin(CIP) resistance, were treated with baicalin for 20 hours. Before and after baicalin treatment, plasmids of A. baumanii were determined with alkaline lysis method, and MICs of GEN and CIP were measured by agar dilution method. RESULTS Baicalin inhibited the growth of A. baumanii. The plasmid curing rate was 37.5%(6/16) in 16 strains of A. baumanii with plasmids, after the treatment with baicalin (1 mg·mL‐1). In 6 strains of A. baumanii with plasmid cured by baicalin, for medium or low level of GEN-resistance strains, MICs of GEN all decreased to sensitive level; for low level of CIP-resistance strains, MICs of CIP all dropped to sensitive level, and for some medium-level of CIP-resistance strains, M!Cs of CIP also dropped, but still above the resistance level. CONCLUSION Baicalin can eliminate plasmids of A. baumanii with low level of antibiotic-resistance, and make these strains recover the susceptibility to GEN and CIP, which shows an adjuvant treatment method for antibiotic-resistant A. baumanii infections.%目的 探讨黄芩苷能否消除鲍曼不动杆菌质粒,并影响抗菌药物对细菌的最低抑菌浓度(MIC).方法 测定黄芩苷处理的鲍曼不动杆菌生长曲线.16株庆大霉素和环丙沙星双重耐药的鲍曼不动杆菌,经黄芩苷作用20h,分别在处理前和处理后应用碱裂解法检测质粒,琼脂稀释法测定抗菌药物MIC.结果 黄芩苷能抑制鲍曼不动杆菌生长.16株鲍曼不动杆菌耐药株均携带质粒,经1mg·mL-1黄芩苷作用后,质粒消除率为37.5%(6/16).6株经黄芩苷消除质粒的细菌,庆大霉素中度水平或低水平耐药株的MIC值均下降至敏感水平;

  15. Carriage of antibiotic-resistant pneumococci in a cohort of a daycare center Portadores nasofaríngeos de neumococo antibiótico-resistente en niños asistentes a guardería

    Directory of Open Access Journals (Sweden)

    Demóstenes Gómez-Barreto

    2002-01-01

    Full Text Available Objective. To define epidemiologic relationships to determine the prevalence and potential risk factors for nasopharyngeal colonization by antibiotic-resistant pneumococci, their serotypes and their antibiotic susceptibility patterns in children attending a daycare center (DCC. Material and Methods. A prospective cohort study was conducted among children (n=53 attending the DCC at Hospital Infantil de México Federico Gómez, which is staffed by 20 employees. Patients were enrolled in the study during a two-year period from September 1997 to September 1999. All the participants were followed prospectively, swabbing them every four months. The strains recovered were typed and screened for susceptibility to several antibiotics. The daycare records were reviewed also. Odds ratios and fisher's exact test: or chi square test of significance were computed from contingency tables as appropriate. Exact 95% confidence intervals were computed for odds ratios. Data analysis was performed using Epi statistics program version 6.04 a. Results. Pneumococci were recovered from 45/53 of the infants at one or more visits. A total of 178 isolates were carried. The carriage rate was 47%. Only 7 adults acquired pneumococci during the study. Types 6,14,19 and 23 were prevalent and represented 77% of the total. Antibiotic-resistant strains were higher to penicillin and erythromycin. Conclusions. Children were frequent carriers of pneumococci, the rate of carriage was high in infancy and tended to decrease with age. The types commonly carried by children were the same as those causing invasive disease. There is a high proportion of carriers with antibiotic-resistant S. pneumoniae strains. Children who have had frequent antimicrobial courses are at particular risk.Objetivo. Analizar longitudinalmente la dinámica de colonización por Streptococcus pneumoniae, determinar la prevalencia, los factores de riesgo potencial para la colonización nasofaríngea con cepas de

  16. Antibiotic-resistant bacteria inhibited by extracts and fractions from Brazilian marine sponges Bactérias resistentes a antibióticos inibidas por extratos e frações de esponjas marinhas do Brasil

    Directory of Open Access Journals (Sweden)

    Palloma R. Marinho

    2010-05-01

    Full Text Available The growing number of bacterial strains resistant to conventional antibiotics has become a serious medical problem in recent years. Marine sponges are a rich source of bioactive compounds, and many species can be useful for the development of new antimicrobial drugs. This study reports the in vitro screening of marine sponges in the search for novel substances against antibiotic-resistant bacteria. Sponge extracts were tested against 44 bacterial strains, including fourteen antibiotic-resistant strains. Ten out of the twelve sponge species studied showed activity in one or more of the bioassays. Aqueous extracts of Cinachyrella sp. and Petromica citrina showed a large action spectrum over resistant-bacteria such as Staphylococcus aureus, coagulase-negative staphylococci and Enterococcus faecalis. Aqueous extract of P. citrina was fractioned and aqueous fraction showed a greatest inhibitory activity on Staphylococcus strains. In addition, this fraction demonstrated a bactericidal effect on exponentially growing S. aureus cells at the MIC (16 µg/mL. The mechanism of action of bioactive fraction is still unclear, but we showed that it affect protein biosynthesis of Staphylococcus. Our results demonstrated for the first time that P. citrina is a potential source of new drugs for the treatment of infections by antibiotic-resistant bacteria.O número crescente de bactérias resistentes aos antibióticos tem se tornado um sério problema médico nos últimos anos. As esponjas marinhas são uma fonte rica em compostos bioativos e muitas espécies podem ser úteis para o desenvolvimento de novos antimicrobianos. Esse estudo descreve uma triagem in vitro de esponjas para a pesquisa de novas substâncias contra bactérias resistentes. Os extratos de esponjas foram testados sobre 44 estirpes bacterianas, incluindo quatorze resistentes a antibióticos. Dez entre doze espécies de esponjas apresentaram atividade em um ou mais bioensaios. Os extratos aquosos

  17. ORGANOPHOSPHATE DEGRADING ENZYMES - PHASE I

    Science.gov (United States)

    Agave BioSystems in collaboration with Carl A. Batt proposes to develop decon-nanoparticles, which will leverage ongoing opportunities in enzyme engineering and the fabrication of functionalized magnetic nanoparticles. Enhanced performance will be engineered into the system t...

  18. Controlled enzyme catalyzed heteropolysaccharide degradation

    DEFF Research Database (Denmark)

    Rasmussen, Louise Enggaard

    The work presented in this PhD thesis has provided a better understanding of the enzyme kinetics and quantitative phenomena of the hydrolysis of xylan substrates by selected pure enzyme preparations. Furthermore, the options for producing specific substituted xylooligosaccharides from selected...... substrates by specific xylanase treatment have been examined. The kinetics of the enzymatic degradation of water-extractable wheat arabinoxylan (WE-AX) during designed treatments with selected monocomponent enzymes was investigated by monitoring the release of xylose and arabinose. The results of different...... between -xylosidase and the α-L-arabinofuranosidases on the xylose release were low as compared to the effect of xylanase addition with β-xylosidase, which increased the xylose release by ~25 times in 30 minutes. At equimolar addition levels of the four enzymes, the xylanase activity was thus rate...

  19. Enzymes: principles and biotechnological applications.

    Science.gov (United States)

    Robinson, Peter K

    2015-01-01

    Enzymes are biological catalysts (also known as biocatalysts) that speed up biochemical reactions in living organisms, and which can be extracted from cells and then used to catalyse a wide range of commercially important processes. This chapter covers the basic principles of enzymology, such as classification, structure, kinetics and inhibition, and also provides an overview of industrial applications. In addition, techniques for the purification of enzymes are discussed.

  20. Heavy enzymes--experimental and computational insights in enzyme dynamics.

    Science.gov (United States)

    Swiderek, Katarzyna; Ruiz-Pernía, J Javier; Moliner, Vicent; Tuñón, Iñaki

    2014-08-01

    The role of protein motions in the chemical step of enzyme-catalyzed reactions is the subject of an open debate in the scientific literature. The systematic use of isotopically substituted enzymes has been revealed as a useful tool to quantify the role of these motions. According to the Born-Oppenheimer approximation, changing the mass of the protein does not change the forces acting on the system but alters the frequencies of the protein motions, which in turn can affect the rate constant. Experimental and theoretical studies carried out in this field are presented in this article and discussed in the framework of Transition State Theory.

  1. Micromotors Powered by Enzyme Catalysis.

    Science.gov (United States)

    Dey, Krishna K; Zhao, Xi; Tansi, Benjamin M; Méndez-Ortiz, Wilfredo J; Córdova-Figueroa, Ubaldo M; Golestanian, Ramin; Sen, Ayusman

    2015-12-01

    Active biocompatible systems are of great current interest for their possible applications in drug or antidote delivery at specific locations. Herein, we report the synthesis and study of self-propelled microparticles powered by enzymatic reactions and their directed movement in substrate concentration gradient. Polystyrene microparticles were functionalized with the enzymes urease and catalase using a biotin-streptavidin linkage procedure. The motion of the enzyme-coated particles was studied in the presence of the respective substrates, using optical microscopy and dynamic light scattering analysis. The diffusion of the particles was found to increase in a substrate concentration dependent manner. The directed chemotactic movement of these enzyme-powered motors up the substrate gradient was studied using three-inlet microfluidic channel architecture. PMID:26587897

  2. Subcellular localization of pituitary enzymes

    Science.gov (United States)

    Smith, R. E.

    1970-01-01

    A cytochemical procedure is reported for identifying subcellular sites of enzymes hydrolyzing beta-naphthylamine substrates, and to study the sites of reaction product localization in cells of various tissues. Investigations using the substrate Leu 4-methoxy-8-naphthylamine, a capture with hexonium pararosaniline, and the final chelation of osmium have identified the hydrolyzing enzyme of rat liver cells; this enzyme localized on cell membranes with intense deposition in the areas of the parcanaliculi. The study of cells in the anterior pituitary of the rat showed the deposition of reaction product on cell membrane; and on the membranes of secretion granules contained within the cell. The deposition of reaction product on the cell membrane however showed no increase or decrease with changes in the physiological state of the gland and release of secretion granules from specific cells.

  3. A structural view of the antibiotic degradation enzyme NDM-1 from a superbug.

    Science.gov (United States)

    Guo, Yu; Wang, Jing; Niu, Guojun; Shui, Wenqing; Sun, Yuna; Zhou, Honggang; Zhang, Yaozhou; Yang, Cheng; Lou, Zhiyong; Rao, Zihe

    2011-05-01

    Gram-negative Enterobacteriaceae with resistance to carbapenem conferred by New Delhi metallo-β-lactamase 1 (NDM-1) are a type of newly discovered antibioticresistant bacteria. The rapid pandemic spread of NDM-1 bacteria worldwide (spreading to India, Pakistan, Europe, America, and Chinese Taiwan) in less than 2 months characterizes these microbes as a potentially major global health problem. The drug resistance of NDM-1 bacteria is largely due to plasmids containing the blaNDM-1 gene shuttling through bacterial populations. The NDM-1 enzyme encoded by the blaNDM-1 gene hydrolyzes β-lactam antibiotics, allowing the bacteria to escape the action of antibiotics. Although the biological functions and structural features of NDM-1 have been proposed according to results from functional and structural investigation of its homologues, the precise molecular characteristics and mechanism of action of NDM-1 have not been clarified. Here, we report the three-dimensional structure of NDM-1 with two catalytic zinc ions in its active site. Biological and mass spectroscopy results revealed that D-captopril can effectively inhibit the enzymatic activity of NDM-1 by binding to its active site with high binding affinity. The unique features concerning the primary sequence and structural conformation of the active site distinguish NDM-1 from other reported metallo-β-lactamases (MBLs) and implicate its role in wide spectrum drug resistance. We also discuss the molecular mechanism of NDM-1 action and its essential role in the pandemic of drug-resistant NDM-1 bacteria. Our results will provide helpful information for future drug discovery targeting drug resistance caused by NDM-1 and related metallo-β-lactamases. PMID:21637961

  4. Biochemical Characterization of Mycobacterium tuberculosis DNA Repair Enzymes – Nfo, XthA and Nei2

    Directory of Open Access Journals (Sweden)

    Sailau Abeldenov

    2014-01-01

    Full Text Available Introduction: Tuberculosis (TB is a human disease caused by Mycobacterium tuberculosis (Mtb. Treatment of TB requires long-term courses of multi-drug therapies to eliminate subpopulations of bacteria, which sometimes persist against antibiotics. Therefore, understanding of the mechanism of Mtb antibiotic-resistance is extremely important. During infection, Mtb overcomes a variety of body defense mechanisms, including treatment with the reactive species of oxygen and nitrogen. The bases in DNA molecule are susceptible to the damages caused by reactive forms of intermediate compounds of oxygen and nitrogen. Most of this damage is repaired by the base excision repair (BER pathway. In this study, we aimed to biochemically characterize three Mtb DNA repair enzymes of BER pathway. Methods: XthA, nfo, and nei genes were identified in mycobacteria by homology search of genomic sequences available in the GenBank database. We used standard methods of genetic engineering  to clone and sequence Mtb genes, which coded Nfo, XthA and Nei2 repair enzymes. The protein products of Mtb genes were expressed and purified in Escherichia coli using affinity tags. The enzymatic activity of purified Nfo, XthA, and Nei2 proteins were measured using radioactively labeled DNA substrates containing various modified residues. Results: The genes end (Rv0670, xthA (Rv0427c, and nei (Rv3297 were PCR amplified using genomic DNA of Mtb H37Rv with primers that contain specific restriction sites. The amplified products were inserted into pET28c(+ expression vector in such a way that the recombinant proteins contain C-terminal histidine tags. The plasmid constructs were verified by sequencing and then transformed into the Escherichia coli BL21 (DE3 strain. Purification of recombinant proteins was performed using Ni2+ ions immobilized affinity column, coupled with the fast performance liquid chromatography machine AKTA. Identification of the isolated proteins was performed by

  5. Enzyme and biochemical producing fungi

    DEFF Research Database (Denmark)

    Lübeck, Peter Stephensen; Lübeck, Mette; Nilsson, Lena;

    2010-01-01

    We are developing a biorefinery concept for biological production of chemicals, drugs, feed and fuels using plant biomass as raw material in well-defined cell-factories. Among the important goals is the discovery of new biocatalysts for production of enzymes, biochemicals and fuels and already our...... screening of a large collection of fungal strains isolated from natural habitats have resulted in identification of strains with high production of hydrolytic enzymes and excretion of organic acids. Our research focuses on creating a fungal platform based on synthetic biology for developing new cell...

  6. Modifying enzyme activity and selectivity by immobilization

    OpenAIRE

    Rodrigues, Rafael C.; Ortiz, Claudia; Berenguer Murcia, Ángel; Torres, Rodrigo; Fernández Lafuente, Roberto

    2013-01-01

    Immobilization of enzymes may produce alterations in their observed activity, specificity or selectivity. Although in many cases an impoverishment of the enzyme properties is observed upon immobilization (caused by the distortion of the enzyme due to the interaction with the support) in some instances such properties may be enhanced by this immobilization. These alterations in enzyme properties are sometimes associated with changes in the enzyme structure. Occasionally, these variations will ...

  7. Thermodynamics of Enzyme-Catalyzed Reactions Database

    Science.gov (United States)

    SRD 74 Thermodynamics of Enzyme-Catalyzed Reactions Database (Web, free access)   The Thermodynamics of Enzyme-Catalyzed Reactions Database contains thermodynamic data on enzyme-catalyzed reactions that have been recently published in the Journal of Physical and Chemical Reference Data (JPCRD). For each reaction the following information is provided: the reference for the data, the reaction studied, the name of the enzyme used and its Enzyme Commission number, the method of measurement, the data and an evaluation thereof.

  8. Enzyme recovery using reversed micelles.

    NARCIS (Netherlands)

    Dekker, M.

    1990-01-01

    The objective of this study was to develop a liquid-liquid extraction process for the recovery of extracellular enzymes. The potentials of reaching this goal by using reversed micelles in an organic solvent have been investigated.Reversed micelles are aggregates of surfactant molecules containing an

  9. Insolubilized enzymes for food synthesis

    Science.gov (United States)

    Marshall, D. L.

    1972-01-01

    Cellulose matrix with numerous enzyme-coated silica particles of colloidal size permanently bound at various sites within matrix was produced that has high activity and possesses requisite physical characteristics for filtration or column operations. Product also allows coupling step in synthesis of edible food to proceed under mild conditions.

  10. The enzymes associated with denitrification

    Science.gov (United States)

    Hochstein, L. I.; Tomlinson, G. A.

    1988-01-01

    The enzymes involved in the reduction of nitrogenous oxides are thought to be intermediates in denitrification processes. This review examines the roles of nitrate reductase, nitrite reductases, nitric oxide reductase, mechanisms of N-N bond formation, and nitrous oxide reductases.

  11. Kathepsine C : Een allosterisch enzyme

    NARCIS (Netherlands)

    Gorter, Jeannette

    1969-01-01

    In chapter I an introduction into allosteric systems is given. In chapter II is a detailed method is described for the applica of Gly-Phe--p. nitroanilide (GPNA) as a substrate for the activity assay of the lysosomal enzyme cathepsin C. It is an allosteric which is activated by Cl-, Br-, 1-, CNS-, N

  12. Udfordringer ved undervisning i enzymer

    DEFF Research Database (Denmark)

    Skriver, Karen; Dandanell, Gert; von Stemann, Jakob Hjorth;

    2015-01-01

    Enzymer er et centralt emne i biokemiundervisning. Det forudsætter og anvender grundlæggende viden inden for og kompetencer i kemi og matematik. Artiklen undersøger hvilke forståelsesvanskeligheder og udfordringer der er knyttet til dette område, såvel som virtuelle øvelsers potentiale i denne...

