WorldWideScience

Sample records for plasmid transduction mediated

  1. Transduction of plasmid DNA in Streptomyces spp. and related genera by bacteriophage FP43.

    Science.gov (United States)

    McHenney, M A; Baltz, R H

    1988-05-01

    A segment (hft) of bacteriophage FP43 DNA cloned into plasmid pIJ702 mediated high-frequency transduction of the resulting plasmid (pRHB101) by FP43 in Streptomyces griseofuscus. The transducing particles contained linear concatemers of plasmid DNA. Lysates of FP43 prepared on S. griseofuscus containing pRHB101 also transduced many other Streptomyces species, including several that restrict plaque formation by FP43 and at least two that produce restriction endonucleases that cut pRHB101 DNA. Transduction efficiencies in different species were influenced by the addition of anti-FP43 antiserum to the transduction plates, the temperature for cell growth before transduction, the multiplicity of infection, and the host on which the transducing lysate was prepared. FP43 lysates prepared on S. griseofuscus(pRHB101) also transduced species of Streptoverticillium, Chainia, and Saccharopolyspora.

  2. Integrins mediating bone signal transduction

    Institute of Scientific and Technical Information of China (English)

    HE Chuanglong; WANG Yuanliang; YANG Lihua; ZHANG Jun

    2004-01-01

    Integrin-mediated adhesions play critical roles in diverse cell functions. Integrins offers a platform on which mechanical stimuli, cytoskeletal organization, biochemical signals can concentrate. Mechanical stimuli transmitted by integrins influence the cytoskeleton, in turn, the cytoskeleton influences cell adhesion via integrins, then cell adhesion results in a series of signal transduction cascades. In skeleton, integrins also have a key role for bone resoption by osteoclasts and reformation by osteoblasts. In present review, the proteins involved in integrin signal transduction and integrin signal transduction pathways were discussed, mainly on the basic mechanisms of integrin signaling and the roles of integrins in bone signal transduction, which may give insight into new therapeutic agents to all kinds of skeletal diseases and new strategies for bone tissue engineering.

  3. Plasmid Transduction Using Bacteriophage Φadh for Expression of CC Chemokines by Lactobacillus gasseri ADH▿

    Science.gov (United States)

    Damelin, Leonard H.; Mavri-Damelin, Demetra; Klaenhammer, Todd R.; Tiemessen, Caroline T.

    2010-01-01

    Vaginal mucosal microfloras are typically dominated by Gram-positive Lactobacillus species, and colonization of vaginal mucosa by exogenous microbicide-secreting Lactobacillus strains has been proposed as a means of enhancing this natural mucosal barrier against human immunodeficiency virus (HIV) infection. We asked whether an alternative strategy could be utilized whereby anti-HIV molecules are expressed within the cervicovaginal milieu by endogenous vaginal Lactobacillus populations which have been engineered in situ via transduction. In this study, we therefore investigated the feasibility of utilizing transduction for the expression of two HIV coreceptor antagonists, the CC chemokines CCL5 and CCL3, in a predominant vaginal Lactobacillus species, Lactobacillus gasseri. Modifying a previously established transduction model, which utilizes L. gasseri ADH and its prophage Φadh, we show that mitomycin C induction of L. gasseri ADH transformants containing pGK12-based plasmids with CCL5 and CCL3 expression and secretion cassettes (under the control of promoters P6 and P59, respectively) and a 232-bp Φadh cos site fragment results in the production of transducing particles which contain 8 to 9 copies of concatemeric plasmid DNA. High-frequency transduction for these particles (almost 6 orders of magnitude greater than that for pGK12 alone) was observed, and transductants were found to contain recircularized expression plasmids upon subsequent culture. Importantly, transductants produced CC chemokines at levels comparable to those produced by electroporation-derived transformants. Our findings therefore lend support to the potential use of transduction in vaginal Lactobacillus species as a novel strategy for the prevention of HIV infection across mucosal membranes. PMID:20418431

  4. Plasmid transduction using bacteriophage Phi(adh) for expression of CC chemokines by Lactobacillus gasseri ADH.

    Science.gov (United States)

    Damelin, Leonard H; Mavri-Damelin, Demetra; Klaenhammer, Todd R; Tiemessen, Caroline T

    2010-06-01

    Vaginal mucosal microfloras are typically dominated by Gram-positive Lactobacillus species, and colonization of vaginal mucosa by exogenous microbicide-secreting Lactobacillus strains has been proposed as a means of enhancing this natural mucosal barrier against human immunodeficiency virus (HIV) infection. We asked whether an alternative strategy could be utilized whereby anti-HIV molecules are expressed within the cervicovaginal milieu by endogenous vaginal Lactobacillus populations which have been engineered in situ via transduction. In this study, we therefore investigated the feasibility of utilizing transduction for the expression of two HIV coreceptor antagonists, the CC chemokines CCL5 and CCL3, in a predominant vaginal Lactobacillus species, Lactobacillus gasseri. Modifying a previously established transduction model, which utilizes L. gasseri ADH and its prophage Phiadh, we show that mitomycin C induction of L. gasseri ADH transformants containing pGK12-based plasmids with CCL5 and CCL3 expression and secretion cassettes (under the control of promoters P6 and P59, respectively) and a 232-bp Phiadh cos site fragment results in the production of transducing particles which contain 8 to 9 copies of concatemeric plasmid DNA. High-frequency transduction for these particles (almost 6 orders of magnitude greater than that for pGK12 alone) was observed, and transductants were found to contain recircularized expression plasmids upon subsequent culture. Importantly, transductants produced CC chemokines at levels comparable to those produced by electroporation-derived transformants. Our findings therefore lend support to the potential use of transduction in vaginal Lactobacillus species as a novel strategy for the prevention of HIV infection across mucosal membranes.

  5. Plasmid mediated quinolone resistance in Enterobacteriaceae

    NARCIS (Netherlands)

    Veldman, K.T.; LS Klinisch Onderzoek Wagenaar

    2014-01-01

    This thesis describes the occurrence of Plasmid Mediated Quinolone Resistance (PMQR) in Salmonella and E. coli from The Netherlands and other European countries. Furthermore, the genetic background of these genes was characterized. Fluoroquinolones are widely used antibiotics in both human and

  6. Plasmid mediated quinolone resistance in Enterobacteriaceae

    NARCIS (Netherlands)

    Veldman, K.T.; LS Klinisch Onderzoek Wagenaar

    2014-01-01

    This thesis describes the occurrence of Plasmid Mediated Quinolone Resistance (PMQR) in Salmonella and E. coli from The Netherlands and other European countries. Furthermore, the genetic background of these genes was characterized. Fluoroquinolones are widely used antibiotics in both human and veter

  7. Plasmid mediated quinolone resistance in Enterobacteriaceae

    NARCIS (Netherlands)

    Veldman, K.T.; LS Klinisch Onderzoek Wagenaar

    2014-01-01

    This thesis describes the occurrence of Plasmid Mediated Quinolone Resistance (PMQR) in Salmonella and E. coli from The Netherlands and other European countries. Furthermore, the genetic background of these genes was characterized. Fluoroquinolones are widely used antibiotics in both human and veter

  8. Plasmid-mediated tetracycline resistance in Haemophilus ducreyi.

    OpenAIRE

    Albritton, W L; Maclean, I W; Slaney, L A; Ronald, A. R.; Deneer, H G

    1984-01-01

    Clinical isolates of Haemophilus ducreyi were shown to be resistant to tetracycline. Resistance was associated in some strains with a 30-megadalton plasmid capable of transferring resistance in conjugative matings with other strains of H. ducreyi and other species of Haemophilus. Restriction endonuclease digestion patterns suggest a relationship between H. ducreyi plasmids and other tetracycline resistance plasmids in Haemophilus. The presence of plasmid-mediated resistance to the tetracyclin...

  9. Prevalence and molecular characterization of plasmid- mediated ...

    African Journals Online (AJOL)

    lactamase genes among nosocomial Staphylococcus aureus drug resistance isolates in Taiwan. .... Table 2: Plasmid profiles of the clinical antibiotic-resistant pathogens. Strain. Profile .... Madec J. Characterization of clinical canine methicillin-.

  10. Plasmid mediated antibiotic resistance in isolated bacteria from burned patients.

    Science.gov (United States)

    Beige, Fahimeh; Baseri Salehi, Majid; Bahador, Nima; Mobasherzadeh, Sina

    2015-01-01

    Nowadays, the treatment of burned patients is difficult because of the high frequency of infection with antibiotic resistance bacteria. This study was conducted to evaluate the level of antibiotic resistance in Gram-negative bacteria and its relation with the existence of plasmid. The samples were collected from two hundred twenty hospitalized burned patients in Isfahan burn hospital during a three-month period (March 2012 to June 2012). The samples were isolated and the Gram-negative bacteria were identified using phenotypic method and API 20E System. Antibiotic susceptibility and plasmid profile were determined by standard Agar disc diffusion and plasmid spin column extraction methods. Totally 117 Gram-negative bacteria were isolated, the most common were Pseudomonas aerugionsa (37.6%), P. fluorescens (25.6%), Acinetobacter baumanii (20/5%) and Klebsiella pneumoniae (7.6%), respectively. The isolates showed high frequency of antibiotic resistance against ceftazidime and co-amoxiclave (100%) and low frequency of antibiotic resistance against amikacin with (70%).The results indicated that 60% of the isolates harboured plasmid. On the other hand, the patients infected with A. baumanii and P. aeruginosa were cured (with 60% frequency) whereas, those infected with P. fluorescens were not cured. Hence, probably antibiotic resistance markers of A. baumanii and P. aeruginosa are plasmid mediated; however, P. fluorescens is chromosomally mediated. Based on our findings, P. aerugionsa is a major causative agent of wound infections and amikacin could be considered as a more effective antibiotic for treatment of the burned patients.

  11. Perspective: Adhesion Mediated Signal Transduction in Bacterial Pathogens

    Science.gov (United States)

    Moorthy, Sudha; Keklak, Julia; Klein, Eric A.

    2016-01-01

    During the infection process, pathogenic bacteria undergo large-scale transcriptional changes to promote virulence and increase intrahost survival. While much of this reprogramming occurs in response to changes in chemical environment, such as nutrient availability and pH, there is increasing evidence that adhesion to host-tissue can also trigger signal transduction pathways resulting in differential gene expression. Determining the molecular mechanisms of adhesion-mediated signaling requires disentangling the contributions of chemical and mechanical stimuli. Here we highlight recent work demonstrating that surface attachment drives a transcriptional response in bacterial pathogens, including uropathogenic Escherichia coli (E. coli), and discuss the complexity of experimental design when dissecting the specific role of adhesion-mediated signaling during infection. PMID:26901228

  12. Reactive oxygen species mediate insulin signal transduction in mouse hypothalamus.

    Science.gov (United States)

    Onoue, Takeshi; Goto, Motomitsu; Tominaga, Takashi; Sugiyama, Mariko; Tsunekawa, Taku; Hagiwara, Daisuke; Banno, Ryoichi; Suga, Hidetaka; Sugimura, Yoshihisa; Arima, Hiroshi

    2016-04-21

    In the hypothalamus, several reports have implied that ROS mediate physiological effects of insulin. In this study, we investigated the mechanisms of insulin-induced ROS production and the effect of ROS on insulin signal transduction in mouse hypothalamic organotypic cultures. Insulin increased intracellular ROS, which were suppressed by NADPH oxidase inhibitor. H2O2 increased phospho-insulin receptor β (p-IRβ) and phospho-Akt (p-Akt) levels. Insulin-induced increases in p-IRβ and p-Akt levels were attenuated by ROS scavenger or NADPH oxidase inhibitor. Our data suggest that insulin-induced phosphorylation of IRβ and Akt is mediated via ROS which are predominantly produced by NADPH oxidase in mouse hypothalamus.

  13. Signal Transduction Model of Magnetic Sensing in Cryptochrome Mediated Photoreception

    Science.gov (United States)

    Todd, Phillise Tiffeny

    While migratory birds have long been known to use the Earth's magnetic field for navigation, the precise biophysical mechanism behind this magnetic sense remains unconfirmed. A leading theory of magnetoreception suggests a chemical compass model with a yet undetermined molecular reaction site and unknown magnetically sensitive reactants. The cryptochrome photoreceptor has emerged as a promising candidate site. This investigation numerically models the first order kinetics of cryptochrome mediated photoreception, in order to evaluate its ability to function as a magnetic sensor and transduce orientation information along a neural pathway. A signal-to-noise ratio is defined to quantify the threshold for the functioning of a cryptochrome-based chemical compass. The model suggests that a flavin-superoxide radical pair in cryptochrome functions as the chemical reactants for magnetoreception. Such a cryptochrome-based signal transduction model reasonably predicts the general light intensity and wavelength effects that have been experimentally observed in migratory birds.

  14. Transduction in Streptomyces hygroscopicus mediated by the temperate bacteriophage SH10.

    Science.gov (United States)

    Süss, F; Klaus, S

    1981-01-01

    The temperate actinophage SH10 mediates generalized transduction in Streptomyces hygroscopicus at low frequency. The efficiency of transduction depends on the average phage input, age of outgrowing spores of the recipient and on the selective marker. The highest EOT was found for the auxotrophic mutants 21(phe-) and 5(try-) (4.2 x 10(-6) and 2.7 x 10(-6), respectively). Transduction of the thermosensitive mutant NG14-216 ts 35 was two orders of magnitude lower (2.5 x 10(-8)). The transductant colonies segregated into stable and unstable clones. Stable transductants were never found to be lysogenic for phage SH10.

  15. Transfer of plasmid-mediated ampicillin resistance from Haemophilus to Neisseria gonorrhoeae requires an intervening organism.

    Science.gov (United States)

    McNicol, P J; Albritton, W L; Ronald, A R

    1986-01-01

    Haemophilus species have been implicated as the source of plasmid-mediated ampicillin resistance in Neisseria gonorrhoeae. Previous attempts to transfer conjugally the resistance plasmids from Haemophilus species to N. gonorrhoeae have met with limited success. Using both biparental and triparental mating systems, it was found that transfer will occur if the commensal Neisseria species, Neisseria cinerea, is used as a transfer intermediate. This organism stably maintains resistance plasmids of Haemophilus and facilitates transfer of these plasmids to N. gonorrhoeae, in a triparental mating system, at a transfer frequency of 10(-8). Both Haemophilus ducreyi and N. gonorrhoeae carry mobilizing plasmids capable of mediating conjugal transfer of the same resistance plasmids. However, restriction endonuclease mapping and DNA hybridization studies indicate that the mobilizing plasmids are distinctly different molecules. Limited homology is present within the transfer region of these plasmids.

  16. Magnetofection Enhances Lentiviral-Mediated Transduction of Airway Epithelial Cells through Extracellular and Cellular Barriers.

    Science.gov (United States)

    Castellani, Stefano; Orlando, Clara; Carbone, Annalucia; Di Gioia, Sante; Conese, Massimo

    2016-11-23

    Gene transfer to airway epithelial cells is hampered by extracellular (mainly mucus) and cellular (tight junctions) barriers. Magnetofection has been used to increase retention time of lentiviral vectors (LV) on the cellular surface. In this study, magnetofection was investigated in airway epithelial cell models mimicking extracellular and cellular barriers. Bronchiolar epithelial cells (H441 line) were evaluated for LV-mediated transduction after polarization onto filters and dexamethasone (dex) treatment, which induced hemicyst formation, with or without magnetofection. Sputum from cystic fibrosis (CF) patients was overlaid onto cells, and LV-mediated transduction was evaluated in the absence or presence of magnetofection. Magnetofection of unpolarized H441 cells increased the transduction with 50 MOI (multiplicity of infection, i.e., transducing units/cell) up to the transduction obtained with 500 MOI in the absence of magnetofection. Magnetofection well-enhanced LV-mediated transduction in mucus-layered cells by 20.3-fold. LV-mediated transduction efficiency decreased in dex-induced hemicysts in a time-dependent fashion. In dome-forming cells, zonula occludens-1 (ZO-1) localization at the cell borders was increased by dex treatment. Under these experimental conditions, magnetofection significantly increased LV transduction by 5.3-fold. In conclusion, these results show that magnetofection can enhance LV-mediated gene transfer into airway epithelial cells in the presence of extracellular (sputum) and cellular (tight junctions) barriers, representing CF-like conditions.

  17. The Role of Cgrp-Receptor Component Protein (Rcp in Cgrp-Mediated Signal Transduction

    Directory of Open Access Journals (Sweden)

    M. A. Prado

    2001-01-01

    Full Text Available The calcitonin gene-related peptide (CGRP-receptor component protein (RCP is a 17-kDa intracellular peripheral membrane protein required for signal transduction at CGRP receptors. To determine the role of RCP in CGRP-mediated signal transduction, RCP was depleted from NIH3T3 cells using antisense strategy. Loss of RCP protein correlated with loss of cAMP production by CGRP in the antisense cells. In contrast, loss of RCP had no effect on CGRP-mediated binding; therefore RCP is not acting as a chaperone for the CGRP receptor. Instead, RCP is a novel signal transduction molecule that couples the CGRP receptor to the cellular signal transduction machinery. RCP thus represents a prototype for a new class of signal transduction proteins that are required for regulation of G protein-coupled receptors.

  18. Microneedle-mediated transcutaneous immunization with plasmid DNA coated on cationic PLGA nanoparticles

    Science.gov (United States)

    Kumar, Amit; Wonganan, Piyanuch; Sandoval, Michael A.; Li, Xinran; Zhu, Saijie; Cui, Zhengrong

    2012-01-01

    Previously, it was shown that microneedle-mediated transcutaneous immunization with plasmid DNA can potentially induce a stronger immune response than intramuscular injection of the same plasmid DNA. In the present study, we showed that the immune responses induced by transcutaneous immunization by applying plasmid DNA onto a skin area pretreated with solid microneedles were significantly enhanced by coating the plasmid DNA on the surface of cationic nanoparticles. In addition, the net surface charge of the DNA-coated nanoparticles significantly affected their in vitro skin permeation and their ability to induce immune responses in vivo. Transcutaneous immunization with plasmid DNA-coated net positively charged anoparticles elicited a stronger immune response than with plasmid DNA-coated net negatively charged nanoparticles or by intramuscular immunization with plasmid DNA alone. Transcutaneous immunization with plasmid DNA-coated net positively charged nanoparticles induced comparable immune responses as intramuscular injection of them, but transcutaneous immunization was able to induce specific mucosal immunity and a more balanced T helper type 1 and type 2 response. The ability of the net positively charged DNA-coated nanoparticles to induce a strong immune response through microneedle-mediated transcutaneous immunization may be attributed to their ability to increase the expression of the antigen gene encoded by the plasmid and to more effectively stimulate the maturation of antigen-presenting cells. PMID:22921518

  19. The Role of Cgrp-Receptor Component Protein (Rcp) in Cgrp-Mediated Signal Transduction

    OpenAIRE

    Prado, M.A.; B. Evans-Bain; Santi, S. L.; Dickerson, I M

    2001-01-01

    The calcitonin gene-related peptide (CGRP)-receptor component protein (RCP) is a 17-kDa intracellular peripheral membrane protein required for signal transduction at CGRP receptors. To determine the role of RCP in CGRP-mediated signal transduction, RCP was depleted from NIH3T3 cells using antisense strategy. Loss of RCP protein correlated with loss of cAMP production by CGRP in the antisense cells. In contrast, loss of RCP had no effect on CGRP-mediated binding; therefore RCP is not acting as...

  20. [Investigation of plasmid-mediated quinolone resistance in Escherichia coli strains].

    Science.gov (United States)

    Aktepe, Orhan Cem; Aşık, Gülşah; Cetinkol, Yeliz; Biçmen, Meral; Gülay, Zeynep

    2012-01-01

    Quinolones are widely used antimicrobial agents, particularly for the treatment of infections caused by gram-negative bacilli such as E.coli. As a consequence, quinolone resistance has been increasing among this species in recent years. Bacterial resistance to quinolones usually results from mutations in the chromosomal genes which encode topoisomerases and also the expression of efflux pumps and loss of porines contributed to development of quinolone resistance. However, recent studies have shown that the spread and increase of quinolone resistance may be due to the transfer of plasmid-mediated genes. To date, three groups of plasmid-mediated quinolone resistance genes, namely qnr, aac(6')-Ib-cr, and qepA, have been described. The aim of this study was to investigate the presence of plasmid-mediated quinolone resistance genes in E.coli clinical isolates. A total of 112 quinolone-resistant E.coli strains isolated from different clinical specimens (84 urine, 16 blood, 10 wound, 2 bronchoalveolar lavage) of which 78 (69.6%) were extended-spectrum beta-lactamase (ESBL) positive, in Afyon Kocatepe University Hospital, Microbiology Laboratory were included in the study. In the isolates, qnrA, qnrB, qnrS, qnrC, qepA, and aac(6')-1b-cr plasmid genes were analysed by polymerase chain reaction (PCR). After aac(6')- 1b determinant was amplified by PCR, all aac(6')-1b positive amplicons were analyzed by digestion with BseGI restriction enzyme to identify aac(6')-1b-cr variant. It was found that, none of the strains horboured qnrA, qnrB, qnrS, qnrC and qepA genes, however, plasmid-mediated quinolone resistance gene aac(6')-1b-cr was found positive in 59.8% (67/112) of the strains. It was notable that 86.6% (58/67) of those isolates were ESBL producers. The rates of quinolone resistance among E.coli isolates infections were high in our region and an increasing trend has been observed in recent years. Our data indicated that the presence of plasmid- mediated resistance genes

  1. Prevalence of plasmid-mediated quinolone resistance determinants in Enterobacteriaceae strains isolated in North-East Italy.

    Science.gov (United States)

    Kocsis, B; Mazzariol, A; Kocsis, E; Koncan, R; Fontana, R; Cornaglia, G

    2013-02-01

    We investigated the prevalence of plasmid-mediated quinolone resistance genes in 756 clinical isolates of Enterobacteriaceae originating from Microbiology Diagnostic Laboratories of North-East Italy. Five point zero two percent of isolates carried a qnr determinant while the aac(6')-Ib-cr determinant was detected in 9·25% of isolates. We also investigated the association between the plasmid-mediated quinolone resistance and the beta-lactamase genes, and characterized the plasmids carrying these determinants of resistance.