  13. Enzymes involved in triglyceride hydrolysis.

    Science.gov (United States)

    Taskinen, M R; Kuusi, T

    1987-08-01

    The lipolytic enzymes LPL and HL play important roles in the metabolism of lipoproteins and participate in lipoprotein interconversions. LPL was originally recognized to be the key enzyme in the hydrolysis of chylomicrons and triglyceride, but it also turned out to be one determinant of HDL concentration in plasma. When LPL activity is high, chylomicrons and VLDL are rapidly removed from circulation and a concomitant rise of the HDL2 occurs. In contrast, low LPL activity impedes the removal of triglyceride-rich particles, resulting in the elevation of serum triglycerides and a decrease of HDL (HDL2). Concordant changes of this kind in LPL and HDL2 are induced by many physiological and pathological perturbations. Finally, the operation of LPL is also essential for the conversion of VLDL to LDL. This apparently clear-cut role of LPL in lipoprotein interconversions is contrasted with the enigmatic actions of HL. The enzyme was originally thought to participate in the catalyses of chylomicron and VLDL remnants generated in the LPL reaction. However, substantial in vitro and in vivo data indicate that HL is a key enzyme in the degradation of plasma HDL (HDL2) in a manner which opposes LPL. A scheme is presented for the complementary actions of the two enzymes in plasma HDL metabolism. In addition, recent studies have attributed a role to HL in the catabolism of triglyceride-rich lipoproteins, particularly those containing apo E. However, this function becomes clinically important only under conditions where the capacity of the LPL-mediated removal system is exceeded. Such a situation may arise when the input of triglyceride-rich particles (chylomicrons and/or VLDL) is excessive or LPL activity is decreased or absent.

  14. Engineering cytochrome p450 enzymes.

    Science.gov (United States)

    Gillam, Elizabeth M J

    2008-01-01

    The last 20 years have seen the widespread and routine application of methods in molecular biology such as molecular cloning, recombinant protein expression, and the polymerase chain reaction. This has had implications not only for the study of toxicological mechanisms but also for the exploitation of enzymes involved in xenobiotic clearance. The engineering of P450s has been performed with several purposes. The first and most fundamental has been to enable successful recombinant expression in host systems such as bacteria. This in turn has led to efforts to solubilize the proteins as a prerequisite to crystallization and structure determination. Lagging behind has been the engineering of enzyme activity, hampered in part by our still-meager comprehension of fundamental structure-function relationships in P450s. However, the emerging technique of directed evolution holds promise in delivering both engineered enzymes for use in biocatalysis and incidental improvements in our understanding of sequence-structure and sequence-function relationships, provided that data mining can extract the fundamental correlations underpinning the data. From the very first studies on recombinant P450s, efforts were directed toward constructing fusions between P450s and redox partners in the hope of generating more efficient enzymes. While this aim has been allowed to lie fallow for some time, this area merits further investigation as does the development of surface-displayed P450 systems for biocatalytic and biosensor applications. The final application of engineered P450s will require other aspects of their biology to be addressed, such as tolerance to heat, solvents, and high substrate and product concentrations. The most important application of these enzymes in toxicology in the near future is likely to be the biocatalytic generation of drug metabolites for the pharmaceutical industry. Further tailoring will be necessary for specific toxicological applications, such as in

  15. A novel bacteriophage Tail-Associated Muralytic Enzyme (TAME from Phage K and its development into a potent antistaphylococcal protein

    Directory of Open Access Journals (Sweden)

    Chikkamadaiah Ravisha

    2011-10-01

    Full Text Available Abstract Background Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. However, the rapid emergence of antibiotic resistance limits the choice of therapeutic options for treating infections caused by this organism. Muralytic enzymes from bacteriophages have recently gained attention for their potential as antibacterial agents against antibiotic-resistant gram-positive organisms. Phage K is a polyvalent virulent phage of the Myoviridae family that is active against many Staphylococcus species. Results We identified a phage K gene, designated orf56, as encoding the phage tail-associated muralytic enzyme (TAME. The gene product (ORF56 contains a C-terminal domain corresponding to cysteine, histidine-dependent amidohydrolase/peptidase (CHAP, which demonstrated muralytic activity on a staphylococcal cell wall substrate and was lethal to S. aureus cells. We constructed N-terminal truncated forms of ORF56 and arrived at a 16-kDa protein (Lys16 that retained antistaphylococcal activity. We then generated a chimeric gene construct encoding Lys16 and a staphylococcal cell wall-binding SH3b domain. This chimeric protein (P128 showed potent antistaphylococcal activity on global clinical isolates of S. aureus including methicillin-resistant strains. In addition, P128 was effective in decolonizing rat nares of S. aureus USA300 in an experimental model. Conclusions We identified a phage K gene that encodes a protein associated with the phage tail structure. The muralytic activity of the phage K TAME was localized to the C-terminal CHAP domain. This potent antistaphylococcal TAME was combined with an efficient Staphylococcus-specific cell-wall targeting domain SH3b, resulting in the chimeric protein P128. This protein shows bactericidal activity against globally prevalent antibiotic resistant clinical isolates of S. aureus and against the genus Staphylococcus in general. In vivo, P128 was efficacious against methicillin

  16. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be...

  17. Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria Atividade de extratos vegetais e fitofármacos sobre bactérias resistentes a antibióticos

    Directory of Open Access Journals (Sweden)

    Gislene G. F. Nascimento

    2000-10-01

    Full Text Available The antimicrobial activity of plant extracts and phytochemicals was evaluated with antibiotic susceptible and resistant microorganisms. In addition, the possible synergistic effects when associated with antibiotics were studied. Extracts from the following plants were utilized: Achillea millifolium (yarrow, Caryophyllus aromaticus (clove, Melissa offficinalis (lemon-balm, Ocimun basilucum (basil, Psidium guajava (guava, Punica granatum (pomegranate, Rosmarinus officinalis (rosemary, Salvia officinalis (sage, Syzygyum joabolanum (jambolan and Thymus vulgaris (thyme. The phytochemicals benzoic acid, cinnamic acid, eugenol and farnesol were also utilized. The highest antimicrobial potentials were observed for the extracts of Caryophyllus aromaticus and Syzygyum joabolanum, which inhibited 64.2 and 57.1% of the tested microorganisms, respectively, with higher activity against antibiotic-resistant bacteria (83.3%. Sage and yarrow extracts did not present any antimicrobial activity. Association of antibiotics and plant extracts showed synergistic antibacterial activity against antibiotic-resistant bacteria. The results obtained with Pseudomonas aeruginosa was particularly interesting, since it was inhibited by clove, jambolan, pomegranate and thyme extracts. This inhibition was observed with the individual extracts and when they were used in lower concentrations with ineffective antibiotics.Foi avaliada a atividade antimicrobiana de extratos vegetais e fitofármacos frente a microrganismos sensíveis e resistentes a antibióticos, bem como observado o possível efeito sinérgico da associação entre antibióticos e extratos vegetais. Foram utilizados os extratos de plantas cujo nomes populares são: tomilho, alecrim, cravo-da-Índia, jambolão, erva cidreira, romã, goiaba, sálvia, manjericão e mil-folhas, e ainda os fitofármacos, ácido benzóico, ácido cinâmico, eugenol e farnesol. Na avaliação da atividade antimicrobiana através do m

  18. Enzyme technology: Key to selective biorefining

    DEFF Research Database (Denmark)

    Meyer, Anne S.

    2014-01-01

    to the reaction is a unique trait of enzyme catalysis. Since enzyme selectivity means that a specific reaction is catalysed between particular species to produce definite products, enzymes are particularly fit for converting specific compounds in mixed biomass streams. Since enzymes are protein molecules...... their rational use in biorefinery processes requires an understanding of the basic features of enzymes and reaction traits with respect to specificity, kinetics, reaction optima, stability and structure-function relations – we are now at a stage where it is possible to use nature’s enzyme structures as starting...... point and then improve the functional traits by targeted mutation of the protein. The talk will display some of our recent hypotheses related to enzyme action, recently obtained results within knowledge-based enzyme improvements as well as cast light on research methods used in optimizing enzyme...

  19. Consumer attitudes to enzymes in food production

    DEFF Research Database (Denmark)

    Søndergaard, Helle Alsted; Grunert, Klaus G.; Scholderer, Joachim

    2005-01-01

    The use of enzymes in food production has potential benefits for both food manufacturers and consumers. A central question is how consumers react to new ways of producing foods with enzymes. This study investigates the formation of consumer attitudes to different enzyme production methods in three...... European countries. Results show that consumers are most positive towards non-GM enzyme production methods. The enzyme production method is by far the most important factor for the formation of buying intentions compared to price and benefits. Results also show that environmental concern and attitudes...... to technological progress are the socio-political attitudes that have the highest predictive value regarding attitudes to enzyme production methods....

  20. Lignolytic Enzymes Production from Selected Mushrooms

    Directory of Open Access Journals (Sweden)

    H.M. Shantaveera Swamy

    2015-06-01

    Full Text Available In this paper, ligninase enzymes produced by selected mushrooms have been reported. We collected mushrooms from Western Ghats, most of them were edible food. Thirty samples isolated were tested using a plate assay through direct agar plate assay by using ABTS, decolourisation containing the fifteen isolates were able to decolourise the dye, indicating a lignin-degrading ability. Spectrophotometric enzyme assays from all selected isolates were carried out to examine the production of Ligninolytic enzymes (Laccase, lignin peroxidase and manganese peroxidase. Ten selected isolates produced all three kinds of enzymes tested. Lignolytic enzymes are groups of enzymes these are actively involved in bioremediation.

  1. Substrate mediated enzyme prodrug therapy.

    Directory of Open Access Journals (Sweden)

    Betina Fejerskov

    Full Text Available In this report, we detail Substrate Mediated Enzyme Prodrug Therapy (SMEPT as a novel approach in drug delivery which relies on enzyme-functionalized cell culture substrates to achieve a localized conversion of benign prodrug(s into active therapeutics with subsequent delivery to adhering cells or adjacent tissues. For proof-of-concept SMEPT, we use surface adhered micro-structured physical hydrogels based on poly(vinyl alcohol, β-glucuronidase enzyme and glucuronide prodrugs. We demonstrate enzymatic activity mediated by the assembled hydrogel samples and illustrate arms of control over rate of release of model fluorescent cargo. SMEPT was not impaired by adhering cells and afforded facile time - and dose - dependent uptake of the in situ generated fluorescent cargo by hepatic cells, HepG2. With the use of a glucuronide derivative of an anticancer drug, SN-38, SMEPT afforded a decrease in cell viability to a level similar to that achieved using parent drug. Finally, dose response was achieved using SMEPT and administration of judiciously chosen concentration of SN-38 glucuronide prodrug thus revealing external control over drug delivery using drug eluting surface. We believe that this highly adaptable concept will find use in diverse biomedical applications, specifically surface mediated drug delivery and tissue engineering.

  2. A DNA tweezer-actuated enzyme nanoreactor.

    Science.gov (United States)

    Liu, Minghui; Fu, Jinglin; Hejesen, Christian; Yang, Yuhe; Woodbury, Neal W; Gothelf, Kurt; Liu, Yan; Yan, Hao

    2013-01-01

    The functions of regulatory enzymes are essential to modulating cellular pathways. Here we report a tweezer-like DNA nanodevice to actuate the activity of an enzyme/cofactor pair. A dehydrogenase and NAD(+) cofactor are attached to different arms of the DNA tweezer structure and actuation of enzymatic function is achieved by switching the tweezers between open and closed states. The enzyme/cofactor pair is spatially separated in the open state with inhibited enzyme function, whereas in the closed state, enzyme is activated by the close proximity of the two molecules. The conformational state of the DNA tweezer is controlled by the addition of specific oligonucleotides that serve as the thermodynamic driver (fuel) to trigger the change. Using this approach, several cycles of externally controlled enzyme inhibition and activation are successfully demonstrated. This principle of responsive enzyme nanodevices may be used to regulate other types of enzymes and to introduce feedback or feed-forward control loops.

  3. Electro-ultrafiltration of industrial enzyme solutions

    DEFF Research Database (Denmark)

    Enevoldsen, Ann Dorrit; Hansen, Erik Børresen; Jonsson, Gunnar Eigil

    2007-01-01

    To reduce the problems with fouling and concentration polarization during crossflow ultrafiltration of industrial enzyme solutions an electric field is applied across the membrane. The filtration performance during electro-ultrafiltration (EUF) has been tested with several enzymes. Results show...

  4. Extracellular enzyme kinetics scale with resource availability

    Science.gov (United States)

    Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimi...

  5. The Application of Enzyme and Yeast

    OpenAIRE

    Zhao, Qing

    2012-01-01

    This bachelor’s thesis concerns the application of enzymes and yeasts for bio-industry. The purpose of this work is to understand the basic knowledge about enzyme and yeast, and meanwhile, to find out their different applications. Through comprehensive study, the knowledge was accumulated which brought a clear understanding for the enzyme structure and yeast microorganism, together with their working principles for the bioprocess. For wood-based industry, the different enzymes used in bi...

  6. Determining Enzyme Activity by Radial Diffusion

    Science.gov (United States)

    Davis, Bill D.

    1977-01-01

    Discusses advantages of radial diffusion assay in determining presence of enzyme and/or rough approximation of amount of enzyme activities. Procedures are included for the preparation of starch-agar plates, and the application and determination of enzyme. Techniques using plant materials (homogenates, tissues, ungerminated embryos, and seedlings)…

  7. Activation of interfacial enzymes at membrane surfaces

    DEFF Research Database (Denmark)

    Mouritsen, Ole G.; Andresen, Thomas Lars; Halperin, Avi;

    2006-01-01

    A host of water-soluble enzymes are active at membrane surfaces and in association with membranes. Some of these enzymes are involved in signalling and in modification and remodelling of the membranes. A special class of enzymes, the phospholipases, and in particular secretory phospholipase A2 (s...

  8. Rhamnogalacturonan I modifying enzymes: an update

    DEFF Research Database (Denmark)

    Silva, Ines R.; Jers, Carsten; Meyer, Anne S.;

    2016-01-01

    Rhamnogalacturonan I (RGI) modifying enzymes catalyse the degradation of the RGI backbone and encompass enzymes specific for either the α1,2-bond linking galacturonic acid to rhamnose or the α1,4-bond linking rhamnose to galacturonic acid in the RGI backbone. The first microbial enzyme found...

  9. 21 CFR 864.4400 - Enzyme preparations.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Enzyme preparations. 864.4400 Section 864.4400...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Specimen Preparation Reagents § 864.4400 Enzyme preparations. (a) Identification. Enzyme preparations are products that are used in the...

  10. Curious cases of the enzymes

    OpenAIRE

    Ulusu, Nuriye Nuray

    2015-01-01

    Life as we know it heavily relies on biological catalysis, in fact, in a very nonromantic version of it, life could be considered as a series of chemical reactions, regulated by the guarding principles of thermodynamics. In ancient times, a beating heart was a good sign of vitality, however, to me, it is actually the presence of active enzymes that counts. Though we do not usually pay attention, the history of enzymology is as old as humanity itself, and dates back to the ancient times. This ...

  11. Curious cases of the enzymes

    OpenAIRE

    Ulusu Nuriye Nuray

    2015-01-01

    J Med Biochem 2015; 34 (3) DOI: 10.2478/jomb-2014-0045 UDK 577. 1 : 61 ISSN 1452-8258 J Med Biochem 34: 271–281, 2015 Review article Pregledni ~lanak CURIOUS CASES OF THE ENZYMES NEOBI^NA ISTORIJA ENZIMA Nuriye Nuray Ulusu Koç University, School of Medicine, Sariyer-Istanbul, Turkey Address for correspondence: N. Nuray Ulusu, PhD Koç University School of Medicine Professor of Biochemistry Rumelifeneri Yolu Sarıyer-Istanbul – Turkey Phone: +90 (212)...

  12. Enzyme Analysis to Determine Glucose Content

    Science.gov (United States)

    Carpenter, Charles; Ward, Robert E.

    Enzyme analysis is used for many purposes in food science and technology. Enzyme activity is used to indicate adequate processing, to assess enzyme preparations, and to measure constituents of foods that are enzyme substrates. In this experiment, the glucose content of corn syrup solids is determined using the enzymes, glucose oxidase and peroxidase. Glucose oxidase catalyzes the oxidation of glucose to form hydrogen peroxide (H2O2), which then reacts with a dye in the presence of peroxidase to give a stable colored product.

  13. Direct Electron Transfer of Enzymes in a Biologically Assembled Conductive Nanomesh Enzyme Platform.

    Science.gov (United States)

    Lee, Seung-Woo; Lee, Ki-Young; Song, Yong-Won; Choi, Won Kook; Chang, Joonyeon; Yi, Hyunjung

    2016-02-24

    Nondestructive assembly of a nanostructured enzyme platform is developed in combination of the specific biomolecular attraction and electrostatic coupling for highly efficient direct electron transfer (DET) of enzymes with unprecedented applicability and versatility. The biologically assembled conductive nanomesh enzyme platform enables DET-based flexible integrated biosensors and DET of eight different enzyme with various catalytic activities.

  14. Relationship Between Enhanced Invracellular Survival Gene Mutation in Mycobacterium Tuberculosis and Aminoglycoside-resistance%结核分枝杆菌eis基因突变与氨基糖苷耐药的研究

    Institute of Scientific and Technical Information of China (English)

    黄芳; 刘家云; 马越云; 苏明权; 郝晓柯

    2011-01-01

    目的:探讨结核分枝杆菌eis基因突变与氨基糖苷耐药之间的相互关系.方法:以本室保存的35株已确定耐一线药物(异烟肼、利福平、乙胺丁醇、链霉素)的结核分支杆菌为研究对象,应用 BECTEC960测定其二线药物(阿米卡星、卡那霉素)的耐药情况,同时应用基因测序的方法测定结核分枝杆菌eis基因突变情况,分析如基因突变与氨基糖苷耐药之间的相互关系.结果:氨基糖苷耐药的部分结核分杆杆菌中.eis基因487位碱基出现突变,相应的 163 位氨基酸密码子由CGT突变为CAT,即由缬氨酸变为异亮氨酸.结论:eis基因V1631突变(缬氨酸变为异亮氨酸)可能与结核分枝杆菌耐氨基糖苷类药物有关.%Objective: To investigate the relationship between enhanced invracellular survival (eis) gene mutations and aminoglycoside-resistance in MTB. Methods: 35 strains of MTB resistant to isoniazid, rifampicin, ethambutol and streptomycin were chosen, the resistance to amikacin and kanamycin were determined by BECTEC 960, eis gene mutations were identified by PCR and sequencing. Results: There was Els gene mutation of G487Ain 4 strains and the amino acid changed from valine to isoleucine. Conclusion: The eis V163I mutation might associate with aminoglycoside-resistance in MTB.

  15. DNA-Based Enzyme Reactors and Systems

    Directory of Open Access Journals (Sweden)

    Veikko Linko

    2016-07-01

    Full Text Available During recent years, the possibility to create custom biocompatible nanoshapes using DNA as a building material has rapidly emerged. Further, these rationally designed DNA structures could be exploited in positioning pivotal molecules, such as enzymes, with nanometer-level precision. This feature could be used in the fabrication of artificial biochemical machinery that is able to mimic the complex reactions found in living cells. Currently, DNA-enzyme hybrids can be used to control (multi-enzyme cascade reactions and to regulate the enzyme functions and the reaction pathways. Moreover, sophisticated DNA structures can be utilized in encapsulating active enzymes and delivering the molecular cargo into cells. In this review, we focus on the latest enzyme systems based on novel DNA nanostructures: enzyme reactors, regulatory devices and carriers that can find uses in various biotechnological and nanomedical applications.

  16. Enzyme extraction by ultrasound from sludge flocs

    Institute of Scientific and Technical Information of China (English)

    YU Guanghui; HE Pinjing; SHAO Liming; ZHU Yishu

    2009-01-01

    Enzymes play essential roles in the biological processes of sludge treatment. In this article, the ultrasound method to extract enzymes from sludge flocs was presented. Results showed that using ultrasound method at 20 kHz could extract more types of enzymes than that ultrasound at 40 kHz and ethylenediamine tetraacetic acid (EDTA) methods. The optimum parameters of ultrasound extraction at 20 kHz were duration of 10 min and power of 480 W. Under the condition, ultrasound could break the cells and extract both the extracellular and intercellular enzymes. Ultrasound power was apparently more susceptive to enzyme extraction than duration, suggesting that the control of power during ultrasound extraction was more important than that of duration. The Pearson correlation analysis between enzyme activities and cation contents revealed that the different types of enzymes had distinct cation binding characteristics.

  17. 糖尿病足分离的铜绿假单胞菌对氨基糖苷类抗生素耐药机制探讨%Study on aminoglycoside antibiotics resistance of Pseudomonas aeruginosa isolated from diabetic foot infections

    Institute of Scientific and Technical Information of China (English)

    乌洪芳; 孙茜; 李玉珠; 张敏; 孟玲玲; 李代清

    2015-01-01

    Objective To investigate the clinical features, phenotypes and genotypes of Pseudomonas aeruginosa (PA) strains isolated from patients with diabetic foot infection (DFI) resisting to aminoglycosides antibiotics (AmAn). Methods The clinical profiles of 209 DFI patients hospitalized in the Tianjin Metabolic Diseases Hospital were collected and ana⁃lyzed. Forty-one PA strains were identified, and their antibiotic resistance profiles were obtained. The DNAs of PA isolates were extracted and applied to amplifications for several aminoglycosides modifying enzyme genes, including aac(3′)-Ⅰ, aac (3′)-Ⅱ, aac(6′)-Ⅰb, aac(6′)-Ⅱ, ant(2′′)-Ⅰand ant(3′′)-Ⅰby PCR method. Combining with the clinical features and the antibiotic resistance profiles, the relationship between genotypes and phenotypes of the PA strains was analyzed. Results Gram positive bacteria (G+) were the majority of the pathogen with 51.67%detection rate. The total detection rate of PA was 19.62%, listed as the top one pathogenic bacterium among gram negative bacteria (47.67%). There was significant difference in the ratio of ulcer area≥4 cm2 between PA group and non-PA group and G+group. There were significantly higher inci⁃dence rate of ischemic ulcer and osteomyelitis in PA group than those of G+group. There were higher clinical characteristics and ulcer depth (SAD) score, and increased hypersensitive C-reactive protein in PA group than those of G+ group. There were 30 strains of PA being resistant to AmAn (73.17%). The predominant drug resistance gene to AmAn was ant(3′′)-Ⅰ(65.85%), and aac(3′)-Ⅰgene was not found from all PA isolates. Conclusion The detection rate of PA isolated from DFI patients was higher, and patients were with the characteristics of larger, deeper and severe ischemia of ulcer area. The phe⁃nomenon of PA resistant to AmAn was more serious, and ant(3′′)-Ⅰgene identified from PA isolates was the most common resistance gene identified to Am

  18. Analysis of antibiotic-resistant gene mecA and pathogenic genes in Staphylococcus aureus%金黄色葡萄球菌耐药基因及致病毒素基因的研究

    Institute of Scientific and Technical Information of China (English)

    王凤玲; 刘静; 杨青

    2009-01-01

    目的 研究金葡菌耐药基因及致病因子中毒休克综合征毒素-Ⅰ(TSST-Ⅰ)基因和杀白细胞毒素 (PVL) 基因的分布特征.方法 收集临床分离的74株金葡菌,PCR法检测毒素基因TSST-Ⅰ、PVL和mecA耐药基因.结果 74株金葡菌 PCR法对其行mecA基因检测,检出率为55.4% (41/74).PVL阳性菌株的分离率为29.7%(22/74),PVL阳性的MRSA为15株(15/41,36.6%), PVL阳性的MSSA为7株(7/33,21.2%),差异无统计学意义(P>0.05).TSST-Ⅰ基因检出率为6.8%, MSSA中未检出TSST-Ⅰ基因.结论 MRSA呈多重耐药性,易造成医院内暴发流行,携带PVL和TSST-Ⅰ的金葡菌其致病力更强,应加强医院感染控制,防止其播散流行.%Objective To investigate the antibiotic-resistant gene mecA and the prevalence of Panton-Valentine leukocidin (PVL) gene, toxic shock syndrome toxin (TSST-Ⅰ) gene in S. aureus. Methods A total of 74 S. aureus were collected from clinical specimens. The mecA, PVL and TSST-Ⅰ genes were detected by PCR.Results PVL gene was identified in 22 S. aureus isolates. The prevalence of PVL was 29.7% in S. aureus, 36.6% in MRSA and 21.2% in MSSA. The difference was not statistically significant (P>0.05). The prevalence of MRSA was 55.4% in 74 S. aureus. The prevalence of TSST-Ⅰ gene was 6.8%. TSST-Ⅰ gene was not detected in MSSA.Conclusions MRSA strains show highly resistant to antibiotics. PVL- and TSST-Ⅰ-positive S. aureus are more pathogenic.

  19. The aminoglycoside resistance methyltransferase Sgm impedes RsmF methylation at an adjacent rRNA nucleotide in the ribosomal A site

    DEFF Research Database (Denmark)

    Cubrilo, Sonja; Babić, Fedora; Douthwaite, Stephen;

    2009-01-01

    methylated nucleotides including m(4)Cm1402 and m(5)C1407. Modification at m(5)C1407 by the methyltransferase RsmF is impeded as Sgm gains access to its adjacent G1405 target on the 30S ribosomal subunit. An Sgm mutant (G135A), which is impaired in S-adenosylmethionine binding and confers lower resistance......Ribosome-targeting antibiotics block protein synthesis by binding at functionally important regions of the bacterial rRNA. Resistance is often conferred by addition of a methyl group at the antibiotic binding site within an rRNA region that is already highly modified with several nucleotide...... methylations. In bacterial rRNA, each methylation requires its own specific methyltransferase enzyme, and this raises the question as to how an extra methyltransferase conferring antibiotic resistance can be accommodated and how it can gain access to its nucleotide target within a short and functionally...

  20. Cellulose degradation by oxidative enzymes

    Directory of Open Access Journals (Sweden)

    Maria Dimarogona

    2012-09-01

    Full Text Available Enzymatic degradation of plant biomass has attracted intensive research interest for the production of economically viable biofuels. Here we present an overview of the recent findings on biocatalysts implicated in the oxidative cleavage of cellulose, including polysaccharide monooxygenases (PMOs or LPMOs which stands for lytic PMOs, cellobiose dehydrogenases (CDHs and members of carbohydrate-binding module family 33 (CBM33. PMOs, a novel class of enzymes previously termed GH61s, boost the efficiency of common cellulases resulting in increased hydrolysis yields while lowering the protein loading needed. They act on the crystalline part of cellulose by generating oxidized and non-oxidized chain ends. An external electron donor is required for boosting the activity of PMOs. We discuss recent findings concerning their mechanism of action and identify issues and questions to be addressed in the future.

  1. Ethanologenic Enzymes of Zymomonas mobilis

    Energy Technology Data Exchange (ETDEWEB)

    Ingram, Lonnie O' Neal

    1999-03-01

    Zymomonas mobilis is a unique microorganism in being both obligately fermentative and utilizing a Entner-Doudoroff pathway for glycolysis. Glycolytic flux in this organism is readily measured as evolved carbon dioxide, ethanol, or glucose consumed and exceeds 1 {micro}mole glucose/min per mg cell protein. To support this rapid glycolysis, approximately 50% of cytoplasmic protein is devoted to the 13 glycolytic and fermentative enzymes which constitute this central catabolic pathway. Only 1 ATP (net) is produced from each glucose metabolized. During the past grant period, we have completed the characterization of 11 of the 13 glycolytic genes from Z. mobilis together with complementary but separate DOE-fimded research by a former post-dot and collaborator, Dr. Tyrrell Conway. Research funded in my lab by DOE, Division of Energy Biosciences can be divided into three sections: A. Fundamental studies; B. Applied studies and utility; and C. Miscellaneous investigations.

  2. Encapsulation of Enzymes and Peptides

    Science.gov (United States)

    Meesters, Gabrie M. H.

    A large part of formulated peptides and proteins, e.g., enzymes used as food ingredients, are formulated in a liquid form. Often, they are dissolved in water to which glycerol or sorbitol is added to reduce the water activity of the liquid, thus reducing the change of microbial growth. Still, there are reasons to formulate them in a solid form. Often, these reasons are stability, since a dry formulation is often much better than liquid formulations, and less transportation cost, since less mass is transported if one gets rid of the liquid; however, most of the times, the reason is that the product is mixed with a solid powder. Here, a liquid addition would lead to lump formation.

  3. Multi-enzyme Process Modeling

    DEFF Research Database (Denmark)

    Andrade Santacoloma, Paloma de Gracia

    The subject of this thesis is to develop a methodological framework that can systematically guide mathematical model building for better understanding of multi-enzyme processes. In this way, opportunities for process improvements can be identified by analyzing simulations of either existing...... features of the process and provides the information required to structure the process model by using a step-by-step procedure with the required tools and methods. In this way, this framework increases efficiency of the model development process with respect to time and resources needed (fast and effective...... in the scientific literature. Reliable mathematical models of such multi-catalytic schemes can exploit the potential benefit of these processes. In this way, the best outcome of the process can be obtained understanding the types of modification that are required for process optimization. An effective evaluation...

  4. Photodynamic inactivation of antibiotic-resistant pathogens

    International Nuclear Information System (INIS)

    Nowadays methicillin-resistant strain Staphylococcus aureus (MRSA) is one of the most widespread multiresistant bacteria. Photodynamic inactivation (PDI) of microorganisms by photosensitizers (PS) may be an effective and alternative therapeutic option against antibiotic resistant bacteria. The effectiveness of new PS cationic porphyrin Zn-TBut4PyP was tested on two strains of S. aureus (MRSA and methicillin-sensitive S. aureus). It is shown that Zn-TBut4PyP has high photodynamic activity against both strains

  5. Molecular dynamics investigation of the ionic liquid/enzyme interface: application to engineering enzyme surface charge.

    Science.gov (United States)

    Burney, Patrick R; Nordwald, Erik M; Hickman, Katie; Kaar, Joel L; Pfaendtner, Jim

    2015-04-01

    Molecular simulations of the enzymes Candida rugosa lipase and Bos taurus α-chymotrypsin in aqueous ionic liquids 1-butyl-3-methylimidazolium chloride and 1-ethyl-3-methylimidazolium ethyl sulfate were used to study the change in enzyme-solvent interactions induced by modification of the enzyme surface charge. The enzymes were altered by randomly mutating lysine surface residues to glutamate, effectively decreasing the net surface charge by two for each mutation. These mutations resemble succinylation of the enzyme by chemical modification, which has been shown to enhance the stability of both enzymes in ILs. After establishing that the enzymes were stable on the simulated time scales, we focused the analysis on the organization of the ionic liquid substituents about the enzyme surface. Calculated solvent charge densities show that for both enzymes and in both solvents that changing positively charged residues to negative charge does indeed increase the charge density of the solvent near the enzyme surface. The radial distribution of IL constituents with respect to the enzyme reveals decreased interactions with the anion are prevalent in the modified systems when compared to the wild type, which is largely accompanied by an increase in cation contact. Additionally, the radial dependence of the charge density and ion distribution indicates that the effect of altering enzyme charge is confined to short range (≤1 nm) ordering of the IL. Ultimately, these results, which are consistent with that from prior experiments, provide molecular insight into the effect of enzyme surface charge on enzyme stability in ILs. PMID:25641162

  6. Enzymes in Fish and Seafood Processing

    Science.gov (United States)

    Fernandes, Pedro

    2016-01-01

    Enzymes have been used for the production and processing of fish and seafood for several centuries in an empirical manner. In recent decades, a growing trend toward a rational and controlled application of enzymes for such goals has emerged. Underlying such pattern are, among others, the increasingly wider array of enzyme activities and enzyme sources, improved enzyme formulations, and enhanced requirements for cost-effective and environmentally friendly processes. The better use of enzyme action in fish- and seafood-related application has had a significant impact on fish-related industry. Thus, new products have surfaced, product quality has improved, more sustainable processes have been developed, and innovative and reliable analytical techniques have been implemented. Recent development in these fields are presented and discussed, and prospective developments are suggested. PMID:27458583

  7. Evaluation of pressure tuning of enzymes

    DEFF Research Database (Denmark)

    Naghshineh, Mahsa

    and high energy consumption. Therefore, searching for an environmentally friendly method of pectin extraction is a task for science and industry. Employment of hydrolytic enzymes may represent a green approach to obtain intact pectin polymer. However, the low stability/activity of enzymes, and low polymer...... yield of enzymatic extraction limits the application of enzyme in pectin production. There is evidence that emerging technology of high hydrostatic pressure processing can result in stabilization and activation of some enzymes. Therefore, the use of high hydrostatic pressure in combination with enzyme...... (cellulase/xylanase: 50/0, 50/25, 50/50, 25/50, and 0/50 U/g lime peel) at ambient pressure, 100 and 200 MPa were used to extract pectin from dried lime peel waste. It was found that pressure level, type and concentration of enzyme significantly influenced pectin yield and degree of esterification (DE...

  8. Prediction of Wild-type Enzyme Characteristics

    DEFF Research Database (Denmark)

    Geertz-Hansen, Henrik Marcus

    of biotechnology, including enzyme discovery and characterization. This work presents two articles on sequence-based discovery and functional annotation of enzymes in environmental samples, and two articles on analysis and prediction of enzyme thermostability and cofactor requirements. The first article presents...... a sequence-based approach to discovery of proteolytic enzymes in metagenomes obtained from the Polar oceans. We show that microorganisms living in these extreme environments of constant low temperature harbour genes encoding novel proteolytic enzymes with potential industrial relevance. The second article...... presents a web server for the processing and annotation of functional metagenomics sequencing data, tailored to meet the requirements of non-bioinformaticians. The third article presents analyses of the molecular determinants of enzyme thermostability, and a feature-based prediction method of the melting...

  9. Enzyme engineering reaches the boiling point

    OpenAIRE

    Arnold, Frances H.

    1998-01-01

    The boiled enzyme was toppled as a standard enzymology control when researchers in the 1970s started uncovering enzymes that loved the heat (1). Identification of a variety of intrinsically hyperstable enzymes from hyperthermophilic organisms, with optimal growth temperatures of 100°C and above, has piqued academic curiosity (e.g., how do these proteins withstand such ‘‘extreme’’ conditions?) and generated considerable interest for their possible applications in biotechnology (2, 3). The real...

  10. Recent advances in sulfotransferase enzyme activity assays

    OpenAIRE

    Paul, Priscilla; Suwan, Jiraporn; Liu, Jian; Dordick, Jonathan S.; Linhardt, Robert J.

    2012-01-01

    Sulfotransferases are enzymes that catalyze the transfer of sulfo groups from a donor, for example 3′-phosphoadenosine 5′-phosphosulfate, to an acceptor, for example the amino or hydroxyl groups of a small molecule, xenobiotic, carbohydrate, or peptide. These enzymes are important targets in the design of novel therapeutics for treatment of a variety of diseases. This review examines assays used for this important class of enzyme, paying particular attention to sulfotransferases acting on car...

  11. PURIFICATION OF GLUTAMINASE ENZYME PRODUCED FROM ERWINIA

    OpenAIRE

    PUSHPINDER PAUL

    2013-01-01

    The purpose of this study was to do Purification of the Glutaminase enzyme produced from free cells of Erwinia species at flask level. Glutaminase can be isolated from a number of sources such as plants, animals and microorganisms. Glutaminase is an important enzyme that serves many functions. It plays a key role in the energy and nitrogen metabolism of mammalian cells. Glutaminase is very important food enzyme used in food industries for flavor enhancement. Glutaminase, in combination with o...