  2. Conjugal transfer of the Sinorhizobium meliloti 1021 symbiotic plasmid is governed through the concerted action of one- and two-component signal transduction regulators.

    Science.gov (United States)

    Nogales, Joaquina; Blanca-Ordóñez, Helena; Olivares, José; Sanjuán, Juan

    2013-03-01

    Conjugal transfer of Sinorhizobium meliloti and Rhizobium etli symbiotic plasmids are repressed by the transcriptional regulator RctA. Here we report on new key players in the signal transduction cascade towards S. meliloti pSym conjugation. We have identified S. meliloti pSymA gene SMa0974 as an orthologue of the R. etli rctB gene which is required to antagonize repression by RctA. In S. meliloti two additional genes, rctR and rctC participate in control of rctB expression. rctR (SMa0955) encodes a protein of the GntR family of transcriptional regulators involved in repression of rctB. A rctR mutant promotes pSymA conjugal transfer and displays increased transcription of tra, virB and rctB genes even in presence of wild-type rctA gene. Among genes repressed by RctR, rctC (SMa0961) encodes a response regulator required to activate rctB transcription and therefore for derepression of plasmid conjugative functions. We conclude that in both R. etli and S. meliloti pSym conjugal transfer is derepressed via rctB, however the regulatory cascades to achieve activation of rctB are probably different. Upstream of rctB, the S. meliloti pSym conjugal transfer is regulated through the concerted action of genes representing one- (rctR) and two-component (rctC) signal transduction systems in response to yet unidentified signals.

  3. Novel plasmids and resistance phenotypes in Yersinia pestis: unique plasmid inventory of strain Java 9 mediates high levels of arsenic resistance.

    Directory of Open Access Journals (Sweden)

    Mark Eppinger

    Full Text Available Growing evidence suggests that the plasmid repertoire of Yersinia pestis is not restricted to the three classical virulence plasmids. The Java 9 strain of Y. pestis is a biovar Orientalis isolate obtained from a rat in Indonesia. Although it lacks the Y. pestis-specific plasmid pMT, which encodes the F1 capsule, it retains virulence in mouse and non-human primate animal models. While comparing diverse Y. pestis strains using subtractive hybridization, we identified sequences in Java 9 that were homologous to a Y. enterocolitica strain carrying the transposon Tn2502, which is known to encode arsenic resistance. Here we demonstrate that Java 9 exhibits high levels of arsenic and arsenite resistance mediated by a novel promiscuous class II transposon, named Tn2503. Arsenic resistance was self-transmissible from Java 9 to other Y. pestis strains via conjugation. Genomic analysis of the atypical plasmid inventory of Java 9 identified pCD and pPCP plasmids of atypical size and two previously uncharacterized cryptic plasmids. Unlike the Tn2502-mediated arsenic resistance encoded on the Y. enterocolitica virulence plasmid; the resistance loci in Java 9 are found on all four indigenous plasmids, including the two novel cryptic plasmids. This unique mobilome introduces more than 105 genes into the species gene pool. The majority of these are encoded by the two entirely novel self-transmissible plasmids, which show partial homology and synteny to other enterics. In contrast to the reductive evolution in Y. pestis, this study underlines the major impact of a dynamic mobilome and lateral acquisition in the genome evolution of the plague bacterium.

  4. Plasmid-mediated quinolone resistance among non-typhi Salmonella enterica isolates, USA

    Science.gov (United States)

    We determined the prevalence of plasmid-mediated quinolone resistance mechanisms among non-Typhi Salmonella (NTS) spp. isolates from humans, food animals, and retail meat in the United States in 2007. Fifty-one (2.4%) of human isolates (n=2165), 5 (1.6%) of isolates from animal isolates (n=1915) an...

  5. DMPD: Signal transduction pathways mediated by the interaction of CpG DNA withToll-like receptor 9. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 14751759 Signal transduction pathways mediated by the interaction of CpG DNA withTo...;16(1):17-22. (.png) (.svg) (.html) (.csml) Show Signal transduction pathways mediated by the interaction of... CpG DNA withToll-like receptor 9. PubmedID 14751759 Title Signal transduction pa

  6. TcpM: a novel relaxase that mediates transfer of large conjugative plasmids from Clostridium perfringens.

    Science.gov (United States)

    Wisniewski, Jessica A; Traore, Daouda A; Bannam, Trudi L; Lyras, Dena; Whisstock, James C; Rood, Julian I

    2016-03-01

    Conjugative transfer of toxin and antibiotic resistance plasmids in Clostridium perfringens is mediated by the tcp conjugation locus. Surprisingly, neither a relaxase gene nor an origin of transfer (oriT) has been identified on these plasmids, which are typified by the 47 kb tetracycline resistance plasmid pCW3. The tcpM gene (previously called intP) encodes a potential tyrosine recombinase that was postulated to be an atypical relaxase. Mutagenesis and complementation studies showed that TcpM was required for wild-type transfer of pCW3 and that a tyrosine residue, Y259, was essential for TcpM activity, which was consistent with the need for a relaxase-mediated hydrophilic attack at the oriT site. Other catalytic residues conserved in tyrosine recombinases were not required for TcpM activity, suggesting that TcpM was not a site-specific recombinase. Mobilization studies led to the identification of the oriT site, which was located in the 391 bp intergenic region upstream of tcpM. The oriT site was localized to a 150 bp region, and gel mobility shift studies showed that TcpM could bind to this region. Based on these studies we postulate that conjugative transfer of pCW3 involves the atypical relaxase TcpM binding to and processing the oriT site to initiate plasmid transfer.

  7. Plasmid-mediated quinolone resistance in Enterobacteriaceae: a systematic review with a focus on Mediterranean countries.

    Science.gov (United States)

    Yanat, B; Rodríguez-Martínez, J-M; Touati, A

    2017-03-01

    Quinolones are a family of synthetic broad-spectrum antimicrobial drugs. These molecules have been widely prescribed to treat various infectious diseases and have been classified into several generations based on their spectrum of activity. Quinolones inhibit bacterial DNA synthesis by interfering with the action of DNA gyrase and topoisomerase IV. Mutations in the genes encoding these targets are the most common mechanisms of high-level fluoroquinolone resistance. Moreover, three mechanisms for plasmid-mediated quinolone resistance (PMQR) have been discovered since 1998 and include Qnr proteins, the aminoglycoside acetyltransferase AAC(6')-Ib-cr, and plasmid-mediated efflux pumps QepA and OqxAB. Plasmids with these mechanisms often encode additional antimicrobial resistance (extended spectrum beta-lactamases [ESBLs] and plasmidic AmpC [pAmpC] ß-lactamases) and can transfer multidrug resistance. The PMQR determinants are disseminated in Mediterranean countries with prevalence relatively high depending on the sources and the regions, highlighting the necessity of long-term surveillance for the future monitoring of trends in the occurrence of PMQR genes.

  8. Measurement of the formation of complexes in tyrosine kinase-mediated signal transduction

    Energy Technology Data Exchange (ETDEWEB)

    Ladbury, John E., E-mail: j.ladbury@biochem.ucl.ac.uk [Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT (United Kingdom)

    2007-01-01

    The use of isothermal titration calorimetry (ITC) provides a full thermodynamic characterization of an interaction in one experiment. The determination of the affinity is an important value; however, the additional layer of information provided by the change in enthalpy and entropy can help in understanding the biology. This is demonstrated with respect to tyrosine kinase-mediated signal transduction. Isothermal titration calorimetry (ITC) provides highly complementary data to high-resolution structural detail. An overview of the methodology of the technique is provided. Ultimately, the correlation of the thermodynamic parameters determined by ITC with structural perturbation observed on going from the free to the bound state should be possible at an atomic level. Currently, thermodynamic data provide some insight as to potential changes occurring on complex formation. Here, this is demonstrated in the context of in vitro quantification of intracellular tyrosine kinase-mediated signal transduction and the issue of specificity of the important interactions. The apparent lack of specificity in the interactions of domains of proteins involved in early signalling from membrane-bound receptors is demonstrated using data from ITC.

  9. Plasmid-Mediated OqxAB Is an Important Mechanism for Nitrofurantoin Resistance in Escherichia coli.

    Science.gov (United States)

    Ho, Pak-Leung; Ng, Ka-Ying; Lo, Wai-U; Law, Pierra Y; Lai, Eileen Ling-Yi; Wang, Ya; Chow, Kin-Hung

    2015-11-09

    Increasing consumption of nitrofurantoin (NIT) for treatment of acute uncomplicated urinary tract infections (UTI) highlights the need to monitor emerging NIT resistance mechanisms. This study investigated the molecular epidemiology of the multidrug-resistant efflux gene oqxAB and its contribution to nitrofurantoin resistance by using Escherichia coli isolates originating from patients with UTI (n = 205; collected in 2004 to 2013) and food-producing animals (n = 136; collected in 2012 to 2013) in Hong Kong. The oqxAB gene was highly prevalent among NIT-intermediate (11.5% to 45.5%) and -resistant (39.2% to 65.5%) isolates but rare (0% to 1.7%) among NIT-susceptible (NIT-S) isolates. In our isolates, the oqxAB gene was associated with IS26 and was carried by plasmids of diverse replicon types. Multilocus sequence typing revealed that the clones of oqxAB-positive E. coli were diverse. The combination of oqxAB and nfsA mutations was found to be sufficient for high-level NIT resistance. Curing of oqxAB-carrying plasmids from 20 NIT-intermediate/resistant UTI isolates markedly reduced the geometric mean MIC of NIT from 168.9 μg/ml to 34.3 μg/ml. In the plasmid-cured variants, 20% (1/5) of isolates with nfsA mutations were NIT-S, while 80% (12/15) of isolates without nfsA mutations were NIT-S (P = 0.015). The presence of plasmid-based oqxAB increased the mutation prevention concentration of NIT from 128 μg/ml to 256 μg/ml and facilitated the development of clinically important levels of nitrofurantoin resistance. In conclusion, plasmid-mediated oqxAB is an important nitrofurantoin resistance mechanism. There is a great need to monitor the dissemination of this transferable multidrug-resistant efflux pump.

  10. Prevalence of plasmid-mediated quinolone resistance determinants among oxyiminocephalosporin-resistant Enterobacteriaceae in Argentina

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    Giovanna Rincon Cruz

    2013-11-01

    Full Text Available High quinolone resistance rates were observed among oxyiminocephalosporin-resistant enterobacteria. In the present study, we searched for the prevalence of plasmid-mediated quinolone resistance (PMQR genes within the 55 oxyiminocephalosporin-resistant enterobacteria collected in a previous survey. The main PMQR determinants were aac(6'-Ib-cr and qnrB, which had prevalence rates of 42.4% and 33.3%, respectively. The aac(6'-Ib-cr gene was more frequently found in CTX-M-15-producing isolates, while qnrB was homogeneously distributed among all CTX-M producers.

  11. A respiratory chain controlled signal transduction cascade in the mitochondrial intermembrane space mediates hydrogen peroxide signaling.

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    Patterson, Heide Christine; Gerbeth, Carolin; Thiru, Prathapan; Vögtle, Nora F; Knoll, Marko; Shahsafaei, Aliakbar; Samocha, Kaitlin E; Huang, Cher X; Harden, Mark Michael; Song, Rui; Chen, Cynthia; Kao, Jennifer; Shi, Jiahai; Salmon, Wendy; Shaul, Yoav D; Stokes, Matthew P; Silva, Jeffrey C; Bell, George W; MacArthur, Daniel G; Ruland, Jürgen; Meisinger, Chris; Lodish, Harvey F

    2015-10-20

    Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) govern cellular homeostasis by inducing signaling. H2O2 modulates the activity of phosphatases and many other signaling molecules through oxidation of critical cysteine residues, which led to the notion that initiation of ROS signaling is broad and nonspecific, and thus fundamentally distinct from other signaling pathways. Here, we report that H2O2 signaling bears hallmarks of a regular signal transduction cascade. It is controlled by hierarchical signaling events resulting in a focused response as the results place the mitochondrial respiratory chain upstream of tyrosine-protein kinase Lyn, Lyn upstream of tyrosine-protein kinase SYK (Syk), and Syk upstream of numerous targets involved in signaling, transcription, translation, metabolism, and cell cycle regulation. The active mediators of H2O2 signaling colocalize as H2O2 induces mitochondria-associated Lyn and Syk phosphorylation, and a pool of Lyn and Syk reside in the mitochondrial intermembrane space. Finally, the same intermediaries control the signaling response in tissues and species responsive to H2O2 as the respiratory chain, Lyn, and Syk were similarly required for H2O2 signaling in mouse B cells, fibroblasts, and chicken DT40 B cells. Consistent with a broad role, the Syk pathway is coexpressed across tissues, is of early metazoan origin, and displays evidence of evolutionary constraint in the human. These results suggest that H2O2 signaling is under control of a signal transduction pathway that links the respiratory chain to the mitochondrial intermembrane space-localized, ubiquitous, and ancient Syk pathway in hematopoietic and nonhematopoietic cells.

  12. Comparative mechanisms of protein transduction mediated by cell-penetrating peptides in prokaryotes.

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    Liu, Betty Revon; Huang, Yue-Wern; Aronstam, Robert S; Lee, Han-Jung

    2015-04-01

    Bacterial and archaeal cell envelopes are complex multilayered barriers that serve to protect these microorganisms from their extremely harsh and often hostile environments. Import of exogenous proteins and nanoparticles into cells is important for biotechnological applications in prokaryotes. In this report, we demonstrate that cell-penetrating peptides (CPPs), both bacteria-expressed nona-arginine peptide (R9) and synthetic R9 (SR9), are able to deliver noncovalently associated proteins or quantum dots into four representative species of prokaryotes: cyanobacteria (Synechocystis sp. PCC 6803), bacteria (Escherichia coli DH5α and Arthrobacter ilicis D-50), and archaea (Thermus aquaticus). Although energy-dependent endocytosis is generally accepted as a hallmark that distinguishes eukaryotes from prokaryotes, cellular uptake of uncomplexed green fluorescent protein (GFP) by cyanobacteria was mediated by classical endocytosis. Mechanistic studies revealed that macropinocytosis plays a critical and major role in CPP-mediated protein transduction in all four prokaryotes. Membrane damage was not observed when cyanobacterial cells were treated with R9/GFP complexes, nor was cytotoxicity detected when bacteria or archaea were treated with SR9/QD complexes in the presence of macropinocytic inhibitors. These results indicate that the uptake of protein is not due to a compromise of membrane integrity in cyanobacteria, and that CPP can be an effective and safe carrier for membrane trafficking in prokaryotic cells. Our investigation provides important new insights into the transport of exogenous proteins and nanoparticles across the complex membrane systems of prokaryotes.

  13. Plasmid-mediated quinolone resistance in typhoidal Salmonellae: A preliminary report from South India

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    V K Geetha

    2014-01-01

    Full Text Available Background: Fluoroquinolones are the drugs extensively employed for the treatment of Salmonella infections. Over the couple of decades that have elapsed since the introduction of fluoroquinolones, resistance to these agents by Enterobacteriaceae family members has become common and widespread. Although fluoroquinolone resistance is mediated by genomic DNA (deoxyribonucleic acid as well as plasmid DNA, the plasmid-mediated quinolone resistance (PMQR facilitates higher level resistance by interacting with genomic mechanism and is capable of horizontal spread. Materials and Methods: During a period of 1-year, 63 typhoidal Salmonellae were isolated from 14,050 blood cultures and one parietal wall abscess. 36 (56.25% were Salmonella Typhi and 27 (42% were Salmonella Paratyphi A. They were all screened for resistance by the disc diffusion method and their minimum inhibitory concentrations were determined using agar dilution, broth dilution and E-strip method. Ciprofloxacin resistant isolates were screened for PMQR determinants by polymerase chain reaction assay. Results: All the 63 isolates were resistant to nalidixic acid. Among the 36 S. Typhi isolates 20 were resistant to ciprofloxacin, of which 14 carried the plasmid gene qnrB and one carried the aac(6′-Ib-cr gene. qnrA and qnrS genes were not detected. Ciprofloxacin resistance was not seen in any of the S. Paratyphi A isolates. Conclusion: The antibiotic sensitivity pattern of typhoidal Salmonellae shows an increasing trend of PMQR. The allele B of qnr gene was found to be the predominant cause of PMQR in this study.

  14. Signal transduction across cellular membranes can be mediated by coupling of the clustering of anchored proteins in both leaflets

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    Yue, Tongtao; Zhang, Xianren

    2012-01-01

    One key question in signal transduction is how the signal is relayed from the outer leaflet of a cellular membrane to the inner leaflet. Using a simulation model, a mechanism for the mediation of signal transduction is proposed here in which the coupling between membrane proteins in different leaflets can be achieved by the clustering of anchored proteins, without recruiting transmembrane proteins. Depending on the hydrophobic length of the anchored proteins, three coupling patterns, including face-to-face clustering, interdigitated clustering, and weak-coupled clustering, are observed in this work. This observation provides a possible explanation of how a particular downstream signaling pathway is selected.

  15. Nitric oxide-mediated bystander signal transduction induced by heavy-ion microbeam irradiation

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    Tomita, Masanori; Matsumoto, Hideki; Funayama, Tomoo; Yokota, Yuichiro; Otsuka, Kensuke; Maeda, Munetoshi; Kobayashi, Yasuhiko

    2015-07-01

    In general, a radiation-induced bystander response is known to be a cellular response induced in non-irradiated cells after receiving bystander signaling factors released from directly irradiated cells within a cell population. Bystander responses induced by high-linear energy transfer (LET) heavy ions at low fluence are an important health problem for astronauts in space. Bystander responses are mediated via physical cell-cell contact, such as gap-junction intercellular communication (GJIC) and/or diffusive factors released into the medium in cell culture conditions. Nitric oxide (NO) is a well-known major initiator/mediator of intercellular signaling within culture medium during bystander responses. In this study, we investigated the NO-mediated bystander signal transduction induced by high-LET argon (Ar)-ion microbeam irradiation of normal human fibroblasts. Foci formation by DNA double-strand break repair proteins was induced in non-irradiated cells, which were co-cultured with those irradiated by high-LET Ar-ion microbeams in the same culture plate. Foci formation was suppressed significantly by pretreatment with an NO scavenger. Furthermore, NO-mediated reproductive cell death was also induced in bystander cells. Phosphorylation of NF-κB and Akt were induced during NO-mediated bystander signaling in the irradiated and bystander cells. However, the activation of these proteins depended on the incubation time after irradiation. The accumulation of cyclooxygenase-2 (COX-2), a downstream target of NO and NF-κB, was observed in the bystander cells 6 h after irradiation but not in the directly irradiated cells. Our findings suggest that Akt- and NF-κB-dependent signaling pathways involving COX-2 play important roles in NO-mediated high-LET heavy-ion-induced bystander responses. In addition, COX-2 may be used as a molecular marker of high-LET heavy-ion-induced bystander cells to distinguish them from directly irradiated cells, although this may depend on the time

  16. Nitric oxide agents impair insulin-mediated signal transduction in rat skeletal muscle

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    Ragoobirsingh Dalip

    2006-05-01

    Full Text Available Abstract Background Evidence demonstrates that exogenously administered nitric oxide (NO can induce insulin resistance in skeletal muscle. We have investigated the modulatory effects of two NO donors, S-nitroso-N-acetyl-D, L-penicillamine (SNAP and S-nitrosoglutathione (GSNO on the early events in insulin signaling in rat skeletal myocytes. Results Skeletal muscle cells from 6–8 week old Sprague-Dawley rats were treated with SNAP or GSNO (25 ng/ml in the presence or absence of glucose (25 mM and insulin (100 nM. Cellular insulin receptor-β levels and tyrosine phosphorylation in IRS-1 were significantly reduced, while serine phosphorylation in IRS-1 was significantly increased in these cells, when compared to the insulin-stimulated control. Reversal to near normal levels was achieved using the NO scavenger, 2-(4-carboxyphenyl-4, 4, 5, 5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO. Conclusion These data suggest that NO is a potent modulator of insulin-mediated signal transduction and may play a significant role in the pathogenesis of type 2 diabetes mellitus.

  17. Molecular hydrogen suppresses FcepsilonRI-mediated signal transduction and prevents degranulation of mast cells.

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    Itoh, Tomohiro; Fujita, Yasunori; Ito, Mikako; Masuda, Akio; Ohno, Kinji; Ichihara, Masatoshi; Kojima, Toshio; Nozawa, Yoshinori; Ito, Masafumi

    2009-11-27

    Molecular hydrogen ameliorates oxidative stress-associated diseases in animal models. We found that oral intake of hydrogen-rich water abolishes an immediate-type allergic reaction in mice. Using rat RBL-2H3 mast cells, we demonstrated that hydrogen attenuates phosphorylation of the FcepsilonRI-associated Lyn and its downstream signal transduction, which subsequently inhibits the NADPH oxidase activity and reduces the generation of hydrogen peroxide. We also found that inhibition of NADPH oxidase attenuates phosphorylation of Lyn in mast cells, indicating the presence of a feed-forward loop that potentiates the allergic responses. Hydrogen accordingly inhibits all tested signaling molecule(s) in the loop. Hydrogen effects have been solely ascribed to exclusive removal of hydroxyl radical. In the immediate-type allergic reaction, hydrogen exerts its beneficial effect not by its radical scavenging activity but by modulating a specific signaling pathway. Effects of hydrogen in other diseases are possibly mediated by modulation of yet unidentified signaling pathways. Our studies also suggest that hydrogen is a gaseous signaling molecule like nitric oxide.

  18. Virus-induced gene silencing reveals signal transduction components required for the Pvr9-mediated hypersensitive response in Nicotiana benthamiana.