  12. The mechanisms of Excited states in enzymes

    DEFF Research Database (Denmark)

    Petersen, Frederic Nicolas Rønne; Bohr, Henrik

    2010-01-01

    Enzyme catalysis is studied on the basis of excited state processes, which are of electronic, vibrational and thermal nature. The ways of achieving the excited state, such as photo-absorption and ligand binding, are discussed and exemplified by various cases of enzymes.......Enzyme catalysis is studied on the basis of excited state processes, which are of electronic, vibrational and thermal nature. The ways of achieving the excited state, such as photo-absorption and ligand binding, are discussed and exemplified by various cases of enzymes....

  13. Enzyme-based antifouling coatings: a review

    DEFF Research Database (Denmark)

    Olsen, Stefan Møller; Pedersen, Leif Toudal; Laursen, M.H.;

    2007-01-01

    for successful development of enzymatic AF coatings. The approaches described in the literature are divided into direct and indirect enzymatic AF, depending on the intended action of the enzymes. Direct antifouling is used when the enzymes themselves are active antifoulants. Indirect antifouling refers...... to the use of enzymes to release an active biocide with AF activity. For direct AF, several patents have been granted, and a commercial product has been launched. However, the achievement of an efficient broad-spectrum AF coating based on a single or a few enzymes has not yet been achieved. An indirect AF...

  14. Enzymes for Enhanced Oil Recovery (EOR)

    Energy Technology Data Exchange (ETDEWEB)

    Nasiri, Hamidreza

    2011-04-15

    Primary oil recovery by reservoir pressure depletion and secondary oil recovery by waterflooding usually result in poor displacement efficiency. As a consequence there is always some trapped oil remaining in oil reservoirs. Oil entrapment is a result of complex interactions between viscous, gravity and capillary forces. Improving recovery from hydrocarbon fields typically involves altering the relative importance of the viscous and capillary forces. The potential of many EOR methods depends on their influence on fluid/rock interactions related to wettability and fluid/fluid interactions reflected in IFT. If the method has the potential to change the interactions favorably, it may be considered for further investigation, i.e. core flooding experiment, pilot and reservoir implementation. Enzyme-proteins can be introduced as an enhanced oil recovery method to improve waterflood performance by affecting interactions at the oil-water-rock interfaces. An important part of this thesis was to investigate how selected enzymes may influence wettability and capillary forces in a crude oil-brine-rock system, and thus possibly contribute to enhanced oil recovery. To investigate further by which mechanisms selected enzyme-proteins may contribute to enhance oil recovery, groups of enzymes with different properties and catalytic functions, known to be interfacially active, were chosen to cover a wide range of possible effects. These groups include (1) Greenzyme (GZ) which is a commercial EOR enzyme and consists of enzymes and stabilizers (surfactants), (2) The Zonase group consists of two types of pure enzyme, Zonase1 and Zonase2 which are protease enzymes and whose catalytic functions are to hydrolyze (breakdown) peptide bonds, (3) The Novozyme (NZ) group consists of three types of pure enzyme, NZ2, NZ3 and NZ6 which are esterase enzymes and whose catalytic functions are to hydrolyze ester bonds, and (4) Alpha-Lactalbumin ( -La) which is an important whey protein. The effect of

  15. The Impact of Enzyme Orientation and Electrode Topology on the Catalytic Activity of Adsorbed Redox Enzymes

    Science.gov (United States)

    McMillan, Duncan G. G.; Marritt, Sophie J.; Kemp, Gemma L.; Gordon-Brown, Piers; Butt, Julea N.; Jeuken, Lars J. C.

    2014-01-01

    It is well established that the structural details of electrodes and their interaction with adsorbed enzyme influences the interfacial electron transfer rate. However, for nanostructured electrodes, it is likely that the structure also impacts on substrate flux near the adsorbed enzymes and thus catalytic activity. Furthermore, for enzymes converting macro-molecular substrates it is possible that the enzyme orientation determines the nature of interactions between the adsorbed enzyme and substrate and therefore catalytic rates. In essence the electrode may impede substrate access to the active site of the enzyme. We have tested these possibilities through studies of the catalytic performance of two enzymes adsorbed on topologically distinct electrode materials. Escherichia coli NrfA, a nitrite reductase, was adsorbed on mesoporous, nanocrystalline SnO2 electrodes. CymA from Shewanella oneidensis MR-1 reduces menaquinone-7 within 200 nm sized liposomes and this reaction was studied with the enzyme adsorbed on SAM modified ultra-flat gold electrodes. PMID:24634538

  16. The Impact of Enzyme Orientation and Electrode Topology on the Catalytic Activity of Adsorbed Redox Enzymes.

    Science.gov (United States)

    McMillan, Duncan G G; Marritt, Sophie J; Kemp, Gemma L; Gordon-Brown, Piers; Butt, Julea N; Jeuken, Lars J C

    2013-11-01

    It is well established that the structural details of electrodes and their interaction with adsorbed enzyme influences the interfacial electron transfer rate. However, for nanostructured electrodes, it is likely that the structure also impacts on substrate flux near the adsorbed enzymes and thus catalytic activity. Furthermore, for enzymes converting macro-molecular substrates it is possible that the enzyme orientation determines the nature of interactions between the adsorbed enzyme and substrate and therefore catalytic rates. In essence the electrode may impede substrate access to the active site of the enzyme. We have tested these possibilities through studies of the catalytic performance of two enzymes adsorbed on topologically distinct electrode materials. Escherichia coli NrfA, a nitrite reductase, was adsorbed on mesoporous, nanocrystalline SnO2 electrodes. CymA from Shewanella oneidensis MR-1 reduces menaquinone-7 within 200 nm sized liposomes and this reaction was studied with the enzyme adsorbed on SAM modified ultra-flat gold electrodes.

  17. Endotoxin contamination of enzyme conjugates used in enzyme-linked immunosorbent assays.

    OpenAIRE

    Bryant, R. E.; Chamovitz, B N; Morse, S A; Apicella, M A; Morthland, V H

    1983-01-01

    The specificity of the enzyme-linked immunosorbent assay(s) is thought to depend on the specificity of the antibody used in the assay system. Therefore, the association of broadly reactive antigens like endotoxin with enzyme conjugates or other enzyme-linked immunosorbent assay reagents has the potential of altering the specificity of reactions in the enzyme-linked immunosorbent assay. Using the Limulus amoebocyte lysate assay, we demonstrated that commercially prepared conjugates of goat ant...

  18. Immobilization to prevent enzyme incompatibility with proteases

    NARCIS (Netherlands)

    Vossenberg, P.; Beeftink, H.H.; Cohen Stuart, M.A.; Tramper, J.

    2011-01-01

    Enzyme incompatibility is a problem in multi-enzyme processes that involve a non-specific protease, such as Alcalase. An example is the one-pot enzymatic synthesis of peptides catalyzed by a lipase and a protease. The incompatibility between lipase B from Candida antarctica (CalB) and Alcalase was s

  19. The use of enzymes for beer brewing

    NARCIS (Netherlands)

    Donkelaar, van Laura H.G.; Mostert, Joost; Zisopoulos, Filippos K.; Boom, Remko M.; Goot, van der Atze Jan

    2016-01-01

    The exergetic performance of beer produced by the conventional malting and brewing process is compared with that of beer produced using an enzyme-assisted process. The aim is to estimate if the use of an exogenous enzyme formulation reduces the environmental impact of the overall brewing process. Th

  20. Enzyme Activity Experiments Using a Simple Spectrophotometer

    Science.gov (United States)

    Hurlbut, Jeffrey A.; And Others

    1977-01-01

    Experimental procedures for studying enzyme activity using a Spectronic 20 spectrophotometer are described. The experiments demonstrate the effect of pH, temperature, and inhibitors on enzyme activity and allow the determination of Km, Vmax, and Kcat. These procedures are designed for teaching large lower-level biochemistry classes. (MR)

  1. A toy quantum analog of enzymes

    CERN Document Server

    Svetlichny, George

    2015-01-01

    We present a quantum system incorporating qualitative aspects of enzyme action in which the possibility of quantum superposition of several conformations of the enzyme-substrate complex is investigated. We present numerical results showing quantum effects that transcend the case of a statistical mixture of conformations.

  2. Orphan enzymes in ether lipid metabolism.

    Science.gov (United States)

    Watschinger, Katrin; Werner, Ernst R

    2013-01-01

    Ether lipids are an emerging class of lipids which have so far not been investigated and understood in every detail. They have important roles as membrane components of e.g. lens, brain and testis, and as mediators such as platelet-activating factor. The metabolic enzymes for biosynthesis and degradation have been investigated to some extent. As most involved enzymes are integral membrane proteins they are tricky to handle in biochemical protocols. The sequence of some ether lipid metabolising enzymes has only recently been reported and other sequences still remain obscure. Defined enzymes without assigned sequence are known as orphan enzymes. One of these enzymes with uncharacterised sequence is plasmanylethanolamine desaturase, a key enzyme for the biosynthesis of one of the most abundant phospholipids in our body, the plasmalogens. This review aims to briefly summarise known functions of ether lipids, give an overview on their metabolism including the most prominent members, platelet-activating factor and the plasmalogens. A special focus is set on the description of orphan enzymes in ether lipid metabolism and on the successful strategies how four previous orphans have recently been assigned a sequence. Only one of these four was characterised by classical protein purification and sequencing, whereas the other three required alternative strategies such as bioinformatic candidate gene selection and recombinant expression or development of an inhibitor and multidimensional metabolic profiling.

  3. Cytochrome P450 enzyme systems in fungi

    NARCIS (Netherlands)

    Brink, H.M. van den; Gorcom, R.F.M. van; Hondel, C.A.M.J.J. van den; Punt, P.J.

    1998-01-01

    The involvement of cytochrome P450 enzymes in many complex fungal bioconversion processes has been characterized in recent years. Accordingly, there is now considerable scientific interest in fungal cytochrome P450 enzyme systems. In contrast to S. cerevisiae, where surprisingly few P450 genes have

  4. Enzyme Catalysis and the Gibbs Energy

    Science.gov (United States)

    Ault, Addison

    2009-01-01

    Gibbs-energy profiles are often introduced during the first semester of organic chemistry, but are less often presented in connection with enzyme-catalyzed reactions. In this article I show how the Gibbs-energy profile corresponds to the characteristic kinetics of a simple enzyme-catalyzed reaction. (Contains 1 figure and 1 note.)

  5. Enzyme adsorption at solid-liquid interfaces.

    NARCIS (Netherlands)

    Duinhoven, S.

    1992-01-01

    Enzymes are proteins with the capacity of catalysing various reactions. Nowadays two types of enzymes, proteases and lipases, are available for use in detergent formulations for household and industrial laundry washing. Proteases are capable of catalysing the hydrolysis of proteins while lipases ena

  6. New rhamnogalacturonan degrading enzymes from Aspergillus aculeatus.

    NARCIS (Netherlands)

    Mutter, M.

    1997-01-01

    Three new  rhamnogalacturonan degrading enzymes were purified from a commercial enzyme preparation, Pectinex Ultra SP, produced by the fungus Aspergillus aculeatus . Pectinex Ultra SP is industrially used in the mash treatment of apples and pears in juice production, increasing juice yield. Rhamnoga

  7. Illustrating Enzyme Inhibition Using Gibbs Energy Profiles

    Science.gov (United States)

    Bearne, Stephen L.

    2012-01-01

    Gibbs energy profiles have great utility as teaching and learning tools because they present students with a visual representation of the energy changes that occur during enzyme catalysis. Unfortunately, most textbooks divorce discussions of traditional kinetic topics, such as enzyme inhibition, from discussions of these same topics in terms of…

  8. Biocatalytic material comprising multilayer enzyme coated fiber

    Science.gov (United States)

    Kim, Jungbae [Richland, WA; Kwak, Ja Hun [Richland, WA; Grate, Jay W [West Richland, WA

    2009-11-03

    The present invention relates generally to high stability, high activity biocatalytic materials and processes for using the same. The materials comprise enzyme aggregate coatings having high biocatalytic activity and stability useful in heterogeneous environment. These new materials provide a new biocatalytic immobilized enzyme system with applications in bioconversion, bioremediation, biosensors, and biofuel cells.

  9. Application of radiopolymerization for immobilization of enzymes

    International Nuclear Information System (INIS)

    Hydrophilic glass-forming monomers were used in an application of irradiation technology for the immobilization of cellulase and cellobiase. Experiments to observe the effect of additives such as silicates and polyethylene glycol in the enzyme entrapment are reported on. In all cases, enzymatic activity was maintained for more than fifteen batch enzyme reactions. (Author)

  10. Enzyme Activity of Cenococcum geophilum Isolates on Enzyme-specific Solid Media

    OpenAIRE

    Obase, Keisuke; Lee, Sang Yong; Chun, Kun Woo; Lee, Jong Kyu

    2011-01-01

    Enzyme activities of Cenococcum geophilum isolates were examined on enzyme-specific solid media. Deoxyribonuclease, phosphatase, and urease were detected in all isolates, whereas cellulase was not detected in any of the isolates. Variations in enzyme activities of amylase, caseinolysis, gelatinase, lipase, and ribonuclease were observed among isolates.

  11. Enzyme activity assay of glycoprotein enzymes based on a boronate affinity molecularly imprinted 96-well microplate.

    Science.gov (United States)

    Bi, Xiaodong; Liu, Zhen

    2014-12-16

    Enzyme activity assay is an important method in clinical diagnostics. However, conventional enzyme activity assay suffers from apparent interference from the sample matrix. Herein, we present a new format of enzyme activity assay that can effectively eliminate the effects of the sample matrix. The key is a 96-well microplate modified with molecularly imprinted polymer (MIP) prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting. Alkaline phosphatase (ALP), a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was taken as a representative target enzyme. The prepared MIP exhibited strong affinity toward the template enzyme (with a dissociation constant of 10(-10) M) as well as superb tolerance for interference. Thus, the enzyme molecules in a complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay, which eliminated the interference from the sample matrix. On the other hand, because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes, the enzyme captured by the MIP was directly used for activity assay. Thus, additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided. Assay of ALP in human serum was successfully demonstrated, suggesting a promising prospect of the proposed method in real-world applications.

  12. Enzyme-Immobilized Microfluidic Process Reactors

    Directory of Open Access Journals (Sweden)

    Hideaki Maeda

    2011-07-01

    Full Text Available Microreaction technology, which is an interdisciplinary science and engineering area, has been the focus of different fields of research in the past few years. Several microreactors have been developed. Enzymes are a type of catalyst, which are useful in the production of substance in an environmentally friendly way, and they also have high potential for analytical applications. However, not many enzymatic processes have been commercialized, because of problems in stability of the enzymes, cost, and efficiency of the reactions. Thus, there have been demands for innovation in process engineering, particularly for enzymatic reactions, and microreaction devices represent important tools for the development of enzyme processes. In this review, we summarize the recent advances of microchannel reaction technologies especially for enzyme immobilized microreactors. We discuss the manufacturing process of microreaction devices and the advantages of microreactors compared to conventional reaction devices. Fundamental techniques for enzyme immobilized microreactors and important applications of this multidisciplinary technology are also included in our topics.

  13. Directed Evolution of Enzymes for Industrial Biocatalysis.

    Science.gov (United States)

    Porter, Joanne L; Rusli, Rukhairul A; Ollis, David L

    2016-02-01

    Enzymes have the potential to catalyse a wide variety of chemical reactions. They are increasingly being sought as environmentally friendly and cost-effective alternatives to conventional catalysts used in industries ranging from bioremediation to applications in medicine and pharmaceutics. Despite the benefits, they are not without their limitations. Many naturally occurring enzymes are not suitable for use outside of their native cellular environments. However, protein engineering can be used to generate enzymes tailored for specific industrial applications. Directed evolution is particularly useful and can be employed even when lack of structural information impedes the use of rational design. The aim of this review is to provide an overview of current industrial applications of enzyme technology and to show how directed evolution can be used to modify and to enhance enzyme properties. This includes a brief discussion on library generation and a more detailed focus on library screening methods, which are critical to any directed evolution experiment.

  14. Evaluation of thermostable enzymes for bioethanol processing

    DEFF Research Database (Denmark)

    Skovgaard, Pernille Anastasia

    of fermentable sugars (glucose) as cellulose is tightly linked to hemicellulose and lignin. Lignocellulose is disrupted during pretreatment, but to degrade cellulose to single sugars, lignocellulolytic enzymes such as cellulases and hemicellulases are needed. Lignocellulolytic enzymes are costly...... for the ioethanol production, but the expenses can be reduced by using thermostable enzymes, which are known for their increased stability and inhibitor olerance. However, the advantage of using thermostable enzymes has not been studied thoroughly and more knowledge is needed for development of bioethanol processes....... Enzymes are added to the bioethanol process after pretreatment. For an efficient sugar and ethanol yield, the solids content of biomass is normally increased, which results in highly viscous slurries that are difficult to mix. Therefore, the first enzymatic challenge is to ensure rapid reduction...

  15. Production of cellulolytic enzymes from ascomycetes

    DEFF Research Database (Denmark)

    Hansen, Gustav Hammerich; Lübeck, Mette; Frisvad, Jens Christian;

    2015-01-01

    Optimizing production of cellulose degrading enzymes is of great interest in order to increase the feasibility of constructing biorefinery facilities for a sustainable supply of energy and chemical products. The ascomycete phylum has a large potential for the production of cellulolytic enzymes....... Although numerous enzymatic profiles have already been unraveled, the research has been covering only a limited number of species and genera, thus leaving many ascomycetes to be analyzed. Such analysis requires choosing appropriate media and cultivation methods that ensure enzyme profiles with high...... specificities and activities. However, the choice of media, cultivation methods and enzyme assays highly affect the enzyme activity profile observed. This review provides an overview of enzymatic profiles for several ascomycetes covering phylogenetically distinct genera and species. The profiles of cellulose...

  16. Highly efficient enzyme encapsulation in a protein nanocage: towards enzyme catalysis in a cellular nanocompartment mimic.

    Science.gov (United States)

    Schoonen, Lise; Nolte, Roeland J M; van Hest, Jan C M

    2016-08-14

    The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions. PMID:27407020

  17. Metagenomics as a Tool for Enzyme Discovery: Hydrolytic Enzymes from Marine-Related Metagenomes.

    Science.gov (United States)

    Popovic, Ana; Tchigvintsev, Anatoly; Tran, Hai; Chernikova, Tatyana N; Golyshina, Olga V; Yakimov, Michail M; Golyshin, Peter N; Yakunin, Alexander F

    2015-01-01

    This chapter discusses metagenomics and its application for enzyme discovery, with a focus on hydrolytic enzymes from marine metagenomic libraries. With less than one percent of culturable microorganisms in the environment, metagenomics, or the collective study of community genetics, has opened up a rich pool of uncharacterized metabolic pathways, enzymes, and adaptations. This great untapped pool of genes provides the particularly exciting potential to mine for new biochemical activities or novel enzymes with activities tailored to peculiar sets of environmental conditions. Metagenomes also represent a huge reservoir of novel enzymes for applications in biocatalysis, biofuels, and bioremediation. Here we present the results of enzyme discovery for four enzyme activities, of particular industrial or environmental interest, including esterase/lipase, glycosyl hydrolase, protease and dehalogenase.

  18. Bacterial enzymes involved in lignin degradation.

    Science.gov (United States)

    de Gonzalo, Gonzalo; Colpa, Dana I; Habib, Mohamed H M; Fraaije, Marco W

    2016-10-20

    Lignin forms a large part of plant biomass. It is a highly heterogeneous polymer of 4-hydroxyphenylpropanoid units and is embedded within polysaccharide polymers forming lignocellulose. Lignin provides strength and rigidity to plants and is rather resilient towards degradation. To improve the (bio)processing of lignocellulosic feedstocks, more effective degradation methods of lignin are in demand. Nature has found ways to fully degrade lignin through the production of dedicated ligninolytic enzyme systems. While such enzymes have been well thoroughly studied for ligninolytic fungi, only in recent years biochemical studies on bacterial enzymes capable of lignin modification have intensified. This has revealed several types of enzymes available to bacteria that enable them to act on lignin. Two major classes of bacterial lignin-modifying enzymes are DyP-type peroxidases and laccases. Yet, recently also several other bacterial enzymes have been discovered that seem to play a role in lignin modifications. In the present review, we provide an overview of recent advances in the identification and use of bacterial enzymes acting on lignin or lignin-derived products. PMID:27544286

  19. Enzyme immobilization by means of ultrafiltration techniques.

    Science.gov (United States)

    Scardi, V; Cantarella, M; Gianfreda, L; Palescandolo, R; Alfani, F; Greco, G

    1980-01-01

    Unstirred, plane membrane, ultrafiltration cells have been used as enzymatic reactor units. Because of the concentration polarization phenomena which take place in the system, at steady-state the enzyme is confined (dynamically immobilized) within an extremely narrow region upstream the ultrafiltration membrane. Correspondingly its concentration attains fairly high values. Kinetic studies have been therefore performed under quite unusual experimental conditions in order to better approximate local enzyme concentration levels in immobilized enzyme systems. Studies have been also carried out on the kinetics of enzyme deactivation in the continuous presence of substrate and reaction products. Once the enzyme concentration profile is completely developed, further injection into the system of suitable amounts of an inert proteic macromolecule (albumin polymers) gives rise to the formation of a gel layer onto the ultrafiltration membrane within which the enzyme is entrapped (statically immobilized). The effect of this immobilization technique has been studied as far as the kinetics of the main reaction, the substrate mass transfer resistances and the enzyme stability are concerned. The rejective properties of such gel layers towards enzymatic molecules have been exploited in producing multilayer, multi-enzymatic reactors. PMID:7417597

  20. Modeling amperometric biosensors based on allosteric enzymes

    Directory of Open Access Journals (Sweden)

    Liutauras Ričkus

    2013-09-01

    Full Text Available Computational modeling of a biosensor with allosteric enzyme layer was investigated in this study. The operation of the biosensor is modeled using non-stationary reaction-diffusion equations. The model involves three regions: the allosteric enzyme layer where the allosteric enzyme reactions as well as then mass transport by diffusion take place, the diffusion region where the mass transport by diffusion and non-enzymatic reactions take place and the convective region in which the analyte concentration is maintained constant. The biosensor response on dependency substrate concentration, cooperativity coefficient and the diffusion layer thickness on the same parameters have been studied.

  1. Dimeric assembly of enterocyte brush border enzymes

    DEFF Research Database (Denmark)

    Danielsen, E M

    1994-01-01

    The noncovalent, dimeric assembly of small intestinal brush border enzymes was studied by sedimentation analysis in density gradients of extracts of pulse-labeled pig jejunal mucosal explants. Like aminopeptidase N (EC 3.4.11.2), sucrase-isomaltase (EC 3.2.1.48-10), aminopeptidase A (EC 3...... appearance of the liposome-reconstituted enzyme [Norén et al. (1986) J. Biol. Chem. 261, 12306-12309], showing only the inner, membrane-anchored domains of the monomers to be in close contact with one another while the outer domains are far apart. In contrast to the other brush border enzymes studied...

  2. Process for preparing multilayer enzyme coating on a fiber

    Science.gov (United States)

    Kim, Jungbae; Kwak, Ja Hun; Grate, Jay W.

    2009-11-03

    A process for preparing high stability, high activity biocatalytic materials is disclosed and processes for using the same. The process involves coating of a material or fiber with enzymes and enzyme aggregate providing a material or fiber with high biocatalytic activity and stability useful in heterogeneous environments. In one illustrative approach, enzyme "seeds" are covalently attached to polymer nanofibers followed by treatment with a reagent that crosslinks additional enzyme molecules to the seed enzymes forming enzyme aggregates thereby improving biocatalytic activity due to increased enzyme loading and enzyme stability. This approach creates a useful new biocatalytic immobilized enzyme system with potential applications in bioconversion, bioremediation, biosensors, and biofuel cells.

  3. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Science.gov (United States)

    2010-04-01

    ... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red blood cell enzyme assay. 864.7100 Section 864... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme...

  4. Potato Peroxidase for the Study of Enzyme Properties.

    Science.gov (United States)

    Shamaefsky, Brian R.

    1993-01-01

    Explains how the surface of a freshly sliced potato can be used for a variety of enzyme action experiments including the influence of pH on enzyme action, the enzyme denaturation potential of boiling water, the inhibition of enzymes by heavy metals, and the effects of salt concentration on enzyme effectiveness. (PR)

  5. Highly efficient enzyme encapsulation in a protein nanocage: towards enzyme catalysis in a cellular nanocompartment mimic

    Science.gov (United States)

    Schoonen, Lise; Nolte, Roeland J. M.; van Hest, Jan C. M.

    2016-07-01

    The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions.The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions. Electronic supplementary information (ESI) available: Experimental procedures for the cloning, expression, and purification of all proteins, as well as supplementary figures and calculations. See DOI: 10.1039/c6nr04181g

  6. ZnO-Based Amperometric Enzyme Biosensors

    Directory of Open Access Journals (Sweden)

    Helong Jiang

    2010-02-01

    Full Text Available Nanostructured ZnO with its unique properties could provide a suitable microenvironment for immobilization of enzymes while retaining their biological activity, and thus lead to an expanded use of this nanomaterial for the construction of electrochemical biosensors with enhanced analytical performance. ZnO-based enzyme electrochemical biosensors are summarized in several tables for an easy overview according to the target biosensing analyte (glucose, hydrogen peroxide, phenol and cholesterol, respectively. Moreover, recent developments in enzyme electrochemical biosensors based on ZnO nanomaterials are reviewed with an emphasis on the fabrications and features of ZnO, approaches for biosensor construction (e.g., modified electrodes and enzyme immobilization and biosensor performances.

  7. Novel enzymes for the degradation of cellulose

    Directory of Open Access Journals (Sweden)

    Horn Svein

    2012-07-01

    Full Text Available Abstract The bulk terrestrial biomass resource in a future bio-economy will be lignocellulosic biomass, which is recalcitrant and challenging to process. Enzymatic conversion of polysaccharides in the lignocellulosic biomass will be a key technology in future biorefineries and this technology is currently the subject of intensive research. We describe recent developments in enzyme technology for conversion of cellulose, the most abundant, homogeneous and recalcitrant polysaccharide in lignocellulosic biomass. In particular, we focus on a recently discovered new type of enzymes currently classified as CBM33 and GH61 that catalyze oxidative cleavage of polysaccharides. These enzymes promote the efficiency of classical hydrolytic enzymes (cellulases by acting on the surfaces of the insoluble substrate, where they introduce chain breaks in the polysaccharide chains, without the need of first “extracting” these chains from their crystalline matrix.

  8. Enzymes: The possibility of production and applications

    Directory of Open Access Journals (Sweden)

    Petronijević Živomir B.

    2003-01-01

    Full Text Available Enzymes are biological catalysts with increasing application in the food pharmaceutical, cosmetic, textile and chemical industry. They are also important as reagents in chemical analysis, leather fabrications and as targets for the design of new drugs. Keeping in mind the growing need to replace classical chemical processes by alternative ones, because of ever growing environmental pollution, it is important that enzyme and other biotechnological processes are economical. Therefore, price decrease and stability and enzyme preparation efficiency increase are required more and more. This paper presents a short review of methods for yield increase and the improvement of the quality of enzyme products as commercial products, as well as a review of the possibilities of their application.

  9. Archaeal Enzymes and Applications in Industrial Biocatalysts.

    Science.gov (United States)

    Littlechild, Jennifer A

    2015-01-01

    Archaeal enzymes are playing an important role in industrial biotechnology. Many representatives of organisms living in "extreme" conditions, the so-called Extremophiles, belong to the archaeal kingdom of life. This paper will review studies carried by the Exeter group and others regarding archaeal enzymes that have important applications in commercial biocatalysis. Some of these biocatalysts are already being used in large scale industrial processes for the production of optically pure drug intermediates and amino acids and their analogues. Other enzymes have been characterised at laboratory scale regarding their substrate specificity and properties for potential industrial application. The increasing availability of DNA sequences from new archaeal species and metagenomes will provide a continuing resource to identify new enzymes of commercial interest using both bioinformatics and screening approaches.

  10. PURIFICATION OF CATALASE ENZYME FROM PLEUROTUS OSTREATUS

    Directory of Open Access Journals (Sweden)

    Susmitha.S

    2014-03-01

    Full Text Available The oyster mushroom Pleurotus ostreatus is the most commonly cultivated mushroom, and are effective for antitumor, antibacterial, anti viral and hematological agents and in immune modulating treatments. Several compounds from oyster mushrooms, potentially beneficial for human health have been isolated and studied. The aim of this research is to purify an enzyme catalase from Pleurotus ostreatus through Sephadox G-75 column, its molecular weight was determined by polyacrylamide gel electrophoresis and the catalase enzyme stability were observed at various temperature and different pH condition. Under denaturing conditions, polyacrylamide gel electrophoresis revealed dissociation of a major component of molecular weight 62,000 kDa, which constituted 90% of the total protein of the stained gel, suggesting that the native enzyme is tetrameric. The optimum temperature and pH for the purified enzyme catalase from Pleurotus ostreatus enzymatic reaction were 30°C and pH 7.5.

  11. Enzyme clustering can induce metabolic channeling

    Science.gov (United States)

    Castellana, Michele

    2015-03-01

    Direct channeling of intermediates via a physical tunnel between enzyme active sites is an established mechanism to improve metabolic efficiency. In this talk, I will present a theoretical model that demonstrates that coclustering multiple enzymes into proximity can yield the full efficiency benefits of direct channeling. The model predicts the separation and size of coclusters that maximize metabolic efficiency, and this prediction is in agreement with the spacing between coclusters in yeast and mammalian cells. The model also predicts that enzyme agglomerates can regulate steady-state flux division at metabolic branch points: we experimentally test this prediction for a fundamental branch point in Escherichia coli, and the results confirm that enzyme colocalization within an agglomerate can accelerate the processing of a shared intermediate by one branch. Our studies establish a quantitative framework to understand coclustering-mediated metabolic channeling and its application to both efficiency improvement and metabolic regulation.

  12. Archaeal Enzymes and Applications in Industrial Biocatalysts

    Directory of Open Access Journals (Sweden)

    Jennifer A. Littlechild

    2015-01-01

    Full Text Available Archaeal enzymes are playing an important role in industrial biotechnology. Many representatives of organisms living in “extreme” conditions, the so-called Extremophiles, belong to the archaeal kingdom of life. This paper will review studies carried by the Exeter group and others regarding archaeal enzymes that have important applications in commercial biocatalysis. Some of these biocatalysts are already being used in large scale industrial processes for the production of optically pure drug intermediates and amino acids and their analogues. Other enzymes have been characterised at laboratory scale regarding their substrate specificity and properties for potential industrial application. The increasing availability of DNA sequences from new archaeal species and metagenomes will provide a continuing resource to identify new enzymes of commercial interest using both bioinformatics and screening approaches.

  13. Microbial Enzymes: Tools for Biotechnological Processes

    Directory of Open Access Journals (Sweden)

    Jose L. Adrio

    2014-01-01

    Full Text Available Microbial enzymes are of great importance in the development of industrial bioprocesses. Current applications are focused on many different markets including pulp and paper, leather, detergents and textiles, pharmaceuticals, chemical, food and beverages, biofuels, animal feed and personal care, among others. Today there is a need for new, improved or/and more versatile enzymes in order to develop more novel, sustainable and economically competitive production processes. Microbial diversity and modern molecular techniques, such as metagenomics and genomics, are being used to discover new microbial enzymes whose catalytic properties can be improved/modified by different strategies based on rational, semi-rational and random directed evolution. Most industrial enzymes are recombinant forms produced in bacteria and fungi.

  14. Supramolecular Tectonics for Enzyme-like Reagents

    Institute of Scientific and Technical Information of China (English)

    MAO; LuYuan

    2001-01-01

    The enzyme-likes and bioactive species were closely related with the life phenomena and served as the reagent of bioassy1,2. In present works, the flow cytometry (FCM) and rapid-scanning stopped-flow (RSSF) spectroscopy combine with the stopped-flow difference UV/Vis spectra, FT-IR and other methods of assay, being used to study the biomimetic reaction and enzyme mimic. Based on catalytic kinetics of enzyme reaction3,4, the reaction mechanisms of the enzyme-likes had been studied and some new methods of kinetic determination were proposed. The study and methods not only provided the basic theoretical models for the life science, but also widened the application fields of biomimetic and analytical chemistry. The main contents of our works and the supramolecular models can be described as follows:  ……

  15. Microbial enzymes: tools for biotechnological processes.

    Science.gov (United States)

    Adrio, Jose L; Demain, Arnold L

    2014-01-16

    Microbial enzymes are of great importance in the development of industrial bioprocesses. Current applications are focused on many different markets including pulp and paper, leather, detergents and textiles, pharmaceuticals, chemical, food and beverages, biofuels, animal feed and personal care, among others. Today there is a need for new, improved or/and more versatile enzymes in order to develop more novel, sustainable and economically competitive production processes. Microbial diversity and modern molecular techniques, such as metagenomics and genomics, are being used to discover new microbial enzymes whose catalytic properties can be improved/modified by different strategies based on rational, semi-rational and random directed evolution. Most industrial enzymes are recombinant forms produced in bacteria and fungi.

  16. Enzyme conductometric biosensor for maltose determination

    OpenAIRE

    Dzyadevych S. V.; Soldatkin O. O.; Saiapina O. Y.; Pyeshkova V. M.

    2009-01-01

    Aim. To develop enzyme conductometric biosensor for maltose determination. Methods. A conductometric transducer consisting of two gold pairs of electrodes was applied. Three-enzyme membrane (glucose oxidase, mutarotase, -glucosidase) immobilized on the surface of the conductometric transducer was used as a bioselective element. Results. A linear range of maltose conductometric biosensor was from 0,002 mM to 1 mM for glucose and maltose detection. The time of maltose analysis in solution was 1...

  17. ZnO-Based Amperometric Enzyme Biosensors

    OpenAIRE

    Helong Jiang; Baoping Wang; Xiaobing Zhang; Zhiwei Zhao; Wei Lei

    2010-01-01

    Nanostructured ZnO with its unique properties could provide a suitable microenvironment for immobilization of enzymes while retaining their biological activity, and thus lead to an expanded use of this nanomaterial for the construction of electrochemical biosensors with enhanced analytical performance. ZnO-based enzyme electrochemical biosensors are summarized in several tables for an easy overview according to the target biosensing analyte (glucose, hydrogen peroxide, phenol and cholesterol)...

  18. Enzymes improve ECF bleaching of pulp

    OpenAIRE

    Lachenal, D.; Bajpai, P. K.; S P Mishra; Sharma, N.; Anand, A; Bajpai, P.

    2006-01-01

    The delignification efficiency of different laccase enzymes was examined on the eucalyptus Kraft pulp. The laccase enzyme from Trametes versicolor showing the highest delignification efficiency was selected and used in the elemental chlorine-free bleaching sequence for improving the pulp bleachability. An appreciable reduction in chlorine dioxide consumption was also obtained. Further reduction in chlorine dioxide consumption was obtained when the same laccase treated pulp was subjected to an...

  19. Production of Protease Enzyme from Wheat Straw

    OpenAIRE

    Mohammed A. Atiya

    2008-01-01

    Protease enzyme production was studied and optimized as a first step to collect information about solid state fermenter) to produce protease enzyme. A local isolated Aspergillus niger was used for this study with constant spores feeding in every experiment at (105/g). Experiments carried out in conical flasks with (250 ml) containing (10 g) of wheat straw as a substrate with different conditions included temperature, pH, hydration ratio, and fermentation time, the results comprised by measuri...