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    Tran, Phu-Tri; Choi, Hoseong; Choi, Doil; Kim, Kook-Hyung

    2016-08-01

    Resistance to pathogens mediated by plant resistance (R) proteins requires different signaling transduction components and pathways. Our previous studies revealed that a potyvirus resistance gene in pepper, Pvr9, confers a hypersensitive response (HR) to pepper mottle virus in Nicotiana benthamiana. Our results show that the Pvr9-mediated HR against pepper mottle virus infection requires HSP90, SGT1, NDR1, but not EDS1. These results suggest that the Pvr9-mediated HR is possibly related to the SA pathway but not the ET, JA, ROS or NO pathways.

  19. The allosteric behavior of Fur mediates oxidative stress signal transduction in Helicobacter pylori

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    Simone ePelliciari

    2015-08-01

    Full Text Available The microaerophilic gastric pathogen Helicobacter pylori is exposed to oxidative stress originating from the aerobic environment, the oxidative burst of phagocytes and the formation of reactive oxygen species, catalyzed by iron excess. Accordingly, the expression of genes involved in oxidative stress defense have been repeatedly linked to the ferric uptake regulator Fur. Moreover, mutations in the Fur protein affect the resistance to metronidazole, likely due to loss-of-function in the regulation of genes involved in redox control. Although many advances in the molecular understanding of HpFur function were made, little is known about the mechanisms that enable Fur to mediate the responses to oxidative stress.Here we show that iron-inducible, apo-Fur repressed genes, such as pfr and hydA, are induced shortly after oxidative stress, while their oxidative induction is lost in a fur knockout strain. On the contrary, holo-Fur repressed genes, such as frpB1 and fecA1, vary modestly in response to oxidative stress. This indicates that the oxidative stress signal specifically targets apo-Fur repressed genes, rather than impairing indiscriminately the regulatory function of Fur. Footprinting analyses showed that the oxidative signal strongly impairs the binding affinity of Fur towards apo-operators, while the binding towards holo-operators is less affected. Further evidence is presented that a reduced state of Fur is needed to maintain apo-repression, while oxidative conditions shift the preferred binding architecture of Fur towards the holo-operator binding conformation, even in the absence of iron. Together the results demonstrate that the allosteric regulation of Fur enables transduction of oxidative stress signals in H. pylori, supporting the concept that apo-Fur repressed genes can be considered oxidation inducible Fur regulatory targets. These findings may have important implications in the study of H. pylori treatment and resistance to

  20. Nasty viruses, costly plasmids, population dynamics, and the conditions for establishing and maintaining CRISPR-mediated adaptive immunity in bacteria.

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    Bruce R Levin

    2010-10-01

    Full Text Available Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR abound in the genomes of almost all archaebacteria and nearly half the eubacteria sequenced. Through a genetic interference mechanism, bacteria with CRISPR regions carrying copies of the DNA of previously encountered phage and plasmids abort the replication of phage and plasmids with these sequences. Thus it would seem that protection against infecting phage and plasmids is the selection pressure responsible for establishing and maintaining CRISPR in bacterial populations. But is it? To address this question and provide a framework and hypotheses for the experimental study of the ecology and evolution of CRISPR, I use mathematical models of the population dynamics of CRISPR-encoding bacteria with lytic phage and conjugative plasmids. The results of the numerical (computer simulation analysis of the properties of these models with parameters in the ranges estimated for Escherichia coli and its phage and conjugative plasmids indicate: (1 In the presence of lytic phage there are broad conditions where bacteria with CRISPR-mediated immunity will have an advantage in competition with non-CRISPR bacteria with otherwise higher Malthusian fitness. (2 These conditions for the existence of CRISPR are narrower when there is envelope resistance to the phage. (3 While there are situations where CRISPR-mediated immunity can provide bacteria an advantage in competition with higher Malthusian fitness bacteria bearing deleterious conjugative plasmids, the conditions for this to obtain are relatively narrow and the intensity of selection favoring CRISPR weak. The parameters of these models can be independently estimated, the assumption behind their construction validated, and the hypotheses generated from the analysis of their properties tested in experimental populations of bacteria with lytic phage and conjugative plasmids. I suggest protocols for estimating these parameters and outline the

  1. An Invertron-Like Linear Plasmid Mediates Intracellular Survival and Virulence in Bovine Isolates of Rhodococcus equi.

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    Valero-Rello, Ana; Hapeshi, Alexia; Anastasi, Elisa; Alvarez, Sonsiray; Scortti, Mariela; Meijer, Wim G; MacArthur, Iain; Vázquez-Boland, José A

    2015-07-01

    We report a novel host-associated virulence plasmid in Rhodococcus equi, pVAPN, carried by bovine isolates of this facultative intracellular pathogenic actinomycete. Surprisingly, pVAPN is a 120-kb invertron-like linear replicon unrelated to the circular virulence plasmids associated with equine (pVAPA) and porcine (pVAPB variant) R. equi isolates. pVAPN is similar to the linear plasmid pNSL1 from Rhodococcus sp. NS1 and harbors six new vap multigene family members (vapN to vapS) in a vap pathogenicity locus presumably acquired via en bloc mobilization from a direct predecessor of equine pVAPA. Loss of pVAPN rendered R. equi avirulent in macrophages and mice. Mating experiments using an in vivo transconjugant selection strategy demonstrated that pVAPN transfer is sufficient to confer virulence to a plasmid-cured R. equi recipient. Phylogenetic analyses assigned the vap multigene family complement from pVAPN, pVAPA, and pVAPB to seven monophyletic clades, each containing plasmid type-specific allelic variants of a precursor vap gene carried by the nearest vap island ancestor. Deletion of vapN, the predicted "bovine-type" allelic counterpart of vapA, essential for virulence in pVAPA, abrogated pVAPN-mediated intramacrophage proliferation and virulence in mice. Our findings support a model in which R. equi virulence is conferred by host-adapted plasmids. Their central role is mediating intracellular proliferation in macrophages, promoted by a key vap determinant present in the common ancestor of the plasmid-specific vap islands, with host tropism as a secondary trait selected during coevolution with specific animal species. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  2. Comparison of nanoparticle-mediated transfection methods for DNA expression plasmids: efficiency and cytotoxicity

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    Nolte Ingo

    2011-10-01

    Full Text Available Abstract Background Reproducibly high transfection rates with low methodology-induced cytotoxic side effects are essential to attain the required effect on targeted cells when exogenous DNA is transfected. Different approaches and modifications such as the use of nanoparticles (NPs are being evaluated to increase transfection efficiencies. Several studies have focused on the attained transfection efficiency after NP-mediated approaches. However, data comparing toxicity of these novel approaches with conventional methods is still rare. Transfection efficiency and methodology-induced cytotoxicity were analysed after transfection with different NP-mediated and conventional approaches. Two eukaryotic DNA-expression-plasmids were used to transfect the mammalian cell line MTH53A applying six different transfection protocols: conventional transfection reagent (FuGENE HD, FHD, FHD in combination with two different sizes of stabilizer-free laser-generated AuNPs (PLAL-AuNPs_S1,_S2, FHD and commercially available AuNPs (Plano-AuNP, and two magnetic transfection protocols. 24 h post transfection efficiency of each protocol was analysed using fluorescence microscopy and GFP-based flow cytometry. Toxicity was assessed measuring cell proliferation and percentage of propidium iodide (PI% positive cells. Expression of the respective recombinant proteins was evaluated by immunofluorescence. Results The addition of AuNPs to the transfection protocols significantly increased transfection efficiency in the pIRES-hrGFPII-eIL-12 transfections (FHD: 16%; AuNPs mean: 28%, whereas the magnet-assisted protocols did not increase efficiency. Ligand-free PLAL-AuNPs had no significant cytotoxic effect, while the ligand-stabilized Plano-AuNPs induced a significant increase in the PI% and lower cell proliferation. For pIRES-hrGFPII-rHMGB1 transfections significantly higher transfection efficiency was observed with PLAL-AuNPs (FHD: 31%; PLAL-AuNPs_S1: 46%; PLAL-AuNPs_S2: 50

  3. Bacterial plasmid-mediated quinolone resistance genes in aquatic environments in China

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    Yan, Lei; Liu, Dan; Wang, Xin-Hua; Wang, Yunkun; Zhang, Bo; Wang, Mingyu; Xu, Hai

    2017-01-01

    Emerging antimicrobial resistance is a major threat to human’s health in the 21st century. Understanding and combating this issue requires a full and unbiased assessment of the current status on the prevalence of antimicrobial resistance genes and their correlation with each other and bacterial groups. In aquatic environments that are known reservoirs for antimicrobial resistance genes, we were able to reach this goal on plasmid-mediated quinolone resistance (PMQR) genes that lead to resistance to quinolones and possibly also to the co-emergence of resistance to β-lactams. Novel findings were made that qepA and aac-(6′)-Ib genes that were previously regarded as similarly abundant with qnr genes are now dominant among PMQR genes in aquatic environments. Further statistical analysis suggested that the correlation between PMQR and β-lactam resistance genes in the environment is still weak, that the correlations between antimicrobial resistance genes could be weakened by sufficient wastewater treatment, and that the prevalence of PMQR has been implicated in environmental, pathogenic, predatory, anaerobic, and more importantly, human symbiotic bacteria. This work provides a comprehensive analysis of PMQR genes in aquatic environments in Jinan, China, and provides information with which combat with the antimicrobial resistance problem may be fought. PMID:28094345

  4. Plasmid-mediated colistin resistance in Escherichia coli from the Arabian Peninsula

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    Ágnes Sonnevend

    2016-09-01

    Conclusions: This is the first report on the presence of the plasmid-coded mcr-1 gene in a variety of multi-resistant clinical isolates from the Arabian Peninsula indicating that several commonly used antibiotics can potentially facilitate the spread of mcr-1 carrying strains, or directly, mcr-1 containing plasmids.

  5. Emergence and Spread of A Plasmid-Mediated Polymyxin Resistance Mechanism, MCR-1: Are Bacteria Winning?

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    Chao Yang

    2015-12-01

    Full Text Available The report of the emergence of mcr-1, the first plasmid-mediated polymyxin resistance mechanism, in Enterobacteriaceae in November 2015 challenged our last psychological line of defense. However, we still trusted that this resistance factor had not spread globally. One month later, in December 2015, the detection of mcr-1 in an Escherichia coliisolate from a septicemic patient in Denmark and in five E. coli isolates from imported chicken meat really defeated us. The worst news was that one of the chicken meat isolates belonged to ST131, a spreading epidemic sequence type. In China, 15%-21% of E. coli strains isolated from raw meat and animals carried mcr-1, and about 1% of patient isolates carried this gene, indicating that E. coli carrying this plasmid is not a rare phenomenon. This gene is transferable by conjugation and can be maintained in Klebsiella pneumonia and Pseudomonas aeruginosa, suggesting the risk of transfer between different bacterial genera. The good news is that the strains carrying mcr-1 do not contain genes for pan-resistance profiles, although some Danish strains contain 15 different resistance genes, including genes for extended-spectrum beta-lactam antibiotics, and gene mutations leading to high-level fluoroquinolone resistance. If the mcr-1-bearing strains acquire multidrug resistance, extensive drug resistance, or pandrug resistance, no antibiotic drugs will be available with which clinicians can treat infected patients. Therefore, the use of antibiotics in both hospitals and the animal breeding industry must be strictly regulated. The origin of mcr-1 may be associated with the wide use of colistin in agriculture. There is no evidence that the Danish mcr-1 gene spread from China. Therefore, it is likely that mcr-1 genes originated in multiple sites simultaneously under the pressure of colistin use, because India and Denmark are the world’ s greatest users of this antibiotic. More surveys must be conducted in different

  6. Plasmid-mediated bioaugmentation of sequencing batch reactors for enhancement of 2,4-dichlorophenoxyacetic acid removal in wastewater using plasmid pJP4.

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    Tsutsui, Hirofumi; Anami, Yasutaka; Matsuda, Masami; Hashimoto, Kurumi; Inoue, Daisuke; Sei, Kazunari; Soda, Satoshi; Ike, Michihiko

    2013-06-01

    Plasmid-mediated bioaugmentation was demonstrated using sequencing batch reactors (SBRs) for enhancing 2,4-dichlorophenoxyacetic acid (2,4-D) removal by introducing Cupriavidus necator JMP134 and Escherichia coli HB101 harboring 2,4-D-degrading plasmid pJP4. C. necator JMP134(pJP4) can mineralize and grow on 2,4-D, while E. coli HB101(pJP4) cannot assimilate 2,4-D because it lacks the chromosomal genes to degrade the intermediates. The SBR with C. necator JMP134(pJP4) showed 100 % removal against 200 mg/l of 2,4-D just after its introduction, after which 2,4-D removal dropped to 0 % on day 7 with the decline in viability of the introduced strain. The SBR with E. coli HB101(pJP4) showed low 2,4-D removal, i.e., below 10 %, until day 7. Transconjugant strains of Pseudomonas and Achromobacter isolated on day 7 could not grow on 2,4-D. Both SBRs started removing 2,4-D at 100 % after day 16 with the appearance of 2,4-D-degrading transconjugants belonging to Achromobacter, Burkholderia, Cupriavidus, and Pandoraea. After the influent 2,4-D concentration was increased to 500 mg/l on day 65, the SBR with E. coli HB101(pJP4) maintained stable 2,4-D removal of more than 95 %. Although the SBR with C. necator JMP134(pJP4) showed a temporal depression of 2,4-D removal of 65 % on day 76, almost 100 % removal was achieved thereafter. During this period, transconjugants isolated from both SBRs were mainly Achromobacter with high 2,4-D-degrading capability. In conclusion, plasmid-mediated bioaugmentation can enhance the degradation capability of activated sludge regardless of the survival of introduced strains and their 2,4-D degradation capacity.

  7. Systematic Prediction of Scaffold Proteins Reveals New Design Principles in Scaffold-Mediated Signal Transduction

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    Hu, Jianfei; Neiswinger, Johnathan; Zhang, Jin; Zhu, Heng; Qian, Jiang

    2015-01-01

    Scaffold proteins play a crucial role in facilitating signal transduction in eukaryotes by bringing together multiple signaling components. In this study, we performed a systematic analysis of scaffold proteins in signal transduction by integrating protein-protein interaction and kinase-substrate relationship networks. We predicted 212 scaffold proteins that are involved in 605 distinct signaling pathways. The computational prediction was validated using a protein microarray-based approach. The predicted scaffold proteins showed several interesting characteristics, as we expected from the functionality of scaffold proteins. We found that the scaffold proteins are likely to interact with each other, which is consistent with previous finding that scaffold proteins tend to form homodimers and heterodimers. Interestingly, a single scaffold protein can be involved in multiple signaling pathways by interacting with other scaffold protein partners. Furthermore, we propose two possible regulatory mechanisms by which the activity of scaffold proteins is coordinated with their associated pathways through phosphorylation process. PMID:26393507

  8. Systematic Prediction of Scaffold Proteins Reveals New Design Principles in Scaffold-Mediated Signal Transduction.

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    Jianfei Hu

    Full Text Available Scaffold proteins play a crucial role in facilitating signal transduction in eukaryotes by bringing together multiple signaling components. In this study, we performed a systematic analysis of scaffold proteins in signal transduction by integrating protein-protein interaction and kinase-substrate relationship networks. We predicted 212 scaffold proteins that are involved in 605 distinct signaling pathways. The computational prediction was validated using a protein microarray-based approach. The predicted scaffold proteins showed several interesting characteristics, as we expected from the functionality of scaffold proteins. We found that the scaffold proteins are likely to interact with each other, which is consistent with previous finding that scaffold proteins tend to form homodimers and heterodimers. Interestingly, a single scaffold protein can be involved in multiple signaling pathways by interacting with other scaffold protein partners. Furthermore, we propose two possible regulatory mechanisms by which the activity of scaffold proteins is coordinated with their associated pathways through phosphorylation process.

  9. Prevalence of Plasmid-Mediated Quinolone Resistance Genes among Ciprofloxacin-Nonsusceptible Escherichia coli and Klebsiella pneumoniae Isolated from Blood Cultures in Korea

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    Hee Young Yang

    2014-01-01

    Full Text Available OBJECTIVES:To analyze the prevalence of plasmid-mediated quinolone resistance (PMQR determinants in ciprofloxacin-nonsusceptible Escherichia coli and Klebsiella pneumoniae isolated from patients at a tertiary care hospital in Korea.

  10. Horizontal Transfer of Plasmid-Mediated Cephalosporin Resistance Genes in the Intestine of Houseflies (Musca domestica).

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    Fukuda, Akira; Usui, Masaru; Okubo, Torahiko; Tamura, Yutaka

    2016-06-01

    Houseflies are a mechanical vector for various types of bacteria, including antimicrobial-resistant bacteria (ARB). If the intestine of houseflies is a suitable site for the transfer of antimicrobial resistance genes (ARGs), houseflies could also serve as a biological vector for ARB. To clarify whether cephalosporin resistance genes are transferred efficiently in the housefly intestine, we compared with conjugation experiments in vivo (in the intestine) and in vitro by using Escherichia coli with eight combinations of four donor and two recipient strains harboring plasmid-mediated cephalosporin resistance genes and chromosomal-encoded rifampicin resistance genes, respectively. In the in vivo conjugation experiment, houseflies ingested donor strains for 6 hr and then recipient strains for 3 hr, and 24 hr later, the houseflies were surface sterilized and analyzed. In vitro conjugation experiments were conducted using the broth-mating method. In 3/8 combinations, the in vitro transfer frequency (Transconjugants/Donor) was ≥1.3 × 10(-4); the in vivo transfer rates of cephalosporin resistance genes ranged from 2.0 × 10(-4) to 5.7 × 10(-5). Moreover, cephalosporin resistance genes were transferred to other species of enteric bacteria of houseflies such as Achromobacter sp. and Pseudomonas fluorescens. These results suggest that houseflies are not only a mechanical vector for ARB but also a biological vector for the occurrence of new ARB through the horizontal transfer of ARGs in their intestine.

  11. Plasmid-mediated quinolone resistance among extended spectrum beta lactase producing Enterobacteriaceae from bloodstream infections.

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    Domokos, Judit; Kristóf, Katalin; Szabó, Dóra

    2016-09-01

    The purpose of this study was to determine prevalence and molecular characterization of plasmid-mediated quinolone resistance (PMQR) genes [qnrA, qnrB, qnrC, qnrD, qnrS, aac(6')-Ib-cr, qepA, and oqxAB] among extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella spp. isolates from bloodcultures in Hungary. A total of 103 isolates were tested for quinolone susceptibility by microdilution method and PMQR genes were detected by polymerase chain reaction. About 40 ESBL-producing E. coli (39%) and 50 ESBL-producing Klebsiella spp. strains (48%) were resistant to ciprofloxacin; 40 ESBL-producing E. coli (39%) and 47 ESBL-producing Klebsiella spp. strains (45%) were resistant to levofloxacin; and 88 strains including 40 ESBL-producing E. coli (39%) and 48 (47%) ESBL-producing Klebsiella spp. were resistant to moxifloxacin. Among the 103 ESBL-producing isolates, 77 (75%) isolates (30 E. coli and 47 Klebsiella spp.) harbored PMQR genes. The most commonly detected gene was aac(6')-Ib-cr (65%). The occurrence of qnrS gene was 6%. Interestingly, qnrA, qnrB, qnrC, qnrD, and qepA were not found in any isolates. Among 77 PMQR-positive isolates, 27 (35.1%) and 1 (1.3%) carried two and three different PMQR genes, respectively. Only Klebsiella spp. harbored more than one PMQR genes. Observing prevalence of PMQR genes in the last 8 years, the increasing incidence of aac(6')-Ib-cr and oqxAB can be seen. Our results highlight high frequency of PMQR genes among ESBL-producing Klebsiella pneumoniae and E. coli isolates with an increasing dynamics in Hungary.

  12. Plasmid-mediated quinolone resistance; interactions between human, animal and environmental ecologies

    Directory of Open Access Journals (Sweden)

    Laurent ePOIREL

    2012-02-01

    Full Text Available Resistance to quinolones and fluoroquinolones is being increasingly reported among human but also veterinary isolates during the last two to three decades, very likely as a consequence of the large clinical usage of those antibiotics. Even if the principle mechanisms of resistance to quinolones are chromosome-encoded, due to modifications of molecular targets (DNA gyrase and topoisomerase IV, decreased outer-membrane permeability (porin defect and overexpression of naturally-occurring efflux, the emergence of plasmid-mediated quinolone resistance (PMQR has been reported since 1998. Although these PMQR determinants confer low-level resistance to quinolones and/or fluoroquinolones, they are a favorable background for selection of additional chromosome-encoded quinolone resistance mechanisms. Different transferable mechanisms have been identified, corresponding to the production of Qnr proteins, of the aminoglycoside acetyltransferase AAC(6’-Ib-cr, or of the QepA-type or OqxAB-type efflux pumps. Qnr proteins protect target enzymes (DNA gyrase and type IV topoisomerase from quinolone inhibition (mostly nalidixic acid. The AAC(6’-Ib-cr determinant acetylates several fluoroquinolones, such as norfloxacin and ciprofloxacin. Finally, the QepA and OqxAB efflux pumps extrude fluoroquinolones from the bacterial cell. A series of studies have identified the environment to be a reservoir of PMQR genes, with farm animals and aquatic habitats being significantly involved. In addition, the origin of the qnr genes has been identified, corresponding to the waterborne species Shewanella sp. Altogether, the recent observations suggest that the aquatic environment might constitute the original source of PMQR genes, that would secondly spread among animal or human isolates.

  13. Erythropoietin-mediated protection of insect brain neurons involves JAK and STAT but not PI3K transduction pathways.

    Science.gov (United States)

    Miljus, N; Heibeck, S; Jarrar, M; Micke, M; Ostrowski, D; Ehrenreich, H; Heinrich, R

    2014-01-31

    The cytokine erythropoietin (Epo) initiates adaptive cellular responses to both moderate environmental challenges and tissue damaging insults in various non-hematopoietic mammalian tissues including the nervous system. Neuroprotective and neuroregenerative functions of Epo in mammals are mediated through receptor-associated Janus kinase 2 and intracellular signaling cascades that modify the transcription of Epo-regulated genes. Signal transducers and activators of transcription (STAT) and phosphoinositol-3-kinase (PI3K) represent key components of two important Epo-induced transduction pathways. Our previous study on insects revealed neuroprotective and regenerative functions of recombinant human Epo (rhEpo) similar to those in mammalian nervous tissues. Here we demonstrate that rhEpo effectively rescues primary cultured locust brain neurons from apoptotic cell death induced by hypoxia or the chemical compound H-7. The Janus kinase inhibitor AG-490 and the STAT inhibitor sc-355797 abolished protective effects of rhEpo on locust brain neurons. In contrast, inhibition of PI3K with LY294002 had no effect on rhEpo-mediated neuroprotection. The results indicate that rhEpo mediates the protection of locust brain neurons through interference with apoptotic pathways by the activation of a Janus kinase-associated receptor and STAT transcription factor(s). The involvement of similar transduction pathways in mammals and insects for the mediation of neuroprotection and support of neural regeneration by Epo indicates that an Epo/Epo receptor-like signaling system with high structural and functional similarity exists in both groups of animals. Epo-like signaling involved in tissue protection appears to be an ancient beneficial function shared by vertebrates and invertebrates.

  14. Plasmid-mediated colistin resistance in Escherichia coli from the Arabian Peninsula.

    Science.gov (United States)

    Sonnevend, Ágnes; Ghazawi, Akela; Alqahtani, Manaf; Shibl, Atef; Jamal, Wafa; Hashmey, Rayhan; Pal, Tibor

    2016-09-01

    Searching for the presence of the mcr-1 gene in colistin resistant Enterobacteriaceae in countries of the Arabian Peninsula. Seventy-five independent, colistin resistant Enterobacteriaceae strains isolated from clinical cases in Bahrain, Kuwait, Oman, Saudi Arabia and the United Arab Emirates were tested by PCR for the mcr-1 gene. mcr-1 positive strains were genotyped, and their antibiotic susceptibility was established. The mcr-1 containing plasmids were mobilized into Escherichia coli K-12 and their sequence was determined. Four E. coli isolates (two from Bahrain, one from Saudi Arabia and one from the United Arab Emirates) were identified carrying the mcr-1 gene on conjugative plasmids. They belonged to global multidrug resistant E. coli clones, i.e. ST648, ST224, ST68 and ST131, respectively. One strain carried the blaNDM-1 carbapenemase gene. Three strains carried mcr-1 on IncI2 type plasmids, one of them also harboring a blaCTX-M-64 gene. In the fourth strain mcr-1 was located on a 240kb IncHI2 plasmid co-harboring 13 other resistance genes. This is the first report on the presence of the plasmid-coded mcr-1 gene in a variety of multi-resistant clinical isolates from the Arabian Peninsula indicating that several commonly used antibiotics can potentially facilitate the spread of mcr-1 carrying strains, or directly, mcr-1 containing plasmids. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. Characterization of the human oncogene SCL/TAL1 interrupting locus (Stil) mediated Sonic hedgehog (Shh) signaling transduction in proliferating mammalian dopaminergic neurons.

    Science.gov (United States)

    Sun, Lei; Carr, Aprell L; Li, Ping; Lee, Jessica; McGregor, Mary; Li, Lei

    2014-07-11

    The human oncogene SCL/TAL1 interrupting locus (Stil) is highly conserved in all vertebrate species. In humans, the expression of Stil is involved in cancer cell survival, apoptosis and proliferation. In this research, we investigated the roles of Stil expression in cell proliferation of mammalian dopaminergic (DA) PC12 cells. Stil functions through the Sonic hedgehog (Shh) signal transduction pathway. Co-immunoprecipitation tests revealed that STIL interacts with Shh downstream components, which include SUFU and GLI1. By examining the expression of Stil, Gli1, CyclinD2 (cell-cycle marker) and PCNA (proliferating cell nuclear antigen), we found that up-regulation of Stil expression (transfection with overexpression plasmids) increased Shh signaling transduction and PC12 cell proliferation, whereas down-regulation of Stil expression (by shRNA) inhibited Shh signaling transduction, and thereby decreased PC12 cell proliferation. Transient transfection of PC12 cells with Stil knockdown or overexpression plasmids did not affect PC12 cell neural differentiation, further indicating the specific roles of Stil in cell proliferation. The results from this research suggest that Stil may serve as a bio-marker for neurological diseases involved in DA neurons, such as Parkinson's disease.

  16. Characterization of the human oncogene SCL/TAL1 interrupting locus (Stil) mediated Sonic hedgehog (Shh) signaling transduction in proliferating mammalian dopaminergic neurons

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Lei [Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556 (United States); Department of Physiology, Nankai University School of Medicine, Tianjin 300071 (China); Carr, Aprell L. [Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556 (United States); Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556 (United States); Li, Ping; Lee, Jessica; McGregor, Mary [Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556 (United States); Li, Lei, E-mail: Li.78@nd.edu [Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556 (United States); Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556 (United States)

    2014-07-11

    Highlights: • Stil is a human oncogene that is conserved in vertebrate species. • Stil functions in the Shh pathway in mammalian cells. • The expression of Stil is required for mammalian dopaminergic cell proliferation. - Abstract: The human oncogene SCL/TAL1 interrupting locus (Stil) is highly conserved in all vertebrate species. In humans, the expression of Stil is involved in cancer cell survival, apoptosis and proliferation. In this research, we investigated the roles of Stil expression in cell proliferation of mammalian dopaminergic (DA) PC12 cells. Stil functions through the Sonic hedgehog (Shh) signal transduction pathway. Co-immunoprecipitation tests revealed that STIL interacts with Shh downstream components, which include SUFU and GLI1. By examining the expression of Stil, Gli1, CyclinD2 (cell-cycle marker) and PCNA (proliferating cell nuclear antigen), we found that up-regulation of Stil expression (transfection with overexpression plasmids) increased Shh signaling transduction and PC12 cell proliferation, whereas down-regulation of Stil expression (by shRNA) inhibited Shh signaling transduction, and thereby decreased PC12 cell proliferation. Transient transfection of PC12 cells with Stil knockdown or overexpression plasmids did not affect PC12 cell neural differentiation, further indicating the specific roles of Stil in cell proliferation. The results from this research suggest that Stil may serve as a bio-marker for neurological diseases involved in DA neurons, such as Parkinson’s disease.

  17. First environmental sample containing plasmid-mediated colistin-resistant ESBL-producing Escherichia coli detected in Norway.

    Science.gov (United States)

    Jørgensen, Silje Bakken; Søraas, Arne; Arnesen, Lotte Stenfors; Leegaard, Truls; Sundsfjord, Arnfinn; Jenum, Pål A

    2017-09-01

    We hereby report the detection of the plasmid borne mcr-1 gene conferring colistin resistance in an extended-spectrum β-lactamase (ESBL) producing Escherichia coli ST10 strain retrieved from seawater at a public beach in Norway. The sample was collected in September 2010 and was investigated by whole-genome sequencing in 2016. This report illustrates that E. coli strains carrying plasmid-mediated colistin resistance genes have also reached areas where this drug is hardly used at all. Surveillance of colistin resistance in environmental, veterinary, and human strains is warranted also in countries where colistin resistance is rare in clinical settings. © 2017 APMIS. Published by John Wiley & Sons Ltd.

  18. Study on the Genotype of Extended-spectrum Beta-lactamases Mediated by Plasmid in Southern China

    Institute of Scientific and Technical Information of China (English)

    陆坚; 唐英春; 文丽霞; 张扣兴; 张天托; 朱家馨; 谈淑卿

    2004-01-01

    To investigate the prevalence and genotype of extended spectrum beta-lactamases (ESBLs) mediated by plasmid in Gram-negative bacteria found in southern China, a total of 1184 clinical isolates of non-repetitive strains of Gram-negative bacteria were collected in 2001 from 5 different cities in southern China. The ESBLs-producing isolates were distinguished by means of the phenotype confimatory test based on the NCCLS criteria and were subjected to plasmid conjugation and electroporation experiments. Those clinical isolates succeeded in plasmid transfers had undergone plasmid conjugation and electro-transformation, plasmid DNA extraction and Pst Ⅰ digest linger-printing analysis, as well as the tmiversal primer PCR amplification of the TEM, SHV, CTX-M, VEB, PER and SFO genes and the DNA sequencing in order to determine the genotypes of ESBLs and their plasmid locations. It was found that the incidence of the ESBLs-producing strains of Gram-negative bacteria was 14.6% (173/1184) with 67 strains of transconjugants and 11 strains of electro-transformants, in which CTX-M-14 type was 33.3% (26/78); CTX-M-3 type was 23.1% (18/78); CTX-M-9 type was 14.1% (11/78); CTX-M-5 type was 6.4% (5/78); CTX-M-13 type was 2.6% (2/78); SHV-5 type was 7.7% (6/78); SHV-12 type was 5.1% (4/78), SHV-2a type was 2.6% (2/78) and unidentified type was 5.1% (4/78). 29.5% of the wild strains also carried broad-spectrum beta-lactamases TEM-1 and SHV-1 types. The above mentioned ESBLs genes were located on transferable plasmids with variable sizes (from 35 to 190 kb). The CTX-M type ESBLs was characterized by high-level of resistance to cefotaxime. It concluded that the CTX-M-type was the most prevalent genotype in clinical isolates of Gram-negative bacteria in southern China, and the SHVtype ranks in the second place. TEM-, VEB-, Toho- and PER-types were not found in these isolates.

  19. Plasmid-Mediated Quinolone Resistance in Escherichia coli Isolates from Wild Birds and Chickens in South Korea.

    Science.gov (United States)

    Oh, Jae-Young; Kwon, Yong-Kuk; Tamang, Migma Dorji; Jang, Hyung-Kwan; Jeong, Ok-Mi; Lee, Hee-Soo; Kang, Min-Su

    2016-01-01

    A total of 2,423 nonduplicate isolates of Escherichia coli recovered from wild birds (n=793) and chickens (n=1,630) in South Korea were investigated for plasmid-mediated quinolone resistance (PMQR) genes. Altogether, 56 isolates with PMQR genes were identified, including 25 (3.2%) from wild birds and 31 (1.9%) from chickens, which were further characterized using molecular methods. Among them, qnrS, aac(6')-Ib-cr, qnrB, and qepA genes were detected in 47 (1.9%), 6 (0.24%), 2 (0.08%), and 1 (0.04%) isolates, respectively. The most prevalent gene, qnrS, was identified in 21 (0.9%) and 26 (1.1%) isolates from wild birds and chickens, respectively. The qnrB gene was identified in two chicken isolates, which included qnrB19 and a novel qnrB44 gene. Plasmid isolation and Southern hybridization revealed that qnrS1 was located on a large (>200 kbp) plasmid. The spread of the PMQR genes was attributed to a combination of horizontal dissemination and clonal expansion. The horizontal dissemination of PMQR genes was mostly mediated by IncK plasmids. Molecular typing demonstrated that the majority of the PMQR-positive isolates were genetically diverse. Only one chicken isolate belonged to ST131, which harbored an additional CMY-2 gene. Our findings suggest that the wild birds could serve as reservoirs of PMQR genes and spread them over long distances through migration. To our knowledge, this is the first report of PMQR genes in Korean wild birds. This study also reports qnrS2, qnrB19, qnrB44, and qepA genes for the first time in animal E. coli isolates from South Korea.

  20. Plasmid-Mediated Dimethoate Degradation by Bacillus licheniformis Isolated From a Fresh Water Fish Labeo rohita

    Directory of Open Access Journals (Sweden)

    Manisha Deb Mandal

    2005-01-01

    Full Text Available The Bacillus licheniformis strain isolated from the intestine of Labeo rohita by an enrichment technique showed capability of utilizing dimethoate as the sole source of carbon. The bacterium rapidly utilized dimethoate beyond 0.6 mg/mL and showed prolific growth in a mineral salts medium containing 0.45 mg/mL dimethoate. The isolated B licheniformis exhibited high level of tolerance of dimethoate (3.5 mg/mL in nutrient broth, while its cured mutant did not tolerate dimethoate beyond 0.45 mg/mL and it was unable to utilize dimethoate. The wild B licheniformis strain transferred dimethoate degradation property to E coli C600 (Nar, F− strain. The transconjugant harbored a plasmid of the same molecular size (approximately 54 kb as that of the donor plasmid; the cured strain was plasmid less. Thus a single plasmid of approximately 54 kb was involved in dimethoate degradation. Genes encoding resistance to antibiotic and heavy metal were also located on the plasmid.

  1. Plasmid-Mediated Dimethoate Degradation by Bacillus licheniformis Isolated From a Fresh Water Fish Labeo rohita

    Science.gov (United States)

    2005-01-01

    The Bacillus licheniformis strain isolated from the intestine of Labeo rohita by an enrichment technique showed capability of utilizing dimethoate as the sole source of carbon. The bacterium rapidly utilized dimethoate beyond 0.6 mg/mL and showed prolific growth in a mineral salts medium containing 0.45 mg/mL dimethoate. The isolated B licheniformis exhibited high level of tolerance of dimethoate (3.5 mg/mL) in nutrient broth, while its cured mutant did not tolerate dimethoate beyond 0.45 mg/mL and it was unable to utilize dimethoate. The wild B licheniformis strain transferred dimethoate degradation property to E coli C600 (Nar, F−) strain. The transconjugant harbored a plasmid of the same molecular size (approximately 54 kb) as that of the donor plasmid; the cured strain was plasmid less. Thus a single plasmid of approximately 54 kb was involved in dimethoate degradation. Genes encoding resistance to antibiotic and heavy metal were also located on the plasmid. PMID:16192686

  2. Plasmid Mediated Antibiotic and Heavy Metal Resistance in Bacillus Strains Isolated From Soils in Rize, Turkey

    Directory of Open Access Journals (Sweden)

    Elif SEVİM

    2015-09-01

    Full Text Available Fifteen Bacillus strains which were isolated from soil samples were examined for resistance to 17 different antibiotics (ampicillin, methicillin, erythromycin, norfloxacin, cephalotine, gentamycin, ciprofloxacin, streptomycin, tobramycin, chloramphenicol, trimethoprim-sulfamethoxazole, tetracycline, vancomycin, oxacilin, neomycin, kanamycin and, novabiocin and to 10 different heavy metals (copper, lead, cobalt, chrome, iron, mercury, zinc, nickel, manganese and, cadmium and for the presence of plasmid DNA. A total of eleven strains (67% were resistant to at least one antibiotic. The most common resistance was observed against methicillin and oxacillin. The most resistance strains were found as Bacillus sp. B3 and Bacillus sp. B11. High heavy metal resistance against copper, chromium, zinc, iron and nickel was detected, but mercury and cobalt resistance was not detected, except for 3 strains (B3, B11, and B12 which showed mercury resistance. It has been determined that seven Bacillus strains have plasmids. The isolated plasmids were transformed into the Bacillus subtilis W168 and it was shown that heavy metal and antibiotic resistance determinants were carried on these plasmids. These results showed that there was a correlation between plasmid content and resistance for both antibiotic and heavy metal resistance

  3. Signal transduction, receptors, mediators and genes: younger than ever - the 13th meeting of the Signal Transduction Society focused on aging and immunology

    Directory of Open Access Journals (Sweden)

    Klotz Lars-Oliver

    2010-02-01

    Full Text Available Abstract The 13th meeting of the Signal Transduction Society was held in Weimar, from October 28 to 30, 2009. Special focus of the 2009 conference was "Aging and Senescence", which was co-organized by the SFB 728 "Environmentally-Induced Aging Processes" of the University of Düsseldorf and the study group 'Signal Transduction' of the German Society for Cell Biology (DGZ. In addition, several other areas of signal transduction research were covered and supported by different consortia associated with the Signal Transduction Society including the long-term associated study groups of the German Society for Immunology and the Society for Biochemistry and Molecular Biology, and for instance the SFB/Transregio 52 "Transcriptional Programming of Individual T Cell Subsets" located in Würzburg, Mainz and Berlin. The different research areas that were introduced by outstanding keynote speakers attracted more than 250 scientists, showing the timeliness and relevance of the interdisciplinary concept and exchange of knowledge during the three days of the scientific program. This report gives an overview of the presentations of the conference.

  4. Kinetic Properties of Four Plasmid-Mediated AmpC β-Lactamases

    Science.gov (United States)

    Bauvois, Cédric; Ibuka, Akiko Shimizu; Celso, Almeida; Alba, Jimena; Ishii, Yoshikazu; Frère, Jean-Marie; Galleni, Moreno

    2005-01-01

    The heterologous production in Escherichia coli, the purification, and the kinetic characterization of four plasmid-encoded class C β-lactamases (ACT-1, MIR-1, CMY-2, and CMY-1) were performed. Except for their instability, these enzymes are very similar to the known chromosomally encoded AmpC β-lactamases. Their kinetic parameters did not show major differences from those obtained for the corresponding chromosomal enzymes. However, the Km values of CMY-2 for cefuroxime, cefotaxime, and oxacillin were significantly decreased compared to those of the chromosomal AmpC enzymes. Finally, the susceptibility patterns of different E. coli hosts producing a plasmid- or a chromosome-encoded class C enzyme toward β-lactam antibiotics are mainly due to the overproduction of the β-lactamase in the periplasmic space of the bacteria rather than to a specific catalytic profile of the plasmid-encoded β-lactamases. PMID:16189104

  5. Transfer of plasmid-mediated resistance to tetracycline in pathogenic bacteria from fish and aquaculture environments.

    Science.gov (United States)

    Guglielmetti, Elena; Korhonen, Jenni M; Heikkinen, Jouni; Morelli, Lorenzo; von Wright, Atte

    2009-04-01

    The transferability of a large plasmid that harbors a tetracycline resistance gene tet(S), to fish and human pathogens was assessed using electrotransformation and conjugation. The plasmid, originally isolated from fish intestinal Lactococcus lactis ssp. lactis KYA-7, has potent antagonistic activity against the selected recipients (Lactococcus garvieae and Listeria monocytogenes), preventing conjugation. Therefore the tetracycline resistance determinant was transferred via electroporation to L. garvieae. A transformant clone was used as the donor in conjugation experiments with three different L. monocytogenes strains. To our knowledge, this is the first study showing the transfer of an antibiotic resistance plasmid from fish-associated lactic bacteria to L. monocytogenes, even if the donor L. garvieae was not the original host of the tetracycline resistance but experimentally created by electroporation. These results demonstrate that the antibiotic resistance genes in the fish intestinal bacteria have the potential to spread both to fish and human pathogens, posing a risk to aquaculture and consumer safety.

  6. Recombinant adeno-associated viral (rAAV) vectors mediate efficient gene transduction in cultured neonatal and adult microglia.

    Science.gov (United States)

    Su, Wei; Kang, John; Sopher, Bryce; Gillespie, James; Aloi, Macarena S; Odom, Guy L; Hopkins, Stephanie; Case, Amanda; Wang, David B; Chamberlain, Jeffrey S; Garden, Gwenn A

    2016-01-01

    Microglia are a specialized population of myeloid cells that mediate CNS innate immune responses. Efforts to identify the cellular and molecular mechanisms that regulate microglia behaviors have been hampered by the lack of effective tools for manipulating gene expression. Cultured microglia are refractory to most chemical and electrical transfection methods, yielding little or no gene delivery and causing toxicity and/or inflammatory activation. Recombinant adeno-associated viral (rAAVs) vectors are non-enveloped, single-stranded DNA vectors commonly used to transduce many primary cell types and tissues. In this study, we evaluated the feasibility and efficiency of utilizing rAAV serotype 2 (rAAV2) to modulate gene expression in cultured microglia. rAAV2 yields high transduction and causes minimal toxicity or inflammatory response in both neonatal and adult microglia. To demonstrate that rAAV transduction can induce functional protein expression, we used rAAV2 expressing Cre recombinase to successfully excise a LoxP-flanked miR155 gene in cultured microglia. We further evaluated rAAV serotypes 5, 6, 8, and 9, and observed that all efficiently transduced cultured microglia to varying degrees of success and caused little or no alteration in inflammatory gene expression. These results provide strong encouragement for the application of rAAV-mediated gene expression in microglia for mechanistic and therapeutic purposes. Neonatal microglia are functionally distinct from adult microglia, although the majority of in vitro studies utilize rodent neonatal microglia cultures because of difficulties of culturing adult cells. In addition, cultured microglia are refractory to most methods for modifying gene expression. Here, we developed a novel protocol for culturing adult microglia and evaluated the feasibility and efficiency of utilizing Recombinant Adeno-Associated Virus (rAAV) to modulate gene expression in cultured microglia.

  7. Plasmid-mediated biodegradation of the anionic surfactant sodium dodecyl sulphate, by Pseudomonas aeruginosa S7.

    Science.gov (United States)

    Yeldho, Deepthi; Rebello, Sharrel; Jisha, M S

    2011-01-01

    Sodium dodecyl sulphate (SDS), an anionic surfactant, has been used extensively due to its low cost and excellent foaming properties. Fifteen different bacterial isolates capable of degrading SDS were isolated from detergent contaminated soil by enrichment culture technique and the degradation efficiency was assessed by Methylene Blue Active Substances (MBAS) assay. The most efficient SDS degrading isolate was selected and identified as Pseudomonas aeruginosa S7. The selected isolate was found to harbor a single 6-kb plasmid. Acridine orange, ethidium bromide, SDS and elevated temperatures of incubation failed to cure the plasmid. The cured derivatives of SDS degrading Pseudomonas aeruginosa were obtained only when ethidium bromide and elevated temperature (40 °C) were used together. Transformation of E. coli DH5α with plasmid isolated from S7 resulted in subsequent growth of the transformants on minimal salt media with SDS (0.1%) as the sole source of carbon. The SDS degradation ability of S7 and the transformant was found to be similar as assessed by Methylene Blue Active Substance Assay. The antibiotic resistance profiles of S7, competent DH5α and transformant were analyzed and it was noted that the transfer of antibiotic resistance correlated with the transfer of plasmid as well as SDS degrading property.

  8. Transgene organisation in potato after particle bombardment-mediated (co-) transformation using plasmids and gene cassettes

    NARCIS (Netherlands)

    Romano, A.; Raemakers, C.J.J.M.; Bernardi, J.; Visser, R.G.F.; Mooibroek, A.