  20. Enzyme Scouring of Cotton Fabrics: A Review

    OpenAIRE

    Mojsov, Kiro

    2012-01-01

    Textile processing is a growing industry that traditionally has used a lot of water, energy and harsh chemicals. Due to the ever-growing costs for water and energy worldwide investigations are carriedout to substitute conventional chemical textile processes by environment-friendly andeconomically attractive bioprocesses using enzymes. Enzymes are used in a broad range of processes in the textileindustry: scouring, bleachclean-up, desizing, denim abrasion andpolishing. The conventional scourin...

  1. Enzymes in textile industry: a review

    OpenAIRE

    Mojsov, Kiro

    2014-01-01

    Textile processing is a growing industry that traditionally has used a lot of water, energy and harsh chemicals. Due to the ever-growing costs for water and energy worldwide investigations are carried out to substitute conventional chemical textile processes by environment-friendly and economically attractive bioprocesses using enzymes. The enzymes used in the textile field are amylases, catalase, and laccase which are used to removing the starch, degrading excess hydrogen peroxide, bleaching...

  2. Measuring enzyme activity in single cells

    OpenAIRE

    Kovarik, Michelle L.; Allbritton, Nancy L.

    2011-01-01

    Seemingly identical cells can differ in their biochemical state, function and fate, and this variability plays an increasingly recognized role in organism-level outcomes. Cellular heterogeneity arises in part from variation in enzyme activity, which results from interplay between biological noise and multiple cellular processes. As a result, single-cell assays of enzyme activity, particularly those that measure product formation directly, are crucial. Recent innovations have yielded a range o...

  3. Lignolytic Enzymes Production from Selected Mushrooms

    OpenAIRE

    H.M. Shantaveera Swamy; Ramalingappa

    2015-01-01

    In this paper, ligninase enzymes produced by selected mushrooms have been reported. We collected mushrooms from Western Ghats, most of them were edible food. Thirty samples isolated were tested using a plate assay through direct agar plate assay by using ABTS, decolourisation containing the fifteen isolates were able to decolourise the dye, indicating a lignin-degrading ability. Spectrophotometric enzyme assays from all selected isolates were carried out to examine the production of Ligninoly...

  4. Microbial Enzymes: Tools for Biotechnological Processes

    OpenAIRE

    Jose L. Adrio; Demain, Arnold L.

    2014-01-01

    Microbial enzymes are of great importance in the development of industrial bioprocesses. Current applications are focused on many different markets including pulp and paper, leather, detergents and textiles, pharmaceuticals, chemical, food and beverages, biofuels, animal feed and personal care, among others. Today there is a need for new, improved or/and more versatile enzymes in order to develop more novel, sustainable and economically competitive production processes. Microbial diversity an...

  5. Building proficient enzymes with foldamer prostheses.

    Science.gov (United States)

    Mayer, Clemens; Müller, Manuel M; Gellman, Samuel H; Hilvert, Donald

    2014-07-01

    Foldamers are non-natural oligomers that adopt stable conformations reminiscent of those found in proteins. To evaluate the potential of foldameric subunits for catalysis, semisynthetic enzymes containing foldamer fragments constructed from α- and β-amino acid residues were designed and characterized. Systematic variation of the α→β substitution pattern and types of β-residue afforded highly proficient hybrid catalysts, thus demonstrating the feasibility of expanding the enzyme-engineering toolkit with non-natural backbones.

  6. Controlled enzyme catalyzed heteropolysaccharide degradation:Xylans

    OpenAIRE

    Rasmussen, Louise Enggaard; Meyer, Anne S.

    2011-01-01

    The work presented in this PhD thesis has provided a better understanding of the enzyme kinetics and quantitative phenomena of the hydrolysis of xylan substrates by selected pure enzyme preparations. Furthermore, the options for producing specific substituted xylooligosaccharides from selected substrates by specific xylanase treatment have been examined. The kinetics of the enzymatic degradation of water-extractable wheat arabinoxylan (WE-AX) during designed treatments with selected monocompo...

  7. Controlling reaction specificity in pyridoxal phosphate enzymes

    OpenAIRE

    Michael D Toney

    2011-01-01

    Pyridoxal 5'-phosphate enzymes are ubiquitous in the nitrogen metabolism of all organisms. They catalyze a wide variety of reactions including racemization, transamination, decarboxylation, elimination, retro-aldol cleavage, Claisen condensation, and others on substrates containing an amino group, most commonly α-amino acids. The wide variety of reactions catalyzed by PLP enzymes is enabled by the ability of the covalent aldimine intermediate formed between substrate and PLP to stabilize carb...

  8. Enzymes as catalysts in polymer chemistry

    OpenAIRE

    Sinigoi, Loris

    2011-01-01

    The use of enzymes in synthetic chemistry is attracting the interest of many researchers thanks to their extraordinary efficiency under mild conditions, high stereo- regio- and chemoselectivity and low environmental impact. Their application in the field of polymer chemistry has provided new synthetic strategies for useful polymers. The advantages coming from the use of enzymes are mainly: i) the possibility to synthesize polymers with novel properties and difficult to produce by conventional...

  9. Semisupervised Gaussian Process for Automated Enzyme Search.

    Science.gov (United States)

    Mellor, Joseph; Grigoras, Ioana; Carbonell, Pablo; Faulon, Jean-Loup

    2016-06-17

    Synthetic biology is today harnessing the design of novel and greener biosynthesis routes for the production of added-value chemicals and natural products. The design of novel pathways often requires a detailed selection of enzyme sequences to import into the chassis at each of the reaction steps. To address such design requirements in an automated way, we present here a tool for exploring the space of enzymatic reactions. Given a reaction and an enzyme the tool provides a probability estimate that the enzyme catalyzes the reaction. Our tool first considers the similarity of a reaction to known biochemical reactions with respect to signatures around their reaction centers. Signatures are defined based on chemical transformation rules by using extended connectivity fingerprint descriptors. A semisupervised Gaussian process model associated with the similar known reactions then provides the probability estimate. The Gaussian process model uses information about both the reaction and the enzyme in providing the estimate. These estimates were validated experimentally by the application of the Gaussian process model to a newly identified metabolite in Escherichia coli in order to search for the enzymes catalyzing its associated reactions. Furthermore, we show with several pathway design examples how such ability to assign probability estimates to enzymatic reactions provides the potential to assist in bioengineering applications, providing experimental validation to our proposed approach. To the best of our knowledge, the proposed approach is the first application of Gaussian processes dealing with biological sequences and chemicals, the use of a semisupervised Gaussian process framework is also novel in the context of machine learning applied to bioinformatics. However, the ability of an enzyme to catalyze a reaction depends on the affinity between the substrates of the reaction and the enzyme. This affinity is generally quantified by the Michaelis constant KM

  10. Enzyme-driven mechanisms in biocorrosion

    OpenAIRE

    Basséguy, Régine

    2007-01-01

    Objectives (abstract of presentation): Recent works carried out in our team concerning enzymes and biocorrosion are presented at the meeting. For aerobic conditions, the direct catalysis of the reduction of oxygen on steel by enzymes or porphyrin was proved and a local electrochemical analysis technique (SVET) was developed to visualize the localization of the catalysis. On anaerobic conditions, the influence of phosphate species and other weak acids on the water reduction on steel was shown....

  11. Protein engineering of enzymes for process applications

    DEFF Research Database (Denmark)

    Woodley, John M

    2013-01-01

    Scientific progress in the field of enzyme modification today enables the opportunity to tune a given biocatalyst for a specific industrial application. Much work has been focused on extending the substrate repertoire and altering selectivity. Nevertheless, it is clear that many new forthcoming...... opportunities will be targeted on modification to enable process application. This article discusses the challenges involved in enzyme modification focused on process requirements, such as the need to fulfill reaction thermodynamics, specific activity under the required conditions, kinetics at required...

  12. Extracellular enzyme kinetics scale with resource availability

    Science.gov (United States)

    Sinsabaugh, Robert L.; Belnap, Jayne; Findlay, Stuart G.; Follstad Shah, Jennifer J.; Hill, Brian H.; Kuehn, Kevin A.; Kuske, Cheryl; Litvak, Marcy E.; Martinez, Noelle G.; Moorhead, Daryl L.; Warnock, Daniel D.

    2014-01-01

    Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimilation of carbon, nitrogen and phosphorus by diverse aquatic and terrestrial microbial communities (1160 cases). Regression analyses were conducted by habitat (aquatic and terrestrial), enzyme class (hydrolases and oxidoreductases) and assay methodology (low affinity and high affinity substrates) to relate potential reaction rates to substrate availability. Across enzyme classes and habitats, the scaling relationships between apparent Vmax and apparent Km followed similar power laws with exponents of 0.44 to 0.67. These exponents, called elasticities, were not statistically distinct from a central value of 0.50, which occurs when the Km of an enzyme equals substrate concentration, a condition optimal for maintenance of steady state. We also conducted an ecosystem scale analysis of ten extracellular hydrolase activities in relation to soil and sediment organic carbon (2,000–5,000 cases/enzyme) that yielded elasticities near 1.0 (0.9 ± 0.2, n = 36). At the metabolomic scale, the elasticity of extracellular enzymatic reactions is the proportionality constant that connects the C:N:P stoichiometries of organic matter and ecoenzymatic activities. At the ecosystem scale, the elasticity of extracellular enzymatic reactions shows that organic matter ultimately limits effective enzyme binding sites. Our findings suggest that one mechanism by which microbial communities maintain homeostasis is regulating extracellular enzyme expression to optimize the short-term responsiveness of substrate acquisition. The analyses also show that, like elemental stoichiometry, the fundamental attributes of enzymatic reactions can be extrapolated from biochemical to community and ecosystem scales.

  13. Visualization of enzyme activities inside earthworm pores

    Science.gov (United States)

    Hoang, Duyen; Razavi, Bahar S.

    2015-04-01

    In extremely dynamic microhabitats as bio-pores made by earthworm, the in situ enzyme activities are assumed as a footprint of complex biotic interactions. Our study focused on the effect of earthworm on the enzyme activities inside bio-pores and visualizing the differences between bio-pores and earthworm-free soil by zymography technique (Spohn and Kuzyakov, 2013). For the first time, we aimed at quantitative imaging of enzyme activities in bio-pores. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). After two weeks when bio-pore systems were formed by earthworms, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine-aminopeptidase, and phosphatase. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. However, the differences in activity of cellobiohydrolase and leucine aminopeptidase between bio-pore and bulk soil were less pronounced. This demonstrated an applicability of zymography approach to monitor and to distinguish the in situ activity of hydrolytic enzymes in soil biopores.

  14. Enzyme activity in the crowded milieu.

    Directory of Open Access Journals (Sweden)

    Tobias Vöpel

    Full Text Available The cytosol of a cell is a concentrated milieu of a variety of different molecules, including small molecules (salts and metabolites and macromolecules such as nucleic acids, polysaccharides, proteins and large macromolecular complexes. Macromolecular crowding in the cytosolic environment is proposed to influence various properties of proteins, including substrate binding affinity and enzymatic activity. Here we chose to use the synthetic crowding agent Ficoll, which is commonly used to mimic cytosolic crowding conditions to study the crowding effect on the catalytic properties of glycolytic enzymes, namely phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, and acylphosphatase. We determined the kinetic parameters of these enzymes in the absence and in the presence of the crowding agent. We found that the Michaelis constant, K(m, and the catalytic turnover number, k(cat, of these enzymes are not perturbed by the presence of the crowding agent Ficoll. Our results support earlier findings which suggested that the Michaelis constant of certain enzymes evolved in consonance with the substrate concentration in the cell to allow effective enzyme function in bidirectional pathways. This conclusion is further supported by the analysis of nine other enzymes for which the K(m values in the presence and absence of crowding agents have been measured.

  15. Immobilization of enzyme on a polymer surface

    Science.gov (United States)

    Shen, Lei; Cheng, Kenneth Chun Kuen; Schroeder, McKenna; Yang, Pei; Marsh, E. Neil G.; Lahann, Joerg; Chen, Zhan

    2016-06-01

    We successfully immobilized enzymes onto polymer surfaces via covalent bonds between cysteine groups of the enzyme and dibromomaleimide functionalities present at the polymer surface. In this work, we used nitroreductase (NfsB) as a model enzyme molecule. The polymers were prepared by chemical vapor deposition (CVD) polymerization, resulting in surfaces with dibromomaleimide groups. NfsB variants were engineered so that each NfsB molecule only has one cysteine group on the enzyme surface. Two different NfsB constructs were studied, with cysteines at the positions of H360 and V424, respectively. A combination of sum frequency generation (SFG) vibrational and attenuated total reflectance Fourier transformed infrared (ATR-FTIR) spectroscopies were used to deduce the orientation of the immobilized enzymes on the surface. It was found that the orientation of the immobilized enzymes is controlled by the position of the cysteine residue in the protein. The NfsB H360C construct exhibited a similar orientational behavior on the polymer surface as compared to that on the self-assembled monolayer surface, but the NsfB V424C construct showed markedly different orientations on the two surfaces.

  16. Activity assessment of microbial fibrinolytic enzymes.

    Science.gov (United States)

    Kotb, Essam

    2013-08-01

    Conversion of fibrinogen to fibrin inside blood vessels results in thrombosis, leading to myocardial infarction and other cardiovascular diseases. In general, there are four therapy options: surgical operation, intake of antiplatelets, anticoagulants, or fibrinolytic enzymes. Microbial fibrinolytic enzymes have attracted much more attention than typical thrombolytic agents because of the expensive prices and the side effects of the latter. The fibrinolytic enzymes were successively discovered from different microorganisms, the most important among which is the genus Bacillus. Microbial fibrinolytic enzymes, especially those from food-grade microorganisms, have the potential to be developed as functional food additives and drugs to prevent or cure thrombosis and other related diseases. There are several assay methods for these enzymes; this may due to the insolubility of substrate, fibrin. Existing assay methods can be divided into three major groups. The first group consists of assay of fibrinolytic activity with natural proteins as substrates, e.g., fibrin plate methods. The second and third groups of assays are suitable for kinetic studies and are based on the determination of hydrolysis of synthetic peptide esters. This review will deal primarily with the microorganisms that have been reported in literature to produce fibrinolytic enzymes and the first review discussing the methods used to assay the fibrinolytic activity.

  17. Bio-inspired enzyme entrapment and cross-linking approaches as alternative tools for enzyme immobilization

    OpenAIRE

    Ardao Palacios, Inés; Demarche, Philippe; Nair, Rakesh; Agathos, Spiros N.; International Workshop on New and Synthetic Bioproduction Systems

    2012-01-01

    Enzyme immobilization has contributed to the widespread use of enzymes as catalysts in many industrial applications mainly due to stability enhancement and easy reuse of the catalysts, which contributes to a reduction of the process costs. However, enzyme immobilization still suffers from different operational constraints, such as activity losses during immobilization and mass-transfer limitations. Novel immobilization techniques with the goal of overcoming these limitations are increasingly ...

  18. REtools: A laboratory program for restriction enzyme work: enzyme selection and reaction condition assistance

    Directory of Open Access Journals (Sweden)

    Boulukos Kim E

    2006-02-01

    Full Text Available Abstract Background Restriction enzymes are one of the everyday tools used in molecular biology. The continuously expanding panel of known restriction enzymes (several thousands renders their optimal use virtually impossible without computerized assistance. Several manufacturers propose on-line sites that assist scientists in their restriction enzyme work, however, none of these sites meet all the actual needs of laboratory workers, and they do not take into account the enzymes actually present in one's own laboratory. Results Using FileMaker Pro, we developed a stand-alone application which can run on both PCs and Macintoshes. We called it REtools, for Restriction Enzyme tools. This program, which references all currently known enzymes (>3500, permits the creation and update of a personalized list of restriction enzymes actually available in one's own laboratory. Upon opening the program, scientists will be presented with a user friendly interface that will direct them to different menus, each one corresponding to different situations that restriction enzyme users commonly encounter. We particularly emphasized the ease of use to make REtools a solution that laboratory members would actually want to use. Conclusion REtools, a user friendly and easily customized program to organize any laboratory enzyme stock, brings a software solution that will make restriction enzyme use and reaction condition determination straightforward and efficient. The usually unexplored potential of isoschizomers also becomes accessible to all, since REtools proposes all possible enzymes similar to the one(s chosen by the user. Finally, many of the commonly overlooked subtleties of restriction enzyme work, such as methylation requirement, unusual reaction conditions, or the number of flanking bases required for cleavage, are automatically provided by REtools.

  19. Functional representation of enzymes by specific peptides.

    Directory of Open Access Journals (Sweden)

    Vered Kunik

    2007-08-01

    Full Text Available Predicting the function of a protein from its sequence is a long-standing goal of bioinformatic research. While sequence similarity is the most popular tool used for this purpose, sequence motifs may also subserve this goal. Here we develop a motif-based method consisting of applying an unsupervised motif extraction algorithm (MEX to all enzyme sequences, and filtering the results by the four-level classification hierarchy of the Enzyme Commission (EC. The resulting motifs serve as specific peptides (SPs, appearing on single branches of the EC. In contrast to previous motif-based methods, the new method does not require any preprocessing by multiple sequence alignment, nor does it rely on over-representation of motifs within EC branches. The SPs obtained comprise on average 8.4 +/- 4.5 amino acids, and specify the functions of 93% of all enzymes, which is much higher than the coverage of 63% provided by ProSite motifs. The SP classification thus compares favorably with previous function annotation methods and successfully demonstrates an added value in extreme cases where sequence similarity fails. Interestingly, SPs cover most of the annotated active and binding site amino acids, and occur in active-site neighboring 3-D pockets in a highly statistically significant manner. The latter are assumed to have strong biological relevance to the activity of the enzyme. Further filtering of SPs by biological functional annotations results in reduced small subsets of SPs that possess very large enzyme coverage. Overall, SPs both form a very useful tool for enzyme functional classification and bear responsibility for the catalytic biological function carried out by enzymes.

  20. Trametes suaveolens as ligninolytic enzyme producer

    Directory of Open Access Journals (Sweden)

    Knežević Aleksandar

    2013-01-01

    Full Text Available Species of the genus Trametes represent one of the most efficient lignin-degraders which can be attributed to a well developed ligninolytic enzyme system. Current trends are screening of ability of new species to produce these enzymes, as well as the optimization of conditions for their overproduction. Therefore, the aim of the study was to evaluate the potential of T. suaveolens to synthesize laccase and Mn-oxidizing peroxidases during fermentation of the selected plant raw materials. Level of enzyme activities was measured on 7, 10 and 14th day of submersion, as well as the solid-state fermentation of wheat straw and oak sawdust in the presence of NH4NO3 in previously determined optimal nitrogen concentration of 25 mM. The enzyme activity was determined spectrophotometrically using ABTS and phenol red as the substrates. The highest level of laccase activity (1087.1 U/L was noted after 7 days of wheat straw solid-state fermentation, while during the submerged cultivation the production of the enzyme was not noted. Submerged cultivation in oak sawdust-enriched medium was the optimal for activity of Mn-dependent peroxidase (1767.7 U/L on day 14 and Mn-independent peroxidase (1113.7 U/L on day 7. Introduction of T. suaveolens to produce ligninolytic enzyme represented the base for further study, as well as the determination of relation between enzyme activity and rate of lignin degradation. It could lead to greater possibility of fungal species selection with high delignification capacity, which could take participation in sustainable production of food, feed, fibres, and energy, environmentally friendly pollution prevention, and bioremediation.

  1. Evolutionary Trajectories of Beta-Lactamase CTX-M-1 Cluster Enzymes: Predicting Antibiotic Resistance

    OpenAIRE

    Angela Novais; Iñaki Comas; Fernando Baquero; Rafael Cantón; Coque, Teresa M.; Andrés Moya; Fernando González-Candelas; Juan-Carlos Galán

    2010-01-01

    Extended-spectrum beta-lactamases (ESBL) constitute a key antibiotic-resistance mechanism affecting Gram-negative bacteria, and also an excellent model for studying evolution in real time. A shift in the epidemiology of ESBLs is being observed, which is characterized by the explosive diversification and increase in frequency of the CTX-M-type beta-lactamases in different settings. This provides a unique opportunity for studying a protein evolutionary radiation by the sequential acquisition of...

  2. A structural view of the antibiotic degradation enzyme NDM-1 from a superbug

    OpenAIRE

    Guo, Yu; Wang, Jing; Niu, Guojun; Shui, Wenqing; Sun, Yuna; Zhou, Honggang; Zhang, Yaozhou; Yang, Cheng; Lou, Zhiyong; Rao, Zihe

    2011-01-01

    Gram-negative Enterobacteriaceae with resistance to carbapenem conferred by New Delhi metallo-β-lactamase 1 (NDM-1) are a type of newly discovered antibioticresistant bacteria. The rapid pandemic spread of NDM-1 bacteria worldwide (spreading to India, Pakistan, Europe, America, and Chinese Taiwan) in less than 2 months characterizes these microbes as a potentially major global health problem. The drug resistance of NDM-1 bacteria is largely due to plasmids containing the blaNDM-1 gene shuttli...

  3. 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. PMID:26537447

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

    OpenAIRE

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

    2015-01-01

    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.

  5. Soil Enzyme Activities with Greenhouse Subsurface Irrigation

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yu-Long; WANG Yao-Sheng

    2006-01-01

    Various environmental conditions determine soil enzyme activities, which are important indicators for changes of soil microbial activity, soil fertility, and land quality. The effect of subsurface irrigation scheduling on activities of three soil enzymes (phosphatase, urease, and catalase) was studied at five depths (0-10, 10-20, 20-30, 30-40, and 40-60 cm) of a tomato greenhouse soil. Irrigation was scheduled when soil water condition reached the maximum allowable depletion(MAD) designed for different treatments (-10, -16, -25, -40, and -63 kPa). Results showed that soil enzyme activities had significant responses to the irrigation scheduling during the period of subsurface irrigation. The neutral phosphatase activity and the catalase activity were found to generally increase with more frequent irrigation (MAD of -10 and -16kPa). This suggested that a higher level of water content favored an increase in activity of these two enzymes. In contrast,the urease activity decreased under irrigation, with less effect for MAD of -40 and -63 kPa. This implied that relatively wet soil conditions were conducive to retention of urea N, but relatively dry soil conditions could result in increasing loss of urea N. Further, this study revealed that soil enzyme activities could be alternative natural bio-sensors for the effect of irrigation on soil biochemical reactions and could help optimize irrigation management of greenhouse crop production.

  6. Structure/function relationships in cellulolytic enzymes

    Institute of Scientific and Technical Information of China (English)

    Marc Claeyssens

    2004-01-01

    @@ Cellulose and hemicellulose (mostly xylan), together with lignin, are the major polymeric constituents of plant cell walls and from the largest reservoir of fixed carbon in nature. The enzymatic hydrolysis of polymeric substances by extracellular enzymes, such as cellulases, hemicellulases and laccases, is preferred to chemical depolymerisation to avoid the production of toxic by-products and waste that are expensive to treat. The monosaccharides released through enzymatic hydrolysis can subsequently be microbially converted to commercial commodities, such as bio-ethanol (fuel extender) or microbial protein as feed supplements. The individual depolymerisering enzymes used, such as cellulases,xylanases and laccases, also have industrial application in (i) biobleaching in the paper and pulp industry, (ii) improvement of animal feed (poultry and ruminants) digestibility in feed industries, and (iii) dough rheology and bread volume in the baking process, and beer viscosity and filtration velocity during brewing. The cloning of the genes, coding for several xylan degrading enzymes, and their expression in Baker' s yeast (Saccharomyces cerevisiae) and filamentous fungi (Aspergillus species)opened the possibility to study the pure enzymes, without contaminating activity.Trichoderma reesei produces several of these enzymes and detailed information on their specificity,synergies and structure/activity relationships is known. An overview will be presented.

  7. Immobilization of enzymes using non-ionic colloidal liquid aphrons (CLAs): Surface and enzyme effects.

    Science.gov (United States)

    Ward, Keeran; Xi, Jingshu; Stuckey, David C

    2015-12-01

    The use of non-ionic colloidal liquid aphrons (CLAs) as a support for enzyme immobilisation was investigated. Formulation required the mixing of an aqueous-surfactant solution with a relatively non-polar solvent-surfactant solution, forming a solvent droplet surrounded by a thin stabilised aqueous film (soapy shell). Studies utilising anionic surfactants have showed increased retention, however, very little have been understood about the forces governing immobilisation. This study seeks to determine the effects of enzyme properties on CLA immobilisation by examining a non-ionic/non-polar solvent system comprised of two non-ionic surfactants, Tween 20 and 80, mineral oil and the enzymes lipase, aprotinin and α-chymotrypsin. From these results it was deduced that hydrophobic interactions strongly governed immobilisation. Confocal Scanning Laser Microscopy (CSLM) revealed that immobilisation was predominantly achieved by surface adsorption attributed to hydrophobic interactions between the enzyme and the CLA surface. Enzyme surface affinity was found to increase when added directly to the formulation (pre-manufacture addition), as opposed to the bulk continuous phase (post-manufacture addition), with α-chymotrypsin and aprotinin being the most perturbed, while lipase was relatively unaffected. The effect of zeta potential on immobilisation showed that enzymes adsorbed better closer to their pI, indicating that charge minimisation was necessary for immobilisation. Finally, the effect of increasing enzyme concentration in the aqueous phase resulted in an increase in adsorption for all enzymes due to cooperativity between protein molecules, with saturation occurring faster at higher adsorption rates.

  8. Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes.

    Science.gov (United States)

    Wei, Hui; Wang, Erkang

    2013-07-21

    Over the past few decades, researchers have established artificial enzymes as highly stable and low-cost alternatives to natural enzymes in a wide range of applications. A variety of materials including cyclodextrins, metal complexes, porphyrins, polymers, dendrimers and biomolecules have been extensively explored to mimic the structures and functions of naturally occurring enzymes. Recently, some nanomaterials have been found to exhibit unexpected enzyme-like activities, and great advances have been made in this area due to the tremendous progress in nano-research and the unique characteristics of nanomaterials. To highlight the progress in the field of nanomaterial-based artificial enzymes (nanozymes), this review discusses various nanomaterials that have been explored to mimic different kinds of enzymes. We cover their kinetics, mechanisms and applications in numerous fields, from biosensing and immunoassays, to stem cell growth and pollutant removal. We also summarize several approaches to tune the activities of nanozymes. Finally, we make comparisons between nanozymes and other catalytic materials (other artificial enzymes, natural enzymes, organic catalysts and nanomaterial-based catalysts) and address the current challenges and future directions (302 references).

  9. Enzyme replacement in Tay-Sachs disease.

    Science.gov (United States)

    von Specht, B U; Geiger, B; Arnon, R; Passwell, J; Keren, G; Goldman, B; Padeh, B

    1979-06-01

    Enzyme replacement therapy was attempted with two Tay-Sachs-diseased individuals--a 14-month-old child and a 7-week-old infant. Treatment consisted of repeated weekly intrathecal injections of pure hexosaminidase A. Injection of this enzyme resulted in almost complete disappearance of GM2 from the serum, but did not bring about dissolution of the GM2 membranous cytoplasmic bodies in the brain, as detected by electronmicroscopy. Both patients tolerated the treatment without apparent clinical complications, but no clear-cut improvement was noted as a result of prolonged injections of hexosaminidase A. Since this treatment was initiated in both an advanced stage and a very early stage of the disease, we conclude that enzyme replacement treatment by this route is not beneficial for patients with Tay-Sachs disease. PMID:572006

  10. Inhibitors of alanine racemase enzyme: a review.

    Science.gov (United States)

    Azam, Mohammed Afzal; Jayaram, Unni

    2016-08-01

    Alanine racemase is a fold type III PLP-dependent amino acid racemase enzyme catalysing the conversion of l-alanine to d-alanine utilised by bacterial cell wall for peptidoglycan synthesis. As there are no known homologs in humans, it is considered as an excellent antibacterial drug target. The standard inhibitors of this enzyme include O-carbamyl-d-serine, d-cycloserine, chlorovinyl glycine, alaphosphin, etc. d-Cycloserine is indicated for pulmonary and extra pulmonary tuberculosis but therapeutic use of drug is limited due to its severe toxic effects. Toxic effects due to off-target affinities of cycloserine and other substrate analogs have prompted new research efforts to identify alanine racemase inhibitors that are not substrate analogs. In this review, an updated status of known inhibitors of alanine racemase enzyme has been provided which will serve as a rich source of structural information and will be helpful in generating selective and potent inhibitor of alanine racemase. PMID:26024289

  11. Assays for investigating deSUMOylation enzymes.

    Science.gov (United States)

    Madu, Ikenna G; Chen, Yuan

    2012-07-01

    Post-translational modifications by the SUMO (Small Ubiquitin-like MOdifier) family of proteins are recently discovered essential regulatory mechanisms. All SUMO proteins are synthesized as larger precursors that are matured by SUMO-specific proteases, known as SENPs, which remove several C-terminal amino acids of SUMO to expose the Gly-Gly motif. SENPs also remove SUMO modifications from target proteins, making this modification highly dynamic. At least six deSUMOylation enzymes, all of which are encoded by essential genes, have been identified in mammals. SENP1 has been shown to play an important role in the development of prostate cancer and in angiogenesis. This unit describes and discusses methods for characterizing the deSUMOylation enzymes. These assays enable the identification of inhibitors of these enzymes and investigation of their mechanism of inhibition in order to develop research tools and future therapeutics.

  12. Enzyme activity in dialkyl phosphate ionic liquids

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, M.F.; Dunn, J.; Li, L.-L.; Handley-Pendleton, J. M.; van der lelie, D.; Wishart, J. F.

    2011-12-01

    The activity of four metagenomic enzymes and an enzyme cloned from the straw mushroom, Volvariellavolvacea were studied in the following ionic liquids, 1,3-dimethylimidazolium dimethyl phosphate, [mmim][dmp], 1-ethyl-3-methylimidazolium dimethyl phosphate, [emim][dmp], 1-ethyl-3-methylimidazolium diethyl phosphate, [emim][dep] and 1-ethyl-3-methylimidazolium acetate, [emim][OAc]. Activity was determined by analyzing the hydrolysis of para-nitrobenzene carbohydrate derivatives. In general, the enzymes were most active in the dimethyl phosphate ionic liquids, followed by acetate. Generally speaking, activity decreased sharply for concentrations of [emim][dep] above 10% v/v, while the other ionic liquids showed less impact on activity up to 20% v/v.

  13. Enzyme-based multiplexer and demultiplexer.

    Science.gov (United States)

    Arugula, Mary A; Bocharova, Vera; Halámek, Jan; Pita, Marcos; Katz, Evgeny

    2010-04-22

    A digital 2-to-1 multiplexer and a 1-to-2 demultiplexer were mimicked by biocatalytic reactions involving concerted operation of several enzymes. Using glucose oxidase (GOx) and laccase (Lac) as the data input signals and variable pH as the addressing signal, ferrocyanide oxidation in the output channel was selectively activated by one from two inputs, thus mimicking the multiplexer operation. A demultiplexer based on the enzyme system composed of GOx, glucose dehydrogenase (GDH) and horseradish peroxidase (HRP) allowed selective activation of different output channels (oxidation of ferrocyanide or reduction of NAD(+)) by the glucose input. The selection of the output channel was controlled by the addressing input of NAD(+). The designed systems represent important novel components of future branched enzyme networks processing biochemical signals for biosensing and bioactuating.

  14. Enzyme conductometric biosensor for maltose determination

    Directory of Open Access Journals (Sweden)

    Dzyadevych S. V.

    2009-08-01

    Full Text Available Aim. To develop enzyme conductometric biosensor for maltose determination. Methods. A conductometric transducer consisting of two gold pairs of electrodes was applied. Three-enzyme membrane (glucose oxidase, mutarotase, -glucosidase immobilized on the surface of the conductometric transducer was used as a bioselective element. Results. A linear range of maltose conductometric biosensor was from 0,002 mM to 1 mM for glucose and maltose detection. The time of maltose analysis in solution was 1–2 minutes. The dependence of biosensor responses to substrate on pH, ionic strength, and buffer capacity of work solution was studied. The data of biosensor selectivity are presented. The developed conductometric biosensor is characterized by high operational stability and signal reproducibility. Conclusion. The enzyme conductometric biosensor for maltose determination has been developed. The analytical characteristics of the maltose biosensor were investigated. The proposed method could be used in food industry to control and optimize production.

  15. Enzyme Computation - Computing the Way Proteins Do

    Directory of Open Access Journals (Sweden)

    Jaime-Alberto Parra-Plaza

    2013-08-01

    Full Text Available It is presented enzyme computation, a computational paradigm based on the molecular activity inside the biological cells, particularly in the capacity of proteins to represent information, of enzymes to transform that information, and of genes to produce both elements according to the dynamic requirements of a given system. The paradigm explodes the rich computational possibilities offered by metabolic pathways and genetic regulatory networks and translates those possibilities into a distributed computational space made up of active agents which communicate through the mechanism of message passing. Enzyme computation has been tested in diverse problems, such as image processing, species classification, symbolic regression, and constraints satisfaction. Also, given its distributed nature, an implementation in dynamical reconfigurable hardware has been possible.

  16. Arabinogalactan proteins: focus on carbohydrate active enzymes

    Directory of Open Access Journals (Sweden)

    Eva eKnoch

    2014-06-01

    Full Text Available Arabinogalactan proteins (AGPs are a highly diverse class of cell surface proteoglycans that are commonly found in most plant species. AGPs play important roles in many cellular processes during plant development, such as reproduction, cell proliferation, pattern formation and growth, and in plant-microbe interaction. However, little is known about the molecular mechanisms of their function. Numerous studies using monoclonal antibodies that recognize different AGP glycan epitopes have shown the appearance of a slightly altered AGP glycan in a specific stage of development in plant cells. Therefore, it is anticipated that the biosynthesis and degradation of AGP glycan is tightly regulated during development. Until recently, however, little was known about the enzymes involved in the metabolism of AGP glycans. In this review, we summarize recent discoveries of carbohydrate active enzymes (CAZy; http://www.cazy.org/ involved in the biosynthesis and degradation of AGP glycans, and we discuss the biological role of these enzymes in plant development.

  17. Engineering of pectinolytic enzymes for enhanced thermostability

    DEFF Research Database (Denmark)

    Larsen, Dorte Møller

    Conversion of waste materials into valuable compounds is promising concerning transformation of byproduct streams such as sugar beet and potato pulp. In order to obtain those compounds with reduced energy consumption, carbohydrate active enzymes can be used as catalysts. Sugar beet and potato pulp...... consist of pectin that can be converted into beneficial polymeric and oligomeric carbohydrates requiring enzymes such as pectin lyases, rhamnogalacturonan I (RGI) lyases, polygalacturonases and galactanases. Enzymatic conversion of such pectinaceous biomasses at high temperatures is advantageous...... as it gives rise to lower substrate viscosity, easier mixing, higher substrate solubility and lowers the risk of contamination. The overall objective of this thesis was to discover enzymes for degradation of RGI structures in pectin and further engineer for enhanced thermostability. The hypotheses were...