    2003-01-01

    Protocols for efficient co-transformation of potato internodes with genes contained in separate plasmids or gene cassettes (i.e., linear PCR fragments comprising a promoter-gene-terminator) using particle bombardment were established. Twenty-eight out of 62 (45%) and 11 out of 65 (17%) plants transf

  9. Electromobility of plasmid DNA in tumor tissues during electric field-mediated gene delivery.

    Science.gov (United States)

    Zaharoff, D A; Barr, R C; Li, C-Y; Yuan, F

    2002-10-01

    Interstitial transport is a crucial step in plasmid DNA-based gene therapy. However, interstitial diffusion of large nucleic acids is prohibitively slow. Therefore, we proposed to facilitate interstitial transport of DNA via pulsed electric fields. To test the feasibility of this approach to gene delivery, we developed an ex vivo technique to quantify the magnitude of DNA movement due to pulsed electric fields in two tumor tissues: B16.F10 (a mouse melanoma) and 4T1 (a mouse mammary carcinoma). When the pulse duration and strength were 50 ms and 233 V/cm, respectively, we found that the average plasmid DNA movements per 10 pulses were 1.47 microm and 0.35 microm in B16.F10 and 4T1 tumors, respectively. The average plasmid DNA movements could be approximately tripled, ie to reach 3.69 microm and 1.01 microm, respectively, when the pulse strength was increased to 465 V/cm. The plasmid DNA mobility was correlated with the tumor collagen content, which was approximately eight times greater in 4T1 than in B16.F10 tumors. These data suggest that electric field can be a powerful driving force for improving interstitial transport of DNA during gene delivery.

  10. Potential insight for drug discovery from high fidelity receptor-mediated transduction mechanisms in insects

    Science.gov (United States)

    Raffa, Robert B.; Raffa, Kenneth F.

    2011-01-01

    Introduction There is a pervasive and growing concern about the small number of new pharmaceutical agents. There are many proposed explanations for this trend that do not involve the drug-discovery process per se, but the discovery process itself has also come under scrutiny. If the current paradigms are indeed not working, where are novel ideas to come from? Perhaps it is time to look to novel sources. Areas covered The receptor-signaling and 2nd-messenger transduction processes present in insects are quite similar to those in mammals (involving G proteins, ion channels, etc.). However, a review of these systems reveals an unprecedented degree of high potency and receptor selectivity to an extent greater than that modeled in most current drug-discovery approaches. Expert opinion A better understanding of insect receptor pharmacology could stimulate novel theoretical and practical ideas in mammalian pharmacology (drug discovery) and, conversely, the application of pharmacology and medicinal chemistry principles could stimulate novel advances in entomology (safer and more targeted control of pest species). PMID:21984882

  11. Bone marrow X kinase-mediated signal transduction in irradiated vascular endothelium.

    Science.gov (United States)

    Tu, Tianxiang; Thotala, Dinesh; Geng, Ling; Hallahan, Dennis E; Willey, Christopher D

    2008-04-15

    Radiation-induced activation of the phosphatidyl inositol-3 kinase/Akt signal transduction pathway requires Akt binding to phosphatidyl-inositol phosphates (PIP) on the cell membrane. The tyrosine kinase bone marrow X kinase (Bmx) binds to membrane-associated PIPs in a manner similar to Akt. Because Bmx is involved in cell growth and survival pathways, it could contribute to the radiation response within the vascular endothelium. We therefore studied Bmx signaling within the vascular endothelium. Bmx was activated rapidly in response to clinically relevant doses of ionizing radiation. Bmx inhibition enhanced the efficacy of radiotherapy in endothelial cells as well as tumor vascular endothelium in lung cancer tumors in mice. Retroviral shRNA knockdown of Bmx protein enhanced human umbilical vascular endothelial cell (HUVEC) radiosensitization. Furthermore, pretreatment of HUVEC with a pharmacologic inhibitor of Bmx, LFM-A13, produced significant radiosensitization of endothelial cells as measured by clonogenic survival analysis and apoptosis as well as functional assays including cell migration and tubule formation. In vivo, LFM-A13, when combined with radiation, resulted in significant tumor microvascular destruction as well as enhanced tumor growth delay. Bmx therefore represents a molecular target for the development of novel radiosensitizing agents.

  12. AAV8 capsid variable regions at the two-fold symmetry axis contribute to high liver transduction by mediating nuclear entry and capsid uncoating

    Energy Technology Data Exchange (ETDEWEB)

    Tenney, Rebeca M.; Bell, Christie L.; Wilson, James M., E-mail: wilsonjm@mail.med.upenn.edu

    2014-04-15

    Adeno-associated virus serotype 8 (AAV8) is a promising vector for liver-directed gene therapy. Although efficient uncoating of viral capsids has been implicated in AAV8's robust liver transduction, much about the biology of AAV8 hepatotropism remains unclear. Our study investigated the structural basis of AAV8 liver transduction efficiency by constructing chimeric vector capsids containing sequences derived from AAV8 and AAV2 – a highly homologous yet poorly hepatotropic serotype. Engineered vectors containing capsid variable regions (VR) VII and IX from AAV8 in an AAV2 backbone mediated near AAV8-like transduction in mouse liver, with higher numbers of chimeric genomes detected in whole liver cells and isolated nuclei. Interestingly, chimeric capsids within liver nuclei also uncoated similarly to AAV8 by 6 weeks after administration, in contrast with AAV2, of which a significantly smaller proportion were uncoated. This study links specific AAV capsid regions to the transduction ability of a clinically relevant AAV serotype. - Highlights: • We construct chimeric vectors to identify determinants of AAV8 liver transduction. • An AAV2-based vector with 17 AAV8 residues exhibited high liver transduction in mice. • This vector also surpassed AAV2 in cell entry, nuclear entry and onset of expression. • Most chimeric vector particles were uncoated at 6 weeks, like AAV8 and unlike AAV2. • Chimera retained heparin binding and was antigenically distinct from AAV2 and AAV8.

  13. Interaction between muscarinic receptor subtype signal transduction pathways mediating bladder contraction

    Science.gov (United States)

    BRAVERMAN, ALAN S.; TALLARIDA, RONALD J.; RUGGIERI, MICHAEL R.

    2012-01-01

    M3 muscarinic receptors mediate cholinergic-induced contraction in most smooth muscles. However, in the denervated rat bladder, M2 receptors participate in contraction because M3-selective antagonists [para-fluoro-hexahydro-sila-diphenidol (p-F-HHSiD) and 4-DAMP] have low affinities. However, the affinity of the M2-selective antagonist methoctramine in the denervated bladder is consistent with M3 receptor mediating contraction. It is possible that two pathways interact to mediate contraction: one mediated by the M2 receptor and one by the M3 receptor. To determine whether an interaction exists, the inhibitory potencies of combinations of methoctramine and p-F-HHSiD for reversing cholinergic contractions were measured. In normal bladders, all combinations gave additive effects. In denervated bladders, synergistic effects were seen with the 10:1 and 1:1 (methoctramine:p-F-HHSiD wt/wt) combinations. After application of the sarcoplasmic reticulum ATPase inhibitor thapsigargin to normal tissue, the 10:1 and 1:1 ratios became synergistic, mimicking denervated tissue. Thus in normal bladders both M2 and M3 receptors can induce contraction. In the denervated bladder, the M2 and the M3 receptors interact in a facilitatory manner to mediate contraction. PMID:12185001

  14. Interaction between muscarinic receptor subtype signal transduction pathways mediating bladder contraction.

    Science.gov (United States)

    Braverman, Alan S; Tallarida, Ronald J; Ruggieri, Michael R

    2002-09-01

    M(3) muscarinic receptors mediate cholinergic-induced contraction in most smooth muscles. However, in the denervated rat bladder, M(2) receptors participate in contraction because M(3)-selective antagonists [para-fluoro-hexahydro-sila-diphenidol (p-F-HHSiD) and 4-DAMP] have low affinities. However, the affinity of the M(2)-selective antagonist methoctramine in the denervated bladder is consistent with M(3) receptor mediating contraction. It is possible that two pathways interact to mediate contraction: one mediated by the M(2) receptor and one by the M(3) receptor. To determine whether an interaction exists, the inhibitory potencies of combinations of methoctramine and p-F-HHSiD for reversing cholinergic contractions were measured. In normal bladders, all combinations gave additive effects. In denervated bladders, synergistic effects were seen with the 10:1 and 1:1 (methoctramine:p-F-HHSiD wt/wt) combinations. After application of the sarcoplasmic reticulum ATPase inhibitor thapsigargin to normal tissue, the 10:1 and 1:1 ratios became synergistic, mimicking denervated tissue. Thus in normal bladders both M(2) and M(3) receptors can induce contraction. In the denervated bladder, the M(2) and the M(3) receptors interact in a facilitatory manner to mediate contraction.

  15. Dissemination of NDM-1-Producing Enterobacteriaceae Mediated by the IncX3-Type Plasmid.

    Directory of Open Access Journals (Sweden)

    Qing Yang

    Full Text Available The emergence and spread of NDM-1-producing Enterobacteriaceae have resulted in a worldwide public health risk that has affected some provinces of China. China is an exceptionally large country, and there is a crucial need to investigate the epidemic of blaNDM-1-positive Enterobacteriaceae in our province. A total of 186 carbapenem-resistant Enterobacteriaceae isolates (CRE were collected in a grade-3 hospital in Zhejiang province. Carbapenem-resistant genes, including blaKPC, blaIMP, blaVIM, blaOXA-48 and blaNDM-1 were screened and sequenced. Ninety isolates were identified as harboring the blaKPC-2 genes, and five blaNDM-1-positive isolates were uncovered. XbaI-PFGE revealed that three blaNDM-1-positive K. pneumoniae isolates belonged to two different clones. S1-PFGE and southern blot suggested that the blaNDM-1 genes were located on IncX3-type plasmids with two different sizes ranging from 33.3 to 54.7 kb (n=4 and 104.5 to 138.9 kb (n=1, respectively, all of which could easily transfer to Escherichia coli by conjugation and electrotransformation. The high-throughput sequencing of two plasmids was performed leading to the identification of a smaller 54-kb plasmid, which had high sequence similarity with a previously reported pCFNDM-CN, and a larger plasmid in which only a 7.8-kb sequence of a common gene environment around blaNDM-1 (blaNDM-1-trpF- dsbC-cutA1-groEL-ΔInsE, was detected. PCR mapping and sequencing demonstrated that four smaller blaNDM-1 plasmids contained a common gene environment around blaNDM-1 (IS5-blaNDM-1-trpF- dsbC-cutA1-groEL. We monitored the CRE epidemic in our hospital and determined that KPC-2 carbapenemase was a major risk to patient health and the IncX3-type plasmid played a vital role in the spread of the blaNDM-1 gene among the CRE.

  16. Lentiviral-mediated gene delivery in human monocyte-derived dendritic cells: optimized design and procedures for highly efficient transduction compatible with clinical constraints.

    Science.gov (United States)

    Rouas, Redouane; Uch, Rathviro; Cleuter, Yvette; Jordier, François; Bagnis, Claude; Mannoni, Patrice; Lewalle, Philippe; Martiat, Philippe; Van den Broeke, Anne

    2002-09-01

    Gene delivery to dendritic cells (DCs) could represent a powerful method of inducing potent, long-lasting immunity. Although recent studies underline the intense interest in lentiviral vector-mediated monocyte-derived DC transduction, efficient gene transfer methods currently require high multiplicities of infection and are not compatible with clinical constraints. We have designed a strategy to optimize the efficiency and clinical relevance of this approach. Initially, using a third generation lentiviral vector expressing green fluorescent protein, we found that modifying the vector design, the DC precursor cell type, and the DC differentiation stage for transduction results in sustained transgene expression in 75-85% of immature DCs (transduction at a multiplicity of infection of 8). This high efficiency was reproducible among different donors irrespective of whether DCs were expanded from fresh or cryopreserved CD14(+) precursors. We then developed procedures that bypass the need for highly concentrated lentiviral preparations and the addition of polybrene to achieve efficient transduction. DCs transduced under these conditions retain their immature phenotype and immunostimulatory potential in both autologous and allogeneic settings. Furthermore, genetically modified DCs maintain their ability to respond to maturation signals and secrete bioactive IL-12, indicating that they are fully functional. Finally, the level of transgene expression is preserved in the therapeutically relevant mature DCs, demonstrating that there is neither promoter-silencing nor loss of transduced cells during maturation. The novel approach described should advance lentiviral-mediated monocyte-derived DC transduction towards a clinical reality.

  17. [The effectiveness of empirical antibiotic therapy of pyelonephritis in patients with type 2 diabetes and without depending on the availability of plasmid-mediated resistance genes].

    Science.gov (United States)

    Chub, O I; Bilchenko, A V

    2015-02-01

    Multi-drug resistance has been increasing in the treatment of urinary tract infections, especially complicated. The prevalence of plasmid-mediated resistance genes among urinary pathogens has nether been studied in Ukraine. So, the aim of our study was to identify the plasmid-mediated resistance genes and to determine their impact on the efficacy of the treatment. A total of 105 adult patients with chronic pyelonephritis were included in the study. Among them, 32 patients were diagnosed with type 2 diabetes mellitus. The diagnosis of pyelonephritis was verified according to the criteria EAU, 2013. Plasmid-mediated resistance genes were determined by polymerase chain reaction (PCR). The prevalence of plasmid-mediated resistance mechanisms among patients with pyelonephritis were 44,4%. ESBLs was the most common isolated genes. Favorable clinical response was seen in 11/31 (35,5%) infected with ESBL-producing organisms compared with 59/74 (79,7%) patients with non-ESBL-producing organisms (ppyelonephritis due to presence of plasmid-mediated resistance genes. Therefore, prоpеr mаnagеment fоr prescriptiоn of аntibiоtics and also idеntificаtiоn of ESBL-prоducing bаcteria in cоmmunitiеs arе impоrtant fоr prevеntion.

  18. Impact of carbondiimide crosslinker used for magnetic carbon nanotube mediated GFP plasmid delivery

    Energy Technology Data Exchange (ETDEWEB)

    Hao Yuzhi; He Chuan; Yang Xiaoyan; Huang Min; Chen Jie [Department of Electrical and Computer Engineering, University of Alberta, Edmonton, AB (Canada); Xu Peng [National Institute of Nanotechnology, Edmonton, AB (Canada); Xing, James [IntelligentNano Incorporated, Edmonton, AB (Canada)

    2011-07-15

    1-ethyl-3-(3-dimethylaminopropyl) carbondiimide hydrochloride (EDC) is commonly used as a crosslinker to help bind biomolecules, such as DNA plasmids, with nanostructures. However, EDC often remains, after a crosslink reaction, in the micro-aperture of the nanostructure, e.g., carbon nanotube. The remaining EDC shows positive green fluorescent signals and makes a nanostructure with a strong cytotoxicity which induces cell death. The toxicity of EDC was confirmed on a breast cancer cell line (MCF-7) and two leukemic cell lines (THP-1 and KG-1). The MCF-7 cells mainly underwent necrosis after treatment with EDC, which was verified by fluorescein isothiocyanate (FITC) annexin V staining, video microscopy and scanning electronic microscopy (SEM). If the EDC was not removed completely, the nanostructures with remaining EDC produced a green fluorescent background that could interfere with flow cytometry (FACS) measurement and result in false information about GFP plasmid delivery. Effective methods to remove residual EDC on macromolecules were also developed.

  19. Plasmid Mediated Resistance to Cephalosporin and Adhesion Properties in E.Coli

    Directory of Open Access Journals (Sweden)

    Salwa Oufrid

    2014-02-01

    Full Text Available Introduction: The objective of this study is to evaluate the relationship between biofilm formation, surface characteristics and the presence of plasmid conferring resistance to cephalosporin Methodology: The plasmid of resistance of Salmonella 3349 was purified and transferred by electroporation to the E. coli DH10B originally incompetent to form biofilm. The physico-chemical surface properties of the three bacteria (E. coli DH10B, Salmonella 3349 and its isogenic transformant 3519EC1 were estimated and compared by the Microbial Adhesion to Solvents test (MAST and angle contact measurement. Cellular densities of bacteria adhered to stainless supports were examined with a scanning electron microscope. Results: The physicochemical properties of bacterial cell surface demonstrated that E.coli DH10B strain was hydrophilic, electron donating and weakly electron accepting than Salmonella 3349 and its transformant 3519EC1 strains. Moreover, there was a weak correlation between the acid-base properties determined by the Microbial Adhesion to Solvents test and angle contact measurement. Analysis of microscopical images of bacterial adhesion indicated that E.coli 3519EC1 and Salmonella 3349 adhered to the stainless surface, whereas the E.coli DH10B does not adhere. Conclusions: The results of this study suggest that the presences of the plasmid of resistance modify the microbial surface properties and biofilm formation.

  20. High Prevalence of Plasmid-Mediated Quinolone Resistance and IncQ Plasmids Carrying qnrS2 Gene in Bacteria from Rivers near Hospitals and Aquaculture in China.

    Directory of Open Access Journals (Sweden)

    Yanping Wen

    Full Text Available Effluents from hospital and aquaculture are considered important sources of quinolone resistance. However, little information is available on the impact of this effluent on nearby rivers. In this study, 188 ciprofloxacin-resistant bacterial isolates obtained from rivers near hospitals and aquaculture were screened for plasmid-mediated quinolone resistance (PMQR genes. Species identification, antibiotic susceptibility testing, and PMQR gene transferability assessment were conducted for PMQR-positive bacteria. Representative qnrS2-encoding plasmids were subsequently sequenced using a primer-walking approach. In total, 44 isolates (23.4% were positive for qnr genes (16 qnrB2, 3 qnrS1, and 25 qnrS2 and 32 isolates (17.0% were positive for aac(6'-Ib-cr. Other PMQR genes were not detected. The qnrB2 and aac(6'-Ib-cr genes had a higher prevalence in aquaculture samples than in hospital samples, and were significantly associated with Enterobacteriaceae (p < 0.05. In contrast, the prevalence of qnrS2 was not site-related, but was significantly associated with Aeromonas spp. (p < 0.05. All PMQR isolates were resistant to three or more classes of antibiotics. Eleven qnrS2-harboring plasmids from Aeromonas spp., including a novel conjugative plasmid pHP18, were selected for sequencing. These plasmids were small in size (6,388-16,197 bp and belonged to the IncQ or IncU plasmid family, with qnrS2 being part of a mobile insertion cassette. Taken together, our findings suggest that aquaculture is a possible source for aac(6'-Ib-cr and qnrB2 dissemination, and demonstrate the ubiquity of qnrS2 in aquatic environments. Finally, Aeromonas spp. served as vectors for qnrS2 with the help of IncQ-type plasmids.

  1. Molecular Mechanisms of Bcl10-Mediated NF-kB Signal Transduction

    Science.gov (United States)

    2006-03-08

    metabolizes lactose into glucose and galactose BinCARD (Bcl10-interacting protein with CARD): a CARD protein capable of inhibiting Bcl10-mediated... crystallized , so its complete structure is unknown. While it possesses an N-terminal CARD based on sequence homology, its C-terminus is of unknown...and function of Bcl10. These data could then be used to support the data gained from a crystallized Bcl10. Currently, this lab is collaborating

  2. Novel Plasmid-Mediated Colistin Resistance Gene mcr-3 in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Wenjuan Yin

    2017-06-01

    Full Text Available The mobile colistin resistance gene mcr-1 has attracted global attention, as it heralds the breach of polymyxins, one of the last-resort antibiotics for the treatment of severe clinical infections caused by multidrug-resistant Gram-negative bacteria. To date, six slightly different variants of mcr-1, and a second mobile colistin resistance gene, mcr-2, have been reported or annotated in the GenBank database. Here, we characterized a third mobile colistin resistance gene, mcr-3. The gene coexisted with 18 additional resistance determinants in the 261-kb IncHI2-type plasmid pWJ1 from porcine Escherichia coli. mcr-3 showed 45.0% and 47.0% nucleotide sequence identity to mcr-1 and mcr-2, respectively, while the deduced amino acid sequence of MCR-3 showed 99.8 to 100% and 75.6 to 94.8% identity to phosphoethanolamine transferases found in other Enterobacteriaceae species and in 10 Aeromonas species, respectively. pWJ1 was mobilized to an E. coli recipient by conjugation and contained a plasmid backbone similar to those of other mcr-1-carrying plasmids, such as pHNSHP45-2 from the original mcr-1-harboring E. coli strain. Moreover, a truncated transposon element, TnAs2, which was characterized only in Aeromonas salmonicida, was located upstream of mcr-3 in pWJ1. This ΔTnAs2-mcr-3 element was also identified in a shotgun genome sequence of a porcine E. coli isolate from Malaysia, a human Klebsiella pneumoniae isolate from Thailand, and a human Salmonella enterica serovar Typhimurium isolate from the United States. These results suggest the likelihood of a wide dissemination of the novel mobile colistin resistance gene mcr-3 among Enterobacteriaceae and aeromonads; the latter may act as a potential reservoir for mcr-3.

  3. Methoxychlor enhances degranulation of murine mast cells by regulating FcεRI-mediated signal transduction.

    Science.gov (United States)

    Yasunaga, Sho; Nishi, Kosuke; Nishimoto, Sogo; Sugahara, Takuya

    2015-01-01

    Methoxychlor, an organochlorine insecticide developed to replace DDT (dichlorodiphenyltrichloroethane), has been reported to induce mast cell degranulation and to enhance IgE-mediated allergic responses. However, the mechanisms underlying these effects are not clear. To clarify potential mechanisms, the effects of methoxychlor on degranulation of mast cells were examined. Degranulation responses were evaluated using RBL-2H3 cells and mouse bone marrow-derived mast cells with either the antigen-induced or calcium ionophore-induced stimulation. Phosphorylation of enzymes related to signaling events associated with mast cell degranulation was analyzed by immunoblotting. Effects on vascular permeability in the passive cutaneous anaphylaxis reaction were evaluated following oral administration of methoxychlor to BALB/c mice. The results indicated that methoxychlor caused increased mast cell degranulation in the presence of antigen, whereas it had no effect on calcium ionophore-induced degranulation of RBL-2H3 cells. Immunoblot analyses demonstrated that the phosphorylation level of phosphoinositide 3-kinase (which plays a central role in mast cell signaling) was increased by methoxychlor during antigen-induced degranulation. In addition, methoxychlor activated the signaling pathway via the high-affinity IgE receptor by inducing phosphorylation of Syk and PLCγ1/2, which transfer the signal for degranulation downstream. Lastly, oral administration of methoxychlor exhibited a tendency to promote vascular permeability in passive cutaneous anaphylaxis model mice. Taken together, the results here suggested that methoxychlor enhanced degranulation through FcεRI-mediated signaling and promoted allergenic symptoms involved in mast cell degranulation.

  4. Multiplex PCR Study of Plasmid-Mediated AmpC Beta-Lactamases Genes in Clinical Isolates of Escherichia coli

    Directory of Open Access Journals (Sweden)

    Maryam Dehghani

    2017-02-01

    Full Text Available Background:   AmpC β-lactamases are important cephalosporinases chromosomally encoded in many of Enterobacteriaceae and a few other organisms where they mediate resistance to cephalothin, cefazolin, cefoxitin and penicillins. The six different families of plasmid-mediated AmpC β-lactamases have been described, but no phenotypic test can discriminate among them. AmpC multiplex PCR has been successfully used to discriminate plasmid-mediated ampC specific families in organisms such as Klebsiella pneumonia and Escherichia coli. The aim of this study was to indicate the prevalence of AmpC β-lactamase genes by specifically designed primers through PCR test.Methods:   243 total clinical urine samples were collected, and 227 isolates were identified as Escherichia coli based on standard biochemical tests. Subsequently, the isolates were screened by disc diffusion and combined disc test for β-lactamase production. Resistant isolates were evaluated by PCR for ampC family determination. Results:  Antibiotic resistance pattern were observed as follows: cefepime (%25, ceftazidime (%31, ceftriaxone (%37, cefotaxime (%38. The ratio of isolates was detected as ESBLs and AmpC producers were 34% and 5.2%, respectively. PCR performed on 12 selected isolates via phenotypic tests and the results revealed that among 12 isolates, 11 contained blaCMY-42. Conclusion:  Unfortunately, antibiotic resistance has become an increasingly critical problem in many countries like Iran and occurrence of isolates co-expressing AmpC-β-lactamases and ESBLs can create serious problems in the future. As antibiotic options in the treatment of AmpC β-lactamases and ESBLs producing organisms are extremely limited, molecular screening by laboratories is suggested to reduce the risk of therapeutic defeat.

  5. Analysis of plasmid-mediated multidrug resistance in Escherichia coli and Klebsiella oxytoca isolates from clinical specimens in Japan.

    Science.gov (United States)

    Ode, Takashi; Saito, Ryoichi; Kumita, Wakako; Sato, Kenya; Okugawa, Shu; Moriya, Kyoji; Koike, Kazuhiko; Okamura, Noboru

    2009-10-01

    This study investigated the relationship of plasmid-mediated quinolone resistance (PMQR) and aminoglycoside resistance among oxyimino-cephalosporin-resistant Escherichia coli (n=46) and Klebsiella oxytoca (n=28) clinical isolates in Japan. Seventy-three isolates appeared to produce an extended-spectrum beta-lactamase (ESBL) and one K. oxytoca isolate produced IMP-1 metallo-beta-lactamase (MBL). Polymerase chain reaction (PCR) and sequencing confirmed that eight CTX-M-9/SHV-12-producing isolates, one IMP-1-producing K. oxytoca isolate, and six ESBL-positive E. coli isolates respectively possessed PMQR genes qnrA1, qnrB6, and aac(6')-Ib-cr. All qnr-positive isolates also carried either aac(6')-Ib or aac(6')-IIc aminoglycoside acetyltransferase genes. Resistance determinants to beta-lactams, quinolones and aminoglycosides were co-transferred with a plasmid of ca. 140 kb. The qnrA1 gene was located downstream of insertion sequence ISCR1 in complex class 1 integrons. A novel qnrA1-carrying class 1 integron with the cassette arrangement aac(6')-IIc-aadA2 as well as a unique class 1 integron with bla(IMP-1)-aac(6')-IIc cassettes on the plasmid carrying qnrB6 were found in K. oxytoca isolates. We describe the identification of qnrB6 and aac(6')-Ib-cr and the close association of qnr with aac(6')-Ib and aac(6')-IIc for the first time in clinical isolates producing ESBL or MBL in Japan.

  6. Prevalence of plasmid mediated pesticide resistant bacterial assemblages in crop fields.

    Science.gov (United States)

    Umamaheswari, S; Murali, M

    2010-11-01

    Three crop fields namely paddy sugarcane and tomato exposed to bavistin [Methyl (1H-benzimidazol-2-yl) carbomate], monocrotophos[Dimethyl(E)-1-methyl-2-(methyl-carbamoyl) vinyl phosphate] and kinado plus [(EZ)-2-chloro-3-dimethoxyphosphinoyloxy-X1, X1-diethylbut-2-enamide], respectively were chosen for the present investigation to know the bacterial population and degradation of pesticides. The chemical nature of the soil and water samples from the pesticide contaminated fields was analysed along with counting of the total heterotrophic bacteria (THB), Staphylococci and Enterococcci population. Mean calcium, phosphate and biological oxygen demand were maximum in tomato field water Field water recorded maximum phophate and silicate content, whereas, sugarcane field water elicited maximum dissolved oxygen content. On the other hand, available phosphate and exchangeable potassium were maximum is sugarcane field soil. Significant variations in the bacterial population were evident between the treatments in sugarcane field soil and tomato field water exposed to monocrotophos and kinado plus, respectively In addition, significant variations between THB, Staphlyococci and Enterococci population were also evinced in both the sugarcane andtomato fields. The dominant pesticide resistant bacteria, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeuroginosa harboured plasmids and the resistant trait observed were found to be plasmid borne.

  7. The Role of Intrinsic Flexibility in Signal Transduction Mediated by the Cell Cycle Regulator, p27Kip1

    Energy Technology Data Exchange (ETDEWEB)

    Galea, Charles A. [St. Jude Children' s Research Hospital; Nourse, Amanda [St. Jude Children' s Research Hospital; Wang, Yuefeng [St. Jude Children' s Research Hospital; Sivakolundu, Sivashankar G. [St. Jude Children' s Research Hospital; Heller, William T [ORNL; Kriwacki, Richard W [University of Tennessee (UT) Health Science Center, Memphis

    2008-02-01

    p27{sup Kip1} (p27), which controls eukaryotic cell division through interactions with cyclin-dependent kinases (Cdks), integrates and transduces promitogenic signals from various nonreceptor tyrosine kinases by orchestrating its own phosphorylation, ubiquitination and degradation. Intrinsic flexibility allows p27 to act as a 'conduit' for sequential signaling mediated by tyrosine and threonine phosphorylation and ubiquitination. While the structural features of the Cdk/cyclin-binding domain of p27 are understood, how the C-terminal regulatory domain coordinates multistep signaling leading to p27 degradation is poorly understood. We show that the 100-residue p27 C-terminal domain is extended and flexible when p27 is bound to Cdk2/cyclin A. We propose that the intrinsic flexibility of p27 provides a molecular basis for the sequential signal transduction conduit that regulates p27 degradation and cell division. Other intrinsically unstructured proteins possessing multiple sites of posttranslational modification may participate in similar signaling conduits.

  8. The cAMP-mediated protein kinase signal transduction pathway is involved in the pyrogenic effect of CRH in rats

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The primary action of corticotropin releasing hormone (CRH) is stimulation of the synthesis and release of adrenocorticotropic hormone and β-endorphin from the pituitary in response to stress. In addition, a number of studies indicate that CRH exerts other physiological actions within the central nervous system which are independent of the pituitary. These include increased body temperature and thermogenesis. However, the intracellular mechanism responsible for pyrogenic action of CRH is still unclear. The purpose of these studies was to determine whether or not cAMP was involved in the pyrogenic action of CRH in the rat. Intracerebroventricular (icv) microinjection of CRH (2.5 μg, 5.0 μg, 10 μg) caused increases in colonic temperature and hypothalamus cAMP level in conscious rats. The pyrogenic effects of CRH were abolished or markedly inhibited by prior injection (icv) of an adenylate cyclase inhibitor, 2,,3,-dideoxyadenosine (DDA, 30 μg) or an inhibitor of cAMP-dependent protein kinase, adenosine-3,,5,-(cyclic) monophosphorothionate (Rp-cAMPs, 15 μg). This is the first report demonstrating the pyrogenic effcet of centrally administration of CRH on the rat via the cAMP-mediated protein kinase signal transduction pathway.

  9. Two-component mediated peroxide sensing and signal transduction in fission yeast.

    Science.gov (United States)

    Quinn, Janet; Malakasi, Panagiota; Smith, Deborah A; Cheetham, Jill; Buck, Vicky; Millar, Jonathan B A; Morgan, Brian A

    2011-07-01

    Two-component related proteins play a major role in regulating the oxidative stress response in the fission yeast, Schizosaccharomyces pombe. For example, the peroxide-sensing Mak2 and Mak3 histidine kinases regulate H(2)O(2)-induced activation of the Sty1 stress-activated protein kinase pathway, and the Skn7-related response regulator transcription factor, Prr1, is essential for activation of the core oxidative stress response genes. Here, we investigate the mechanism by which the S. pombe two-component system senses H(2)O(2), and the potential role of two-component signaling in the regulation of Prr1. Significantly, we demonstrate that PAS and GAF domains present in the Mak2 histidine kinase are essential for redox-sensing and activation of Sty1. In addition, we find that Prr1 is required for the transcriptional response to a wide range of H(2)O(2) concentrations and, furthermore, that two-component regulation of Prr1 is specifically required for the response of cells to high levels of H(2)O(2). Significantly, this provides the first demonstration that the conserved two-component phosphorylation site on Skn7-related proteins influences resistance to oxidative stress and oxidative stress-induced gene expression. Collectively, these data provide new insights into the two-component mediated sensing and signaling mechanisms underlying the response of S. pombe to oxidative stress.

  10. The signal transduction mediated by erythropoietin and proinflammatory cytokines in the JAK/STAT pathway in the children with cerebral palsy.

    Science.gov (United States)

    Tao, Weiyuan; Wen, Fang; Zhang, Hong; Liu, Guheng

    2009-03-01

    It is well established that erythropoietin (EPO) is a pleiotropic cytokine, which has a brain-derived neuroprotective effect in the central nervous system (CNS). Immune abnormality has a close relationship with cerebral palsy (CP), and may be even involved in the development of CP. There is evidence that the amount of EPO in CP children is lower than in normal children, but the levels of proinflammatory cytokines, such as interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha, are higher in the CP children. The signal transduction mediated by EPO that has a neuroprotective effect and mediated by proinflammatory cytokines that lead to brain damage shares the common JAK/STAT pathway. Under acute stress, the JAK/STAT pathway is occupied by massive proinflammatory cytokines, and the negative feedback inhibition factors like suppressor of cytokine signaling (SOCS) proteins are simultaneously activated, which exist in reciprocal inhibition to EPO in the JAK/STAT pathway. As a result, the signal transduction mediated by EPO is prevented or reduced, and the neuroprotective effect of EPO is eventually weakened. In this review, a novel approach to CP treatment through neurodevelopmental treatment (NDT) is put forward by analysis of the interrelationship of signal transduction mediated by EPO and proinflammatory cytokines in the JAK/STAT pathway and their roles in the development of CP, and some reasonable ideas for CP treatment are provided.

  11. Single primer-mediated circular polymerase chain reaction for hairpin DNA cloning and plasmid editing.

    Science.gov (United States)

    Huang, Jiansheng; Khan, Inamullah; Liu, Rui; Yang, Yan; Zhu, Naishuo

    2016-05-01

    We developed and validated a universal polymerase chain reaction (PCR) method, single primer circular (SPC)-PCR, using single primer to simultaneously insert and amplify a short hairpin sequence into a vector with a high success rate. In this method, the hairpin structure is divided into two parts and fused into a vector by PCR. Then, a single primer is used to cyclize the chimera into a mature short hairpin RNA (shRNA) expression vector. It is not biased by loop length or palindromic structures. Six hairpin DNAs with short 4-nucleotide loops were successfully cloned. Moreover, SPC-PCR was also applied to plasmid editing within 3 h with a success rate higher than 95%.

  12. Microbiological characterization of plasmid-mediated AmpC ß-lactamases and E. coli hyperproducers: how and why ?

    Directory of Open Access Journals (Sweden)

    Annibale Raglio

    2010-03-01

    Full Text Available The aim of this study is the evaluation of phenotypic method for the detection of plasmid-mediated AmpC producing Enterobacteriaceae by agar diffusion.We developed a phenotypic method with double disk test (CLSI and evaluation of synergism between Cloxacillin and/or Boronic Acid with cefotaxime and ceftazidime and cefepime with amoxicillin/clavulanic acid. As reference method for AmpC detection we used a multiplex PCR according to Perez-Perez. Among 7476 Enterobacteriaceae we detected 45 strains: 37 (82.2% plasmid-mediated AmpC producers, 6 (13.3% E. coli hyperproducers and 2 E. coli (4.5% positive for both.The AmpC phenotypic test was positive for all the isolates, showing a typical ghost zone between cloxacillin and cephalosporins or boronic acid and cephalosporins.The AmpC multiplex PCR confirmed that 28 P. mirabilis and 7 E. coli harboured a gene belonging to the bla-CMY-LAT family. Sequencing defined the presence of CMY-16 in all P. mirabilis, CMY-2 in E. coli, DHA-1 in 3 K. pneumoniae and FOX in 1 K. pneumoniae and allowed us to identify eight strains as E. coli hyperproducer: six E. coli yielded no amplicon and 2 were also producer of CMY-2. In this study the phenotypic method showed a sensitivity and a specificity of 100%.Waiting for the indication of international authorities, we think this phenotypic screening method could be useful in the routine of microbiological laboratories.

  13. Laboratory surveillance for prospective plasmid-mediated AmpC beta-lactamases in the Kinki region of Japan.

    Science.gov (United States)

    Yamasaki, Katsutoshi; Komatsu, Masaru; Abe, Noriyuki; Fukuda, Saori; Miyamoto, Yugo; Higuchi, Takeshi; Ono, Tamotsu; Nishio, Hisaaki; Sueyoshi, Noriyuki; Kida, Kaneyuki; Satoh, Kaori; Toyokawa, Masahiro; Nishi, Isao; Sakamoto, Masako; Akagi, Masahiro; Nakai, Isako; Kofuku, Tomomi; Orita, Tamaki; Wada, Yasunao; Jikimoto, Takumi; Kinoshita, Shohiro; Miyamoto, Kazuaki; Hirai, Itaru; Yamamoto, Yoshimasa

    2010-09-01

    Extended-spectrum beta-lactamases, plasmid-mediated AmpC beta-lactamases (PABLs), and plasmid-mediated metallo-beta-lactamases confer resistance to many beta-lactams. In Japan, although several reports exist on the prevalence of extended-spectrum beta-lactamases and metallo-beta-lactamases, the prevalence and characteristics of PABLs remain unknown. To investigate the production of PABLs, a total of 22,869 strains of 4 enterobacterial species, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis, were collected during six 6-month periods from 17 clinical laboratories in the Kinki region of Japan. PABLs were detected in 29 (0.13%) of 22,869 isolates by the 3-dimensional test, PCR analysis, and DNA sequencing analysis. PABL-positive isolates were detected among isolates from 13 laboratories. Seventeen of 13,995 (0.12%) E. coli isolates, 8 of 5,970 (0.13%) K. pneumoniae isolates, 3 of 1,722 (0.17%) K. oxytoca isolates, and 1 of 1,182 (0.08%) P. mirabilis isolates were positive for PABLs. Of these 29 PABL-positive strains, 20 (69.0%), 6 (20.7%), 2 (6.9%), and 1 (3.4%) carried the genes for CMY-2, DHA-1, CMY-8, and MOX-1 PABLs, respectively. Pattern analysis of randomly amplified polymorphic DNA and pulsed-field gel electrophoretic analysis revealed that the prevalence of CMY-2-producing E. coli strains was not due to epidemic strains and that 3 DHA-1-producing K. pneumoniae strains were identical, suggesting their clonal relatedness. In conclusion, the DHA-1 PABLs were predominantly present in K. pneumoniae strains, but CMY-2 PABLs were predominantly present in E. coli strains. The present findings will provide significant information to assist in preventing the emergence and further spread of PABL-producing bacteria.

  14. Competition triggers plasmid-mediated enhancement of substrate utilisation in Pseudomonas putida.

    Directory of Open Access Journals (Sweden)

    Hiren Joshi

    Full Text Available Competition between species plays a central role in the activity and structure of communities. Stable co-existence of diverse organisms in communities is thought to be fostered by individual tradeoffs and optimization of competitive strategies along resource gradients. Outside the laboratory, microbes exist as multispecies consortia, continuously interacting with one another and the environment. Survival and proliferation of a particular species is governed by its competitive fitness. Therefore, bacteria must be able to continuously sense their immediate environs for presence of competitors and prevailing conditions. Here we present results of our investigations on a novel competition sensing mechanism in the rhizosphere-inhabiting Pseudomonas putida KT2440, harbouring gfpmut3b-modified Kan(R TOL plasmid. We monitored benzyl alcohol (BA degradation rate, along with GFP expression profiling in mono species and dual species cultures. Interestingly, enhanced plasmid expression (monitored using GFP expression and consequent BA degradation were observed in dual species consortia, irrespective of whether the competitor was a BA degrader (Pseudomonas aeruginosa or a non-degrader (E. coli. Attempts at elucidation of the mechanistic aspects of induction indicated the role of physical interaction, but not of any diffusible compounds emanating from the competitors. This contention is supported by the observation that greater induction took place in presence of increasing number of competitors. Inert microspheres mimicking competitor cell size and concentration did not elicit any significant induction, further suggesting the role of physical cell-cell interaction. Furthermore, it was also established that cell wall compromised competitor had minimal induction capability. We conclude that P. putida harbouring pWW0 experience a competitive stress when grown as dual-species consortium, irrespective of the counterpart being BA degrader or not. The immediate

  15. TLA-1: a new plasmid-mediated extended-spectrum beta-lactamase from Escherichia coli.

    Science.gov (United States)

    Silva, J; Aguilar, C; Ayala, G; Estrada, M A; Garza-Ramos, U; Lara-Lemus, R; Ledezma, L

    2000-04-01

    Escherichia coli R170, isolated from the urine of an infected patient, was resistant to expanded-spectrum cephalosporins, aztreonam, ciprofloxacin, and ofloxacin but was susceptible to amikacin, cefotetan, and imipenem. This particular strain contained three different plasmids that encoded two beta-lactamases with pIs of 7.0 and 9.0. Resistance to cefotaxime, ceftazidime, aztreonam, trimethoprim, and sulfamethoxazole was transferred by conjugation from E. coli R170 to E. coli J53-2. The transferred plasmid, RZA92, which encoded a single beta-lactamase, was 150 kb in length. The cefotaxime resistance gene that encodes the TLA-1 beta-lactamase (pI 9.0) was cloned from the transconjugant by transformation to E. coli DH5alpha. Sequencing of the bla(TLA-1) gene revealed an open reading frame of 906 bp, which corresponded to 301 amino acid residues, including motifs common to class A beta-lactamases: (70)SXXK, (130)SDN, and (234)KTG. The amino acid sequence of TLA-1 shared 50% identity with the CME-1 chromosomal class A beta-lactamase from Chryseobacterium (Flavobacterium) meningosepticum; 48.8% identity with the VEB-1 class A beta-lactamase from E. coli; 40 to 42% identity with CblA of Bacteroides uniformis, PER-1 of Pseudomonas aeruginosa, and PER-2 of Salmonella typhimurium; and 39% identity with CepA of Bacteroides fragilis. The partially purified TLA-1 beta-lactamase had a molecular mass of 31.4 kDa and a pI of 9.0 and preferentially hydrolyzed cephaloridine, cefotaxime, cephalothin, benzylpenicillin, and ceftazidime. The enzyme was markedly inhibited by sulbactam, tazobactam, and clavulanic acid. TLA-1 is a new extended-spectrum beta-lactamase of Ambler class A.

  16. TLA-1: a New Plasmid-Mediated Extended-Spectrum β-Lactamase from Escherichia coli

    Science.gov (United States)

    Silva, J.; Aguilar, C.; Ayala, G.; Estrada, M. A.; Garza-Ramos, U.; Lara-Lemus, R.; Ledezma, L.