  18. Novel Industrial Enzymes from Uncultured Arctic Microorganisms

    DEFF Research Database (Denmark)

    Vester, Jan Kjølhede

    Many industrial and biotechnological processes make use of cold-active enzymes or could benefit from the use, as the reduced temperature can be beneficial in multiple ways. Such processes may save energy and production costs, improve hygiene, maintain taste and other organoleptic properties......, and reduce the risk of contaminations. Cold- and alkaline-active enzymes can be found in microorganisms adapted to living in natural environments with these conditions, which are extremely rare but found in the unique ikaite columns from SW Greenland (4-6 °C, pH >10). It is estimated that less than 1...... on the diversity of microorganisms from the ikaite columns as well as bioprospecting for enzyme activities using both culture dependent and independent methods. Two cold-active β-galactosidases and one extremely cold-active α-amylase, all related to Clostridia, were characterized in more details....

  19. Extracting enzyme processivity from kinetic assays

    Science.gov (United States)

    Barel, Itay; Reich, Norbert O.; Brown, Frank L. H.

    2015-12-01

    A steady-state analysis for the catalytic turnover of molecules containing two substrate sites is presented. A broad class of Markovian dynamic models, motivated by the action of DNA modifying enzymes and the rich variety of translocation mechanisms associated with these systems (e.g., sliding, hopping, intersegmental transfer, etc.), is considered. The modeling suggests an elementary and general method of data analysis, which enables the extraction of the enzyme's processivity directly and unambiguously from experimental data. This analysis is not limited to the initial velocity regime. The predictions are validated both against detailed numerical models and by revisiting published experimental data for EcoRI endonuclease acting on DNA.

  20. Translational control of an intestinal microvillar enzyme

    DEFF Research Database (Denmark)

    Danielsen, E M; Cowell, G M; Sjöström, H;

    1986-01-01

    The rates of biosynthesis of adult and foetal pig small-intestinal aminopeptidase N (EC 3.4.11.2) were compared to determine at which level the expression of the microvillar enzyme is developmentally controlled. In organ-cultured explants, the rate of biosynthesis of foetal aminopeptidase N is only...... about 3% of the adult rate. The small amount synthesized occurs in a high-mannose-glycosylated, membrane-bound, form that is processed to the mature, complex-glycosylated, form at a markedly slower rate than that of the adult enzyme. Extracts of total RNA from adult and foetal intestine contained...

  1. Immobilized enzyme reactor chromatography: Optimization of protein retention and enzyme activity in monolithic silica stationary phases

    Energy Technology Data Exchange (ETDEWEB)

    Besanger, Travis R. [Department of Chemistry, McMaster University, 1280 Main St. West, Hamilton, Ont. L8S 4M1 (Canada); Hodgson, Richard J. [Department of Chemistry, McMaster University, 1280 Main St. West, Hamilton, Ont. L8S 4M1 (Canada); Green, James R.A. [Department of Chemistry, McMaster University, 1280 Main St. West, Hamilton, Ont. L8S 4M1 (Canada); Brennan, John D. [Department of Chemistry, McMaster University, 1280 Main St. West, Hamilton, Ont. L8S 4M1 (Canada)]. E-mail: brennanj@mcmaster.ca

    2006-03-30

    Our group recently reported on the application of protein-doped monolithic silica columns for immobilized enzyme reactor chromatography, which allowed screening of enzyme inhibitors present in mixtures using mass spectrometry for detection. The enzyme was immobilized by entrapment within a bimodal meso/macroporous silica material prepared by a biocompatible sol-gel processing route. While such columns proved to be useful for applications such as screening of protein-ligand interactions, significant amounts of entrapped proteins leached from the columns owing to the high proportion of macropores within the materials. Herein, we describe a detailed study of factors affecting the morphology of protein-doped bioaffinity columns and demonstrate that specific pH values and concentrations of poly(ethylene glycol) can be used to prepare essentially mesoporous columns that retain over 80% of initially loaded enzyme in an active and accessible form and yet still retain sufficient porosity to allow pressure-driven flow in the low {mu}L/min range. Using the enzyme {gamma}-glutamyl transpeptidase ({gamma}-GT), we further evaluated the catalytic constants of the enzyme entrapped in capillary columns with different silica morphologies as a function of flowrate and backpressure using the enzyme reactor assay mode. It was found that the apparent activity of the enzyme was highest in mesoporous columns that retained high levels of enzyme. In such columns, enzyme activity increased by {approx}2-fold with increases in both flowrate (from 250 to 1000 nL/min) and backpressure generated (from 500 to 2100 psi) during the chromatographic activity assay owing to increases in k {sub cat} and decreases in K {sub M}, switching from diffusion controlled to reaction controlled conditions at ca. 2000 psi. These results suggest that columns with minimal macropore volumes (<5%) are advantageous for the entrapment of soluble proteins for bioaffinity and bioreactor chromatography.

  2. Regulation of Enzyme Activity through Interactions with Nanoparticles

    OpenAIRE

    Bin Zhang; Bing Yan; Zhaochun Wu

    2009-01-01

    The structure and function of an enzyme can be altered by nanoparticles (NPs). The interaction between enzyme and NPs is governed by the key properties of NPs, such as structure, size, surface chemistry, charge and surface shape. Recent representative studies on the NP-enzyme interactions and the regulation of enzyme activity by NPs with different size, composition and surface modification are reviewed.

  3. Seeing & Feeling How Enzymes Work Using Tangible Models

    Science.gov (United States)

    Lau, Kwok-chi

    2013-01-01

    This article presents a tangible model used to help students tackle some misconceptions about enzyme actions, particularly the induced-fit model, enzyme-substrate complementarity, and enzyme inhibition. The model can simulate how substrates induce a change in the shape of the active site and the role of attraction force during enzyme-substrate…

  4. 21 CFR 862.2500 - Enzyme analyzer for clinical use.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Enzyme analyzer for clinical use. 862.2500 Section... Instruments § 862.2500 Enzyme analyzer for clinical use. (a) Identification. An enzyme analyzer for clinical use is a device intended to measure enzymes in plasma or serum by nonkinetic or kinetic measurement...

  5. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Insoluble glucose isomerase enzyme preparations... enzyme preparations. (a) Insoluble glucose isomerase enzyme preparations are used in the production of... additional requirements for enzyme preparations in the Food Chemicals Codex, 3d Ed. (1981), p. 107, which...

  6. 21 CFR 864.9400 - Stabilized enzyme solution.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Stabilized enzyme solution. 864.9400 Section 864... and Blood Products § 864.9400 Stabilized enzyme solution. (a) Identification. A stabilized enzyme... enzyme solutions include papain, bromelin, ficin, and trypsin. (b) Classification. Class II...

  7. Encapsulation of Biocatalysts (Cell/Enzyme) with High Retaining Activity

    OpenAIRE

    Liu, Tao

    2015-01-01

    Enzymes are always considered as great gifts from nature since they are holding brilliant properties, including high activity, selectivity and specificity. Nowadays, a variety of enzymes have been applied to many industry processes. However, challenges are still needed to be addressed while applying enzymes. It is worth to point out that enzymes are sensitive to the change of ambient conditions. Most of enzymes are unstable and work under certain sort of temperature and pH conditions. Since e...

  8. Angiotensin converting enzyme inhibitor induced hyperkalaemic paralysis

    OpenAIRE

    Dutta., D; Fischler, M; McClung, A

    2001-01-01

    Secondary hyperkalaemic paralysis is a rare condition often mimicking the Guillain-Barré syndrome. There have been a few case reports of hyperkalaemia caused by renal failure, trauma, and drugs where the presentation has been with muscle weakness. A case of hyperkalaemic paralysis caused by an angiotensin converting enzyme inhibitor is reported.


Keywords: hyperkalaemia; paralysis; ACE inhibitors

  9. Enzyme Specific Activity in Functionalized Nanoporous Supports

    Energy Technology Data Exchange (ETDEWEB)

    Lei, Chenghong; Soares, Thereza A.; Shin, Yongsoon; Liu, Jun; Ackerman, Eric J.

    2008-03-26

    Enzyme specific activity can be increased or decreased to a large extent by changing protein loading density in functionalized nanoporous support, where organophosphorus hydrolase can display a constructive orientation and thus leave a completely open entrance for substrate even at higher protein loading density, but glucose oxidase can not.

  10. Enzyme specific activity in functionalized nanoporous supports

    Energy Technology Data Exchange (ETDEWEB)

    Lei Chenghong; Soares, Thereza A; Shin, Yongsoon; Liu Jun; Ackerman, Eric J [Pacific Northwest National Laboratory, PO Box 999, Richland, WA 99352 (United States)], E-mail: Eric.Ackerman@pnl.gov

    2008-03-26

    Here we reveal that enzyme specific activity can be increased substantially by changing the protein loading density (P{sub LD}) in functionalized nanoporous supports so that the enzyme immobilization efficiency (I{sub e}, defined as the ratio of the specific activity of the immobilized enzyme to the specific activity of the free enzyme in solution) can be much higher than 100%. A net negatively charged glucose oxidase (GOX) and a net positively charged organophosphorus hydrolase (OPH) were entrapped spontaneously in NH{sub 2}- and HOOC-functionalized mesoporous silica (300 A, FMS) respectively. The specific activity of GOX entrapped in FMS increased with decreasing P{sub LD}. With decreasing P{sub LD}, I{sub e} of GOX in FMS increased from<35% to>150%. Unlike GOX, OPH in HOOC-FMS showed increased specific activity with increasing P{sub LD}. With increasing P{sub LD}, the corresponding I{sub e} of OPH in FMS increased from 100% to>200%. A protein structure-based analysis of the protein surface charges directing the electrostatic interaction-based orientation of the protein molecules in FMS demonstrates that substrate access to GOX molecules in FMS is limited at high P{sub LD}, consequently lowering the GOX specific activity. In contrast, substrate access to OPH molecules in FMS remains open at high P{sub LD} and may promote a more favorable confinement environment that enhances the OPH activity.

  11. An NMR Study of Enzyme Activity.

    Science.gov (United States)

    Peterman, Keith E.; And Others

    1989-01-01

    A laboratory experiment designed as a model for studying enzyme activity with a basic spectrometer is presented. Included are background information, experimental procedures, and a discussion of probable results. Stressed is the value of the use of Nuclear Magnetic Resonance in biochemistry. (CW)

  12. Artificial concurrent catalytic processes involving enzymes.

    Science.gov (United States)

    Köhler, Valentin; Turner, Nicholas J

    2015-01-11

    The concurrent operation of multiple catalysts can lead to enhanced reaction features including (i) simultaneous linear multi-step transformations in a single reaction flask (ii) the control of intermediate equilibria (iii) stereoconvergent transformations (iv) rapid processing of labile reaction products. Enzymes occupy a prominent position for the development of such processes, due to their high potential compatibility with other biocatalysts. Genes for different enzymes can be co-expressed to reconstruct natural or construct artificial pathways and applied in the form of engineered whole cell biocatalysts to carry out complex transformations or, alternatively, the enzymes can be combined in vitro after isolation. Moreover, enzyme variants provide a wider substrate scope for a given reaction and often display altered selectivities and specificities. Man-made transition metal catalysts and engineered or artificial metalloenzymes also widen the range of reactivities and catalysed reactions that are potentially employable. Cascades for simultaneous cofactor or co-substrate regeneration or co-product removal are now firmly established. Many applications of more ambitious concurrent cascade catalysis are only just beginning to appear in the literature. The current review presents some of the most recent examples, with an emphasis on the combination of transition metal with enzymatic catalysis and aims to encourage researchers to contribute to this emerging field.

  13. Nanostructure enzyme assemblies for biomass conversion

    Science.gov (United States)

    Biomass represents a vast resource for production of the world’s fuel and chemical feedstock needs. The use of enzymes to effect these bioconversions offers an alternative that is potentially more specific and environmentally-friendly than harsher chemical methodologies. Some species of anaerobic ...

  14. Sertraline-induced pseudocholinesterase enzyme deficiency

    Directory of Open Access Journals (Sweden)

    Beyazit Zencirci

    2010-11-01

    Full Text Available Beyazit ZencirciMOSTAS Private Health Hospital, Department of Anesthesiology, Kahramanmaras, TurkeyAbstract: A 47-year-old Turkish male was scheduled for laparoscopic cholecystectomy under general anesthesia. The patient had 2 operations 28 and 19 years ago under general anesthesia. It was learned that the patient was administered succinylcholine during both of these previous operations and that he did not have a history of prolonged recovery or postoperative apnea. The patient had been using sertraline for 3 years before the operation. Pseudocholinesterase is a drug-metabolizing enzyme responsible for hydrolysis of the muscle-relaxant drugs mivacurium and succinylcholine. Deficiency of this enzyme from any cause can lead to prolonged apnea and paralysis following administration of mivacurium and succinylcholine. The diagnosis of pseudocholinesterase enzyme deficiency can be made after careful clinic supervision and peripheral nerve stimulator monitoring. A decrease in the activity of pseudocholinesterase enzyme and a decline in the block effect over time will help verify the diagnosis. Our patient’s plasma cholinesterase was found to have low activity. Instead of pharmacological interventions that may further complicate the situation in such cases, the preferred course of action should be to wait until the block effect declines with the help of sedation and mechanical ventilation. In our case, the prolonged block deteriorated in the course of time before any complications developed.Keywords: mivacurium, pseudocholinesterase deficiency, sertraline

  15. Enzyme histochemical study on bone tumors.

    Directory of Open Access Journals (Sweden)

    Yoshida,Haruhiko

    1982-12-01

    Full Text Available A total of 19 cases with bone tumors, including six osteosarcomas. three giant cell tumors of bone, one malignant fibrous histiocytoma, four nonossifying fibromas, four chondromas and one chondrosarcoma, were examined as to enzyme histochemistry; the enzymes consisted of alkaline phosphatase (ALPase, acid phosphatase (ACPase, nonspecific esterase (NSE, adenosine triphosphatase (ATPase, 5'-nucleotidase (5'-Nucl and beta-glucuronidase (beta-Gl. Osteosarcoma was strongly positive for ALPase followed by 5'-Nucl. Giant cell tumor, malignant fibrous histiocytoma and nonossifying fibroma showed enzyme histochemistry similar to each other: multinucleated giant cells and round cells in these tumors were strongly positive for ACPase, NSE, ATPase and 5'-Nucl simulating osteoclasts and histiocytes, whereas spindle cells were positive for ATPase and 5'-Nucl in their cytoplasm and weakly positive for ACPase. Chondroma and chondrosarcoma were focally positive for ACPase and NSE; the ACPase was sensitive to tartaric acid treatment. These observations showed that ALPase activity is very characteristic to osteosarcoma, and is useful for its diagnosis. From enzyme histochemistry, giant cell tumor, malignant fibrous histiocytoma and nonossifying fibroma can be regarded as a histiocyte-derived tumor of bone in contrast to osteosarcoma and cartilaginous tumors.

  16. Mimicking respiratory phosphorylation using purified enzymes.

    Science.gov (United States)

    von Ballmoos, Christoph; Biner, Olivier; Nilsson, Tobias; Brzezinski, Peter

    2016-04-01

    The enzymes of oxidative phosphorylation is a striking example of the functional association of multiple enzyme complexes, working together to form ATP from cellular reducing equivalents. These complexes, such as cytochrome c oxidase or the ATP synthase, are typically investigated individually and therefore, their functional interplay is not well understood. Here, we present methodology that allows the co-reconstitution of purified terminal oxidases and ATP synthases in synthetic liposomes. The enzymes are functionally coupled via proton translocation where upon addition of reducing equivalents the oxidase creates and maintains a transmembrane electrochemical proton gradient that energizes the synthesis of ATP by the F1F0 ATP synthase. The method has been tested with the ATP synthases from Escherichia coli and spinach chloroplasts, and with the quinol and cytochrome c oxidases from E. coli and Rhodobacter sphaeroides, respectively. Unlike in experiments with the ATP synthase reconstituted alone, the setup allows in vitro ATP synthesis under steady state conditions, with rates up to 90 ATP×s(-1)×enzyme(-1). We have also used the novel system to study the phenomenon of "mild uncoupling" as observed in mitochondria upon addition of low concentrations of ionophores (e.g. FCCP, SF6847) and the recoupling effect of 6-ketocholestanol. While we could reproduce the described effects, our data with the in vitro system does not support the idea of a direct interaction between a mitochondrial protein and the uncoupling agents as proposed earlier. PMID:26707617

  17. A Comprehensive Enzyme Kinetic Exercise for Biochemistry

    Science.gov (United States)

    Barton, Janice S.

    2011-01-01

    This article describes a comprehensive treatment of experimental enzyme kinetics strongly coupled to electronic data acquisition and use of spreadsheets to organize data and perform linear and nonlinear least-squares analyses, all in a manner that promotes development of important reasoning skills. Kinetic parameters are obtained for the stable…

  18. Enzyme kinetic characterization of protein tyrosine phosphatases

    DEFF Research Database (Denmark)

    Peters, Günther H.J.; Branner, S.; Møller, K. B.;

    2003-01-01

    Protein tyrosine phosphatases (PTPs) play a central role in cellular signaling processes, resulting in an increased interest in modulating the activities of PTPs. We therefore decided to undertake a detailed enzyme kinetic evaluation of various transmembrane and cytosolic PTPs (PTPalpha, PTPbeta...

  19. Modelling Fungal Fermentations for Enzyme Production

    DEFF Research Database (Denmark)

    Albæk, Mads Orla; Gernaey, Krist; Hansen, Morten S.;

    We have developed a process model of fungal fed-batch fermentations for enzyme production. In these processes, oxygen transfer rate is limiting and controls the substrate feeding rate. The model has been shown to describe cultivations of both Aspergillus oryzae and Trichoderma reesei strains in 550...

  20. A Qualitative Approach to Enzyme Inhibition

    Science.gov (United States)

    Waldrop, Grover L.

    2009-01-01

    Most general biochemistry textbooks present enzyme inhibition by showing how the basic Michaelis-Menten parameters K[subscript m] and V[subscript max] are affected mathematically by a particular type of inhibitor. This approach, while mathematically rigorous, does not lend itself to understanding how inhibition patterns are used to determine the…