    2000-01-01

    Escherichia coli R170, isolated from the urine of an infected patient, was resistant to expanded-spectrum cephalosporins, aztreonam, ciprofloxacin, and ofloxacin but was susceptible to amikacin, cefotetan, and imipenem. This particular strain contained three different plasmids that encoded two β-lactamases with pIs of 7.0 and 9.0. Resistance to cefotaxime, ceftazidime, aztreonam, trimethoprim, and sulfamethoxazole was transferred by conjugation from E. coli R170 to E. coli J53-2. The transferred plasmid, RZA92, which encoded a single β-lactamase, was 150 kb in length. The cefotaxime resistance gene that encodes the TLA-1 β-lactamase (pI 9.0) was cloned from the transconjugant by transformation to E. coli DH5α. Sequencing of the blaTLA-1 gene revealed an open reading frame of 906 bp, which corresponded to 301 amino acid residues, including motifs common to class A β-lactamases: 70SXXK, 130SDN, and 234KTG. The amino acid sequence of TLA-1 shared 50% identity with the CME-1 chromosomal class A β-lactamase from Chryseobacterium (Flavobacterium) meningosepticum; 48.8% identity with the VEB-1 class A β-lactamase from E. coli; 40 to 42% identity with CblA of Bacteroides uniformis, PER-1 of Pseudomonas aeruginosa, and PER-2 of Salmonella typhimurium; and 39% identity with CepA of Bacteroides fragilis. The partially purified TLA-1 β-lactamase had a molecular mass of 31.4 kDa and a pI of 9.0 and preferentially hydrolyzed cephaloridine, cefotaxime, cephalothin, benzylpenicillin, and ceftazidime. The enzyme was markedly inhibited by sulbactam, tazobactam, and clavulanic acid. TLA-1 is a new extended-spectrum β-lactamase of Ambler class A. PMID:10722503

  17. The angiogenic response to PLL-g-PEG-mediated HIF-1α plasmid DNA delivery in healthy and diabetic rats.

    Science.gov (United States)

    Thiersch, Markus; Rimann, Markus; Panagiotopoulou, Vasiliki; Öztürk, Ece; Biedermann, Thomas; Textor, Marcus; Lühmann, Tessa C; Hall, Heike

    2013-05-01

    Impaired angiogenesis is a major clinical problem and affects wound healing especially in diabetic patients. Improving angiogenesis is a reasonable strategy to increase diabetes-impaired wound healing. Recently, our lab described a system of transient gene expression due to pegylated poly-l-lysine (PLL-g-PEG) polymer-mediated plasmid DNA delivery in vitro. Here we synthesized peptide-modified PLL-g-PEG polymers with two functionalities, characterized them in vitro and utilized them in vivo via a fibrin-based delivery matrix to induce dermal wound angiogenesis in diabetic rats. The two peptides were 1) a TG-peptide to covalently bind these nanocondensates to the fibrin matrix (TG-peptide) for a sustained release and 2) a polyR peptide to improve cellular uptake of these nanocondensates. In order to induce angiogenesis in vivo we condensed modified and non-modified polymers with plasmid DNA encoding a truncated form of the therapeutic candidate gene hypoxia-inducible transcription factor 1α (HIF-1α). HIF-1α is the primarily oxygen-dependent regulated subunit of the heterodimeric transcription factor HIF-1, which controls angiogenesis among other physiological pathways. The truncated form of HIF-1α lacks the oxygen-dependent degradation domain (ODD) and therefore escapes degradation under normoxic conditions. PLL-g-PEG polymer-mediated HIF-1α-ΔODD plasmid DNA delivery was found to lead to a transiently induced gene expression of angiogenesis-related genes Acta2 and Pecam1 as well as the HIF-1α target gene Vegf in vivo. Furthermore, HIF-1α gene delivery was shown to enhance the number endothelial cells and smooth muscle cells - precursors for mature blood vessels - during wound healing. We show that - depending on the selection of the therapeutic target gene - PLL-g-PEG nanocondensates are a promising alternative to viral DNA delivery approaches, which might pose a risk to health. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Occurrence of the Plasmid-Mediated Fluoroquinolone Resistance qepA1 Gene in Two Clonal Clinical Isolates of CTX-M-15-Producing Escherichia coli from Algeria.

    Science.gov (United States)

    Yanat, Betitera; Dali Yahia, Radia; Yazi, Leila; Machuca, Jesús; Díaz-De-Alba, Paula; Touati, Abdelaziz; Pascual, Álvaro; Rodríguez-Martínez, José-Manuel

    2016-10-13

    QepA is a plasmid-mediated quinolone resistance determinant of low prevalence described worldwide, mainly in Enterobacteriaceae. This study describes, for the first time in Algeria, two clonally related, QepA-producing Escherichia coli clinical isolates positive for CTX-M-15. The clonal spread of these multidrug-resistant isolates is a major public health concern.

  19. Detection of mcr-1 encoding plasmid-mediated colistin-resistant Escherichia coli isolates from human bloodstream infection and imported chicken meat, Denmark 2015

    DEFF Research Database (Denmark)

    Hasman, H.; Hammerum, A. M.; Hansen, F.

    2015-01-01

    The plasmid-mediated colistin resistance gene, mcr-1, was detected in an Escherichia coli isolate from a Danish patient with bloodstream infection and in five E. coli isolates from imported chicken meat. One isolate from chicken meat belonged to the epidemic spreading sequence type ST131. In addi...

  20. Characterization of Plasmid-Mediated AmpC and Carbapenemases among Iranain Nosocomial Isolates of Klebsiella pneumoniae Using Phenotyping and Genotyping Methods

    NARCIS (Netherlands)

    A. Japoni-Nejad (Alireza); E. Ghaznavi Rad (Ehsanollah); A.F. van Belkum (Alex)

    2014-01-01

    textabstractObjectives: Plasmid-mediated AmpC β-lactamases (PMABLs) and carbapenemases are emerging groups of antimicrobial-resistance determinants. The aims of the study were to evaluate the occurrence of PMABLs and carbapenemases in clinical isolates of Klebsiella pneumoniae and compare the test p

  1. Characterization of Plasmid-Mediated AmpC and Carbapenemases among Iranain Nosocomial Isolates of Klebsiella pneumoniae Using Phenotyping and Genotyping Methods

    NARCIS (Netherlands)

    A. Japoni-Nejad (Alireza); E. Ghaznavi Rad (Ehsanollah); A.F. van Belkum (Alex)

    2014-01-01

    textabstractObjectives: Plasmid-mediated AmpC β-lactamases (PMABLs) and carbapenemases are emerging groups of antimicrobial-resistance determinants. The aims of the study were to evaluate the occurrence of PMABLs and carbapenemases in clinical isolates of Klebsiella pneumoniae and compare the test p

  2. Thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR.

    Science.gov (United States)

    Le, Yilin; Chen, Huayou; Zagursky, Robert; Wu, J H David; Shao, Weilan

    2013-08-01

    Polymerase chain reaction (PCR) is a powerful method to produce linear DNA fragments. Here we describe the Tma thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR. In this thermostable DNA ligase-mediated whole-plasmid amplification method, the resultant DNA nick between the 5' end of the PCR primer and the extended newly synthesized DNA 3' end of each PCR cycle is ligated by Tma DNA ligase, resulting in circular plasmid DNA product that can be directly transformed. The template plasmid DNA is eliminated by 'selection marker swapping' upon transformation. When performed under an error-prone condition with Taq DNA polymerase, PPCP allows one-step construction of mutagenesis libraries based on in situ error-prone PCR so that random mutations are introduced into the target gene without altering the expression vector plasmid. A significant difference between PPCP and previously published methods is that PPCP allows exponential amplification of circular DNA. We used this method to create random mutagenesis libraries of a xylanase gene and two cellulase genes. Screening of these libraries resulted in mutant proteins with desired properties, demonstrating the usefulness of in situ error-prone PPCP for creating random mutagenesis libraries for directed evolution.

  3. Influence of ultrasound microbubble-mediated MCD-microRNA interference plasmid on heart function in the rat with myocardial infarction

    Directory of Open Access Journals (Sweden)

    Hong-quan WU

    2012-05-01

    Full Text Available Objective  To evaluate the effect of the targeted inhibition of myocardial malonyl-CoA decarboxylase (MCD by ultrasound microbubble mediated MCD-microRNA interference plasmids on cardiac function in rats with myocardial infarction (MI and its possible mechanism. Methods  The MCD expression plasmid and four MCD-microRNA interference plasmids to be screened were co-transfected into HEK293 cells. The levels of MCD mRNA were determined by real-time fluorescence quantitative PCR, and interference sequence with the highest rate of suppression was selected. MI model was reproduced by ligating the left anterior descending coronary artery. The 28 rats with myocardial infarction were randomly assigned into four experimental groups: MI + saline (group 1, MI + plasmid (group 2, MI + ultrasound + plasmid (group 3, MI + ultrasound + microbubble + plasmid (group 4, 7 rats in each group. An alternative group of sham-operated + saline served as control. The mixture of the selected interference plasmids and lipid microbubbles were co-transfected once every four days into rats with MI for 4 weeks mediated by ultrasound. After transfection for 28 days, the changes in left ventricular ejection fraction (LVEF, short axis fractional shortening (FS, left ventricular end-diastolic diameter (LVIDd were examined by echocardiography, and MCD and lactic acid levels were determined by high-performance liquid chromatography method and enzyme-linked immunosorbent assay respectively. Results  One MCD-microRNA interference plasmid with the highest inhibition rate, which was 82%, was selected according to the result of real-time fluorescence quantitative PCR. In animal experiments, the levels of LVEF and FS in the group 4 were both higher than those in other MI intervention groups (P 0.05, but it was significantly lower than that in the control group (P < 0.05. Conclusion  That MCD-microRNA interference plasmids were targetedly transfected into infarcted myocardium as

  4. Widespread distribution of CTX-M and plasmid-mediated AmpC β-lactamases in Escherichia coli from Brazilian chicken meat

    Directory of Open Access Journals (Sweden)

    Larissa Alvarenga Batista Botelho

    2015-04-01

    Full Text Available The dissemination of plasmid-mediated antimicrobial resistance genes may pose a substantial public health risk. In the present work, the occurrences of blaCTX-M and plasmid-mediated ampC and qnr genes were investigated in Escherichia coli from 16 chicken carcasses produced by four commercial brands in Brazil. Of the brands tested, three were exporters, including one of organic chicken. Our study assessed 136 E. coli isolates that were grouped into 77 distinct biotypes defined by their origin, resistance profiling, the presence of β-lactamase and plasmid-mediated quinolone resistance genes and enterobacterial repetitive intergenic consensus-polimerase chain reaction typing. The blaCTX-M-15, blaCTX-M-2 and blaCTX-M-8 genes were detected in one, 17 and eight different biotypes, respectively (45 isolates. Twenty-one biotypes (46 isolates harboured blaCMY-2. Additionally, blaCMY-2 was identified in isolates that also carried either blaCTX-M-2 or blaCTX-M-8. The qnrB and/or qnrS genes occurred in isolates carrying each of the four types of β-lactamase determinants detected and also in oxyimino-cephalosporin-susceptible strains. Plasmid-mediated extended-spectrum β-lactamase (ESBL and AmpC determinants were identified in carcasses from the four brands tested. Notably, this is the first description of blaCTX-M-15 genes in meat or food-producing animals from South America. The blaCTX-M-8, blaCTX-M-15 and blaCMY-2 genes were transferable in conjugation experiments. The findings of the present study indicate that plasmid-mediated ESBL and AmpC-encoding genes are widely distributed in Brazilian chicken meat.

  5. Dissemination of a clone carrying a fosA3-harbouring plasmid mediates high fosfomycin resistance rate of KPC-producing Klebsiella pneumoniae in China.

    Science.gov (United States)

    Jiang, Yan; Shen, Ping; Wei, Zeqing; Liu, Lilin; He, Fang; Shi, Keren; Wang, Yanfei; Wang, Haiping; Yu, Yunsong

    2015-01-01

    Fosfomycin has been proposed as an adjunct to other active agents for treating KPC-producing Klebsiella pneumoniae infections. This study aimed to investigate the prevalence of fosfomycin resistance and plasmid-mediated resistance determinants among KPC-producing K. pneumoniae isolates from clinical samples in China. In total, 278 KPC-producing and 80 extended-spectrum β-lactamase (ESBL)-producing (non-KPC-producing) clinical K. pneumoniae isolates were collected in 12 hospitals from 2010 to 2013. Fosfomycin susceptibility testing was carried out using the agar dilution method. Phylogenetic clonal patterns were revealed by pulsed-field gel electrophoresis (PFGE). Isolates were screened for plasmid-mediated fosfomycin resistance genes (fosA, fosA3 and fosC2) by PCR amplification. A plasmid was completely sequenced by next-generation sequencing. The fosfomycin resistance rate in KPC-producers (60.8%; 169/278) was significantly higher than in ESBL-producers (12.5%; 10/80). In addition, 94 KPC-producing isolates were positive for fosA3 and most of them were clonally related. A 23939-bp plasmid (pFOS18) co-harbouring fosA3 and bla(KPC-2) was completely sequenced, revealing that the fosA3 gene was flanked by two copies of IS26; however, bla(KPC-2) was located on a Tn3-Tn4401 integration structure. Although the fosA3 and blaKPC-2 genes are located on different transposon systems, they are able to spread together worldwide through plasmid transfer. Dissemination of the clone carrying the fosA3-harbouring plasmid mediates the high fosfomycin resistance rate of KPC-producing K. pneumoniae in China. Fosfomycin as an alternative option for treating infections caused by KPC-producing K. pneumoniae should not be recommended in hospitals in which fosfomycin-resistant clonal dissemination is emerging.

  6. Plasmid-mediated resistance to tetracyclines among Neisseria gonorrhoeae strains isolated in Poland between 2012 and 2013

    Science.gov (United States)

    Młynarczyk-Bonikowska, Beata; Kujawa, Marlena; Malejczyk, Magdalena; Młynarczyk, Grażyna

    2016-01-01

    Introduction One of two main mechanisms of resistance in tetracycline-resistant Neisseria gonorrhoeae (TRNG) is associated with the presence of TetM protein responsible for actively blocking of the tetracycline target site in the 30S ribosomal subunit. This mechanism is encoded by conjugative plasmids. The second mechanism is chromosomal in nature and due to mutations in specific genes. Aim To determine the incidence and type of tetM determinants in TRNG strains isolated from patients presenting with gonorrhea infection to the Dermatology and Venereology Clinic in Warsaw in 2012–2013. Material and methods Tetracycline and doxycycline susceptibility was determined by E-Tests. The presence and type of the tetM gene were determined by polymerase chain reaction. Results Tetracycline resistance was detected in 50.8% of the evaluated strains. The TRNG strains containing the tetM plasmid constituted 13.8% of all the evaluated strains. Dutch type tetM constituted 12.3% and American type tetM 1.5% of all the evaluated strains. In the remaining TRNG strains, resistance to tetracyclines was presumably chromosome-encoded. The minimal inhibitory concentration (MIC) of tetracycline ranged from 0.25 to 32.0 mg/l, MIC50 = 2.0 mg/l, MIC90 = 32.0 mg/l. The MIC of doxycycline ranged from 0.25 to 32.0 mg/l, MIC50 = 4.0 mg/l, MIC90 = 16.0 mg/l. Conclusions Unlike most of European countries, in 2012–2013 in Poland, the Dutch type tetM was found to be much more common than the American type. Minimal inhibitory concentration values of tetracycline and doxycycline were similar, with doxycycline exhibiting a somewhat lower effectiveness in vitro than tetracycline towards chromosome-mediated tetracycline resistant strains of N. gonorrhoeae. PMID:28035227

  7. Release of GTP Exchange Factor Mediated Down-Regulation of Abscisic Acid Signal Transduction through ABA-Induced Rapid Degradation of RopGEFs

    Science.gov (United States)

    Waadt, Rainer; Schroeder, Julian I.

    2016-01-01

    The phytohormone abscisic acid (ABA) is critical to plant development and stress responses. Abiotic stress triggers an ABA signal transduction cascade, which is comprised of the core components PYL/RCAR ABA receptors, PP2C-type protein phosphatases, and protein kinases. Small GTPases of the ROP/RAC family act as negative regulators of ABA signal transduction. However, the mechanisms by which ABA controls the behavior of ROP/RACs have remained unclear. Here, we show that an Arabidopsis guanine nucleotide exchange factor protein RopGEF1 is rapidly sequestered to intracellular particles in response to ABA. GFP-RopGEF1 is sequestered via the endosome-prevacuolar compartment pathway and is degraded. RopGEF1 directly interacts with several clade A PP2C protein phosphatases, including ABI1. Interestingly, RopGEF1 undergoes constitutive degradation in pp2c quadruple abi1/abi2/hab1/pp2ca mutant plants, revealing that active PP2C protein phosphatases protect and stabilize RopGEF1 from ABA-mediated degradation. Interestingly, ABA-mediated degradation of RopGEF1 also plays an important role in ABA-mediated inhibition of lateral root growth. The presented findings point to a PP2C-RopGEF-ROP/RAC control loop model that is proposed to aid in shutting off ABA signal transduction, to counteract leaky ABA signal transduction caused by “monomeric” PYL/RCAR ABA receptors in the absence of stress, and facilitate signaling in response to ABA. PMID:27192441

  8. Gαi2-PROTEIN MEDIATED SIGNAL TRANSDUCTION: A CNS MOLECULAR MECHANISM COUNTERING THE DEVELOPMENT OF SODIUM-DEPENDENT HYPERTENSION

    Science.gov (United States)

    Wainford, Richard D; Carmichael, Casey Y; Pascale, Crissey L; Kuwabara, Jill T

    2014-01-01

    Excess dietary salt-intake is an established cause of hypertension. At present our understanding of the neuro-pathophysiology of salt-sensitive hypertension is limited by a lack of identification of the central nervous system mechanisms that modulate sympathetic outflow and blood pressure in response to dietary salt-intake. We hypothesized that impairment of brain Gαi2 protein-gated signal transduction pathways would result in increased sympathetically mediated renal sodium retention, thus promoting the development of salt-sensitive hypertension. To test this hypothesis, naïve or renal denervated Dahl salt-resistant and Dahl salt-sensitive rats were assigned to receive a continuous intracerebroventricular control scrambled or a targeted Gαi2 oligodeoxynucleotide infusion, and naïve Brown Norway and 8-congenic Dahl salt-sensitive rats, were fed a 21-day normal or high-salt diet. High salt-intake did not alter blood pressure, suppressed plasma norepinephrine, and evoked a site-specific increase in hypothalamic paraventricular nucleus Gαi2 protein levels in naïve Brown-Norway, Dahl salt-resistant and scrambled oligodeoxynucleotide-infused Dahl salt-resistant, but not Dahl salt-sensitive rats. In Dahl salt-resistant rats Gαi2 down-regulation evoked rapid renal nerve-dependent hypertension, sodium retention and sympathoexcitation. In Dahl salt-sensitive rats, Gαi2 down-regulation exacerbated salt-sensitive hypertension via a renal nerve-dependent mechanism. Congenic-8 Dahl salt-sensitive rats exhibited sodium-evoked paraventricular nucleus specific Gαi2 protein up-regulation and attenuated hypertension, sodium retention and global sympathoexcitation compared to Dahl salt-sensitive rats. These data demonstrate that paraventricular nucleus Gαi2 protein-gated pathways represent a conserved central molecular pathway mediating sympathoinhibitory renal-nerve dependent responses evoked to maintain sodium homeostasis and a salt-resistant phenotype. Impairment of this

  9. COX19 mediates the transduction of a mitochondrial redox signal from SCO1 that regulates ATP7A-mediated cellular copper efflux

    Science.gov (United States)

    Leary, Scot C.; Cobine, Paul A.; Nishimura, Tamiko; Verdijk, Robert M.; de Krijger, Ronald; de Coo, René; Tarnopolsky, Mark A.; Winge, Dennis R.; Shoubridge, Eric A.

    2013-01-01

    SCO1 and SCO2 are metallochaperones whose principal function is to add two copper ions to the catalytic core of cytochrome c oxidase (COX). However, affected tissues of SCO1 and SCO2 patients exhibit a combined deficiency in COX activity and total copper content, suggesting additional roles for these proteins in the regulation of cellular copper homeostasis. Here we show that both the redox state of the copper-binding cysteines of SCO1 and the abundance of SCO2 correlate with cellular copper content and that these relationships are perturbed by mutations in SCO1 or SCO2, producing a state of apparent copper overload. The copper deficiency in SCO patient fibroblasts is rescued by knockdown of ATP7A, a trans-Golgi, copper-transporting ATPase that traffics to the plasma membrane during copper overload to promote efflux. To investigate how a signal from SCO1 could be relayed to ATP7A, we examined the abundance and subcellular distribution of several soluble COX assembly factors. We found that COX19 partitions between mitochondria and the cytosol in a copper-dependent manner and that its knockdown partially rescues the copper deficiency in patient cells. These results demonstrate that COX19 is necessary for the transduction of a SCO1-dependent mitochondrial redox signal that regulates ATP7A-mediated cellular copper efflux. PMID:23345593

  10. [Study on the effect of Klotho gene interferred by plasmid-mediated short hairpin RNA (shRNA) on sinoatrial node pacing channel gene].

    Science.gov (United States)

    Cai, Yingying; Wang, Han; Hou, Yanbin; Fang, Chenli; Tian, Peng; Wang, Guihua; Li, Lu; Deng, Juelin

    2013-06-01

    The study was aimed to assess the effect of Klotho gene and sinoatrial node pacing channel gene (HCN4 and HCN2) for studying sick sinus syndrome, with Klotho gene under the interference of Plasmid-mediated short hairpin RNA. Twenty-five C57BL/6J mice were divided into four groups, i. e, plasmid shRNA 24h group, plasmid shRNA 12h group, sodium chloride 24h group and sodium chloride 12h group. Plasmid shRNA 50microL (1microg/microL) and sodium chloride 50microl were respectively injected according to mice vena caudalis into those in plasmid shRNA group and sodium chloride group. After 12h or 24h respectively, all mice were executed and their sinoatrial node tissues were cut. The mRNA of Klotho, HCN4 and HCN2 gene were detected by RT-PCR. The results of RT-PCR showed that Klotho, HCN4 and HCN2 mRNA levels were lower compared with those in sodium chloride 12h group after 12h interference interval. The results indicated that there might be the a certain relationship between Klotho gene and sinoatrial node pacing channel gene.

  11. An Efficient Agrobacterium-Mediated Transformation of Strawberry cv. Camarosa by a Dual Plasmid System

    Directory of Open Access Journals (Sweden)

    Fatemeh Haddadi

    2015-02-01

    Full Text Available An Agrobacterium-mediated transformation method was applied to introduce the luciferase reporter gene under the control of the CaMV35S promoter in the pGreen0049 binary vector into strawberry cv. Camarosa. The in vitro regeneration system of strawberry leaves to be used in the transformation was optimized using different TDZ concentrations in MS medium. TDZ at 16 µM showed the highest percentage (100% of shoot formation and the highest mean number of shoots (24 produced per explant. Studies on the effects of different antibiotics, namely timentin, cefotaxime, carbenicillin and ampicillin, on shoot regeneration of strawberry leaf explants showed the best shoot regeneration in the presence of 300 mg/L timentin and 150 mg/L cefotaxime. Assessment of the different factors affecting Agrobacterium mediated-transformation of strawberry with the luciferase gene showed the highest efficiency of putative transformant production (86% in the treatment with no preculture, bacterial OD600 of 0.6 and the addition of 150 mg/L cefotaxime in the pre-selection and selection media. The presence of the luciferase gene in the plant genome was verified by the luciferase reporter gene assay, nested PCR amplification and dot blot of genomic DNA isolated from the young leaves of each putatively transformed plantlet.

  12. An efficient Agrobacterium-mediated transformation of strawberry cv. Camarosa by a dual plasmid system.

    Science.gov (United States)

    Haddadi, Fatemeh; Aziz, Maheran Abd; Abdullah, Siti Nor Akmar; Tan, Soon Guan; Kamaladini, Hossein

    2015-02-23

    An Agrobacterium-mediated transformation method was applied to introduce the luciferase reporter gene under the control of the CaMV35S promoter in the pGreen0049 binary vector into strawberry cv. Camarosa. The in vitro regeneration system of strawberry leaves to be used in the transformation was optimized using different TDZ concentrations in MS medium. TDZ at 16 µM showed the highest percentage (100%) of shoot formation and the highest mean number of shoots (24) produced per explant. Studies on the effects of different antibiotics, namely timentin, cefotaxime, carbenicillin and ampicillin, on shoot regeneration of strawberry leaf explants showed the best shoot regeneration in the presence of 300 mg/L timentin and 150 mg/L cefotaxime. Assessment of the different factors affecting Agrobacterium mediated-transformation of strawberry with the luciferase gene showed the highest efficiency of putative transformant production (86%) in the treatment with no preculture, bacterial OD600 of 0.6 and the addition of 150 mg/L cefotaxime in the pre-selection and selection media. The presence of the luciferase gene in the plant genome was verified by the luciferase reporter gene assay, nested PCR amplification and dot blot of genomic DNA isolated from the young leaves of each putatively transformed plantlet.

  13. Detection of plasmid-mediated IMP-1 metallo-β-lactamase and quinolone resistance determinants in an ertapenem-resistant Enterobacter cloacae isolate

    Institute of Scientific and Technical Information of China (English)

    Li-rong CHEN; Hong-wei ZHOU; Jia-chang CAI; Rong ZHANG; Gong-xiang CHEN

    2009-01-01

    Objective: To investigate the mechanism of carbapenem resistance and the occurrence of plasmid-mediated quinolone resistance determinants qnr and aac(6')-Ib-cr in a clinical isolate of Enterobacter cloacae. Methods: An ertapenem-resistant E. cloacae ZY106, which was isolated from liquor puris of a female gastric cancer patient in a Chinese hospital, was investigated. Antibiotic susceptibilities were determined by agar dilution method. Conjugation experiments, isoelectric focusing, polymerase chain reaction (PCR), and DNA sequence analyses of plasmid-mediated carbapenemases and quinolone resistance determinants were preformed to confirm the genotype. Outer membrane proteins (OMPs) were examined by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Urea-SDS-PAGE). Results: Minimum inhibitory concentrations (MCs) of imipenem, mer-openem, and ertapenem for ZY106 were 2,4, and 16 ug/ml, respectively. Conjugation studies with Escherichia coli resulted in the transfer of significantly reduced carbapenem susceptibility. ZY106 produced IMP-1 metallo-p-lactamase and CTX-M-3 extended-spectrum P-lactamase, and E. coli transconjugant produced IMP-1. Plasmid-mediated quinolone resistance determinant qnrSI was detected in ZY106. Transfer of the qnrSI-encoding-plasmid into E. coli by conjugation resulted in intermediate resistance to ciprofloxacin in E. coli transconjugant. Urea-SDS-PAGE analysis of OMPs showed that ZY106 lacked an OMP of approximately 38 KDa. Conclusion: It is the first IMP-1-producing Enterobacteriaceae in China and the first report of a clinical isolate that harbors both blaIMP and qnrS genes as well. The blaIMP-1, blaCTX-M-3, and qnrSl are encoded at three different plasmids. IMP-1 combined with the loss of an OMP possibly resulted in ertapenem resistance and reduced imipenem and mero-penem susceptibility in E. cloacae.

  14. Crystal structure of Mox-1, a unique plasmid-mediated class C β-lactamase with hydrolytic activity towards moxalactam.

    Science.gov (United States)

    Oguri, Takuma; Furuyama, Takamitsu; Okuno, Takashi; Ishii, Yoshikazu; Tateda, Kazuhiro; Bonomo, Robert A; Shimizu-Ibuka, Akiko

    2014-07-01

    Mox-1 is a unique plasmid-mediated class C β-lactamase that hydrolyzes penicillins, cephalothin, and the expanded-spectrum cephalosporins cefepime and moxalactam. In order to understand the unique substrate profile of this enzyme, we determined the X-ray crystallographic structure of Mox-1 β-lactamase at a 1.5-Å resolution. The overall structure of Mox-1 β-lactamase resembles that of other AmpC enzymes, with some notable exceptions. First, comparison with other enzymes whose structures have been solved reveals significant differences in the composition of amino acids that make up the hydrogen-bonding network and the position of structural elements in the substrate-binding cavity. Second, the main-chain electron density is not observed in two regions, one containing amino acid residues 214 to 216 positioned in the Ω loop and the other in the N terminus of the B3 β-strand corresponding to amino acid residues 303 to 306. The last two observations suggest that there is significant structural flexibility of these regions, a property which may impact the recognition and binding of substrates in Mox-1. These important differences allow us to propose that the binding of moxalactam in Mox-1 is facilitated by the avoidance of steric clashes, indicating that a substrate-induced conformational change underlies the basis of the hydrolytic profile of Mox-1 β-lactamase.

  15. Responses of plasmid-mediated quinolone resistance genes and bacterial taxa to (fluoro)quinolones-containing manure in arable soil.

    Science.gov (United States)

    Xiong, Wenguang; Sun, Yongxue; Ding, Xueyao; Zhang, Yiming; Zhong, Xiaoxia; Liang, Wenfei; Zeng, Zhenling

    2015-01-01

    The aim of the present study was to investigate the fate of plasmid-mediated quinolone resistance (PMQR) genes and the disturbance of soil bacterial communities posed by (fluoro)quinolones (FQNs)-containing manure in arable soil. Representative FQNs (enrofloxacin (ENR), ciprofloxacin (CIP) and norfloxacin (NOR)), PMQR genes (qepA, oqxA, oqxB, aac(6')-Ib-cr and qnrS) and bacterial communities in untreated soil, +manure and +manure+FQNs groups were analyzed using culture independent methods. The significantly higher abundance of oqxA, oqxB and aac(6')-Ib-cr, and significantly higher abundance of qnrS in +manure group than those in untreated soil disappeared at day 30 and day 60, respectively. All PMQR genes (oqxA, oqxB, aac(6')-Ib-cr and qnrS) dissipated 1.5-1.7 times faster in +manure group than those in +manure+FQNs group. The disturbance of soil bacterial communities posed by FQNs-containing manure was also found. The results indicated that significant effects of PMQR genes (oqxA, oqxB, aac(6')-Ib and qnrS) on arable soils introduced by manure disappeared 2 month after manure application. FQNs introduced by manure slowed down the dissipation of PMQR genes. The presence of high FQNs provided a selective advantage for species affiliated to the phylum including Acidobacteria, Verrucomicrobia and Planctomycetes while suppressing Proteobacteria and Actinobacteria.

  16. Genetic environments of the transferable plasmid-mediated blaCTX-M-3 gene in Serratia marcescens isolates.

    Science.gov (United States)

    Chu, Pei-Yu; Peng, Chien-Fang

    2014-01-01

    In this study, genetic environments of the transferable plasmid-mediated blaCTX-M-3 gene were characterized among 14 isolates of cefotaxime-resistant Serratia marcescens using PCR and BLAST DNA sequence analysis. A total of 3 types of genetic architectures in the regions surrounding this blaCTX-M-3 gene were identified. Type I architecture was characterized by the presence of a complete insertion sequence of tnpA-ISEcp1, identified as interrupting a reverse IS26 sequence in the upstream region of the blaCTX-M-3 gene. A reverse-directional orf477 fragment was located downstream of the blaCTX-M-3 gene, which was in the same direction of the mucA gene. A common region containing the orf513 element was located upstream of the mucA gene. Moreover, a copy of the 3'-CS2 element was located immediately upstream of the orf513 element. A novel complex class 1 integron was characterized by the presence of the dfrA19 gene, which was flanked by two copies of class 1 integrons. This is the first report to describe the dfrA19 gene within a novel complex class 1 integron in S. marcescens isolates from Taiwan. This novel complex class 1 integron structure was located distantly upstream of the blaCTX-M-3 gene.

  17. Effects of osmotic stress on the activity of MAPKs and PDGFR-beta-mediated signal transduction in NIH-3T3 fibroblasts

    DEFF Research Database (Denmark)

    Nielsen, M-B; Christensen, Søren Tvorup; Hoffmann, E K

    2008-01-01

    Signaling in cell proliferation, cell migration, and apoptosis is highly affected by osmotic stress and changes in cell volume, although the mechanisms underlying the significance of cell volume as a signal in cell growth and death are poorly understood. In this study, we used NIH-3T3 fibroblasts...... in a serum- and nutrient-free inorganic medium (300 mosM) to analyze the effects of osmotic stress on MAPK activity and PDGF receptor (PDGFR)-beta-mediated signal transduction. We found that hypoosmolarity (cell swelling at 211 mosM) induced the phosphorylation and nuclear translocation of ERK1/2, most...

  18. Plasmid mediated antibiotic resistance ofVibrio cholerae O1 biotype El Tor serotype Ogawa associated with an outbreak in Kolkata, India

    Institute of Scientific and Technical Information of China (English)

    Shyamapada Mandal; Manisha DebMandal; Nishith Kumar Pal

    2010-01-01

    Objective:To determine the antibiotic resistance ofVibrio cholerae (V. cholerae)O1 biotype El Tor serotype Ogawa isolates involved in an outbreak of watery diarrhea in Kolkata, and to explore the role of plasmid in mediating antibiotic resistance.Methods: Antibiotic susceptibility and minimum inhibitory concentration(MIC) values of antibiotics for the isolated V. choleraeO1 Ogawa (n=12) were determined by disk diffusion and agar dilution methods, respectively, using ampicillin (Am), chloramphenicol (C), trimethoprim (Tm), tetracycline (T), erythromycine (Er), nalidixic acid (Nx), ciprofloxacin (Cp), amikacin (Ak) and cefotaxime (Cf). Plasmid curing of multidrug resistant(MDR)V. choleraeO1 Ogawa strains was done following ethidium bromide treatment. Following electrophoresis, the plasmidDNAs, extracted from the isolatedMDRV. choleraeO1 Ogawa strains and their cured derivatives, were visualized and documented in‘gel doc’ system.Results: The outbreak causingV. choleraeO1 Ogawa isolates wereMDR as determined by disk diffusion susceptibility test, andMIC determination. The isolates showed three different drug resistance patterns: AmTmTErNx (for6 isolates), TmTErCp (for 5 isolates), and AmTmNx (for one isolate), and showed uniform sensitivity to C, Ak and Cf. The loss of plasmids with the concomitant loss of resistance to Am, Tm, T and Er of the isolates occurred following ethidium bromide treatment.Conclusions: The current findings suggest that theV. choleraeO1Ogawa associated with the cholera outbreak wereMDR, and resistance to Am, Tm, T and Er among the isolates were plasmid mediated.

  19. Plasmid interference for curing antibiotic resistance plasmids in vivo.

    Science.gov (United States)

    Kamruzzaman, Muhammad; Shoma, Shereen; Thomas, Christopher M; Partridge, Sally R; Iredell, Jonathan R

    2017-01-01

    Antibiotic resistance increases the likelihood of death from infection by common pathogens such as Escherichia coli and Klebsiella pneumoniae in developed and developing countries alike. Most important modern antibiotic resistance genes spread between such species on self-transmissible (conjugative) plasmids. These plasmids are traditionally grouped on the basis of replicon incompatibility (Inc), which prevents coexistence of related plasmids in the same cell. These plasmids also use post-segregational killing ('addiction') systems, which poison any bacterial cells that lose the addictive plasmid, to guarantee their own survival. This study demonstrates that plasmid incompatibilities and addiction systems can be exploited to achieve the safe and complete eradication of antibiotic resistance from bacteria in vitro and in the mouse gut. Conjugative 'interference plasmids' were constructed by specifically deleting toxin and antibiotic resistance genes from target plasmids. These interference plasmids efficiently cured the corresponding antibiotic resistant target plasmid from different Enterobacteriaceae in vitro and restored antibiotic susceptibility in vivo to all bacterial populations into which plasmid-mediated resistance had spread. This approach might allow eradication of emergent or established populations of resistance plasmids in individuals at risk of severe sepsis, enabling subsequent use of less toxic and/or more effective antibiotics than would otherwise be possible, if sepsis develops. The generalisability of this approach and its potential applications in bioremediation of animal and environmental microbiomes should now be systematically explored.

  20. Agrobacterium-mediated plant transformation by novel mini-T vectors in conjunction with a high-copy vir region helper plasmid.

    Science.gov (United States)

    Zyprian, E; Kado, C I

    1990-08-01

    A new binary vector system for Agrobacterium-mediated plant transformation was developed. A set of four mini-T vectors comprised of T-DNA border sequences from nopaline-type Ti-plasmid pTiC58 flanking a chimaeric hygromycin-resistance gene for selection of transformants and up to eight unique restriction sites for cloning foreign DNA was constructed on a broad-host replicon containing the oriV of plasmid pSa. In two of the constructs these multiple cloning sites are flanked by a strong promoter to activate transcription of inserted DNA in planta. High-efficiency transformation was prompted by a high-copy, stable virulence helper plasmid pUCD2614, which contains a cloned virulence region of pTiC58 and tandem copies of the par locus of plasmid pTAR. Southern blot hybridization and genetic analyses of the progeny of transformed plants showed that the hygromycin resistance gene was stably inherited.

  1. Detection of plasmid-mediated AmpC β-lactamase in Escherichia coli and Klebsiella pneumoniae

    Directory of Open Access Journals (Sweden)

    N O Yilmaz

    2013-01-01

    Full Text Available Background: Detecting plasmid-mediated AmpC (pAmpC β-lactamase-producing organism is important for optimal infection control and providing accurate and effective treatment option for physicians. Objectives: The aim of this study was to investigate the prevalence of pAmpC β-lactamase and compare the results of boronic acid (BA disk test with other phenotypic tests detecting AmpC positive isolates. Materials and Methods: A total of 273 clinical isolates of Klebsiella pneumoniae (n: 82 and Escherichia coli (n: 191 were analysed. The presence of pAmpC β-lactamase was determined by BA disk test, cefoxitin (FOX screening test, modified three dimensional test (M3DT, and multiplex polymerase chain reaction (PCR. Pulsed-field gel electrophoresis was performed to evaluate the genetic similarities between isolates. To detect extended spectrum β-lactamases (ESBL in the presence of AmpC β-lactamase, ESBL confirmation test was carried out with and without BA solution. Results: Of the 273 strains tested, 127 strains were found FOX resistant, 114 were positive by M3DT, 108 were positive in BA disk test, and the multiplex PCR detected 24 pAmpC β-lactamase-positive isolate. The prevalence of AmpC-producing strains was 10.9% in E. coli and 3.6% in K. pneumoniae in the tested population by PCR. CIT and MOX group genes were predominant type in these strains. Conclusion: These results emphasize that clinical laboratories should consider testing the presence of pAmpC enzymes particularly in FOX-resistant isolates, and BA disk test will improve detection of this emerging resistance phenotype.

  2. Prevalence of plasmid-mediated quinolone resistance and aminoglycoside resistance determinants among carbapeneme non-susceptible Enterobacter cloacae.

    Directory of Open Access Journals (Sweden)

    Shifeng Huang

    Full Text Available BACKGROUND: Simultaneous resistance to aminoglycosides and fluoroquinolones in carbapeneme non-susceptible (CNS isolates will inevitably create problems. The present study was performed to characterize the prevalence of the plasmid-mediated quinolone resistance determinants (QRDs and aminoglycoside resistance determinants (ARDs among the CNS Enterobacter cloacae (E. cloacae isolates in a Chinese teaching hospital, and to acquire their molecular epidemiological characteristics. METHODS: The β-lactamases genes (including class A carbapenemase genes bla(KPC and bla(SME, metallo-β-lactamase genes (MBLs bla(IMP, bla(VIM and bla(NDM, and extended spectrum β-lactamases (ESBLs,bla(CTX-M, bla(TEM and bla(SHV, QRDs (including qnrA, qnrB, qnrS and aac(6'-Ib-cr and ARDs (including aac(6'-Ib, armA and rmtB of these 35 isolates were determined by PCR and sequenced bidirectionally. The clonal relatedness was investigated by pulsed-field gel electrophoresis (PFGE. RESULTS: Of the 35 isolates, 9 (25.7% harbored a carbapenemase gene; 23 (65.7% carried ESBLs; 24 (68.6% were QRD positive; and 27 (77.1% were ARD positive. Among the 5 bla(IMP-8 positive strains, 4 (80% contained both ESBL and QRD genes, and all the 5 (100% harbored ARD genes. Of the 23 ESBLs positive isolates, 6 (26.1% were carbapenemase positive, 14 (60.9% were QRD positive, and 18 (78.3% were ARD positive. PFGE revealed genetic diversity among the 35 isolates, indicating that the high prevalence of CNS E. cloacae isolates was not caused by clonal dissemination. CONCLUSION: QRD and ARD genes were highly prevalent among the CNS E. cloacae isolates. Multiple resistant genes were co-expressed in the same isolates. The CNS E. cloacae isolate co-expressing bla(NDM-1, bla(IMP-26, qnrA1 and qnrS1 was first reported.

  3. Occurrence of plasmid-mediated quinolone resistance and virulence genes in avian Escherichia coli isolates from Algeria.

    Science.gov (United States)

    Laarem, Meradi; Barguigua, Abouddihaj; Nayme, Kaotar; Akila, Abdi; Zerouali, Khalid; El Mdaghri, Naima; Timinouni, Mohammed

    2017-02-28

    The emergence and spread of quinolone-resistant Escherichia coli in poultry products puts consumers at risk of exposure to the strains of E. coli that resist antibiotic treatment. The objective of this study was to define the prevalence and virulence potential of poultry-associated nalidixic acid (NAL)-resistant E. coli in the Annaba city, Algeria. In total, 33 samples of retail chicken meat were purchased from various butcher shops and examined for bacterial contamination with NAL-resistant E. coli. These isolates were subjected to antimicrobial susceptibility testing and were also investigated for the presence of plasmid-mediated quinolone resistance (PMQR) genes and virulence genes using conventional polymerase chain reaction (PCR) and DNA sequencing. Phylogenetic grouping of the NAL-resistant E. coli isolates was determined by the conventional multiplex PCR method. Twenty-nine (87.8%) products yielded NAL-resistant E. coli. Antibiograms revealed that 96.55% of NAL-resistant E. coli isolates were multidrug resistant (MDR). Resistance was most frequently observed against sulfamethoxazole-trimethoprim (96.6%), tetracycline (96.6%), ciprofloxacin (72%), and amoxicillin (65.5%). Group A was the most prevalent phylogenetic group, followed by groups D, B1, and B2. The PMQR determinants were detected in three isolates with qnrB72 and qnrS1 type identified. Four (13.8%) isolates carried one of the Shiga toxin E. coli-associated genes stx1, stx2, and ehxA alleles. The high prevalence of NAL-resistant E. coli isolated from retail chicken meat with detection of MDR E. coli harboring Shiga toxin genes in this study gives a warning signal for possible occurrence of foodborne infections with failure in antibiotic treatment.

  4. Expansion of plasmid mediated blaACT-2 among Pseudomonas aeruginosa associated with postoperative infection and its transcriptional response under cephalosporin stress.

    Directory of Open Access Journals (Sweden)

    Birson Ingti, Deepjyoti Paul, Anand Prakash Maurya

    2017-06-01

    Full Text Available Objectives: Organisms harboring multiple plasmid mediated β-lactamases are major concerns in nosocomial infections. Among these plasmid mediated β-lactamases, ACT (EBC family is a clinically important enzyme capable of hydrolyzing broad spectrum cephalosporins. Therefore, the present study was undertaken to determine the prevalence of ACT determinant along with other co-existing β-lactamase genes in P. aeruginosa strains. Methods: A total of 176 Pseudomonas isolates were phenotypically screened for the presence of AmpC β-lactamase by M3DET Method followed by Molecular detection using PCR assay. Transcriptional evaluation of blaACT-2 gene was analyzed by RT-PCR and its transferability was performed by transformation and conjugation. Results: Present study demonstrates the presence of ACT-2 allele among 12 strains of P. aeruginosa. Co-existence of other β-lactamase genes were encountered among ACT-2 harboring strains which includes CTX-M (n=2, SHV (n=3, TEM (n=2, VEB (n=2, OXA-10 (n=1, CIT (n=2 and DHA (n=3. Fingerprinting by REP PCR revealed the isolates harboring ACT-2 to be distinct and these isolates showed high resistance to expanded-spectrum cephalosporins and even to carbapenem group of drugs. This ACT-2 allele was encoded in the plasmid (L/M, FIA, FIB Inc. Group and conjugatively transferable. Transcriptional analysis revealed a significant increase in ACT-2 expression (483 fold when induced by ceftriaxone at 4 µg/ml followed by ceftazidime at 8 µg/ml (31 fold and cefotaxime 4 µg/ml (8 fold. Conclusion: In this study detection of ACT-2 plasmid mediated AmpC β-lactamase along with other β-lactamase genes in clinical isolates of P. aeruginosa represents a serious therapeutic challenge. Therefore, revision in antimicrobial policy is required for effective treatment of patients infected with pathogen expressing this mechanism. J Microbiol Infect Dis 2017; 7(2: 75-82

  5. Genetic organization of plasmid-mediated Qnr determinants in cefotaxime-resistant Enterobacter cloacae isolates in Korea.

    Science.gov (United States)

    Kim, Yang Soo; Kim, Eun Sil; Jeong, Jin-Yong

    2010-11-01

    Because of the strong association between qnr genes and plasmids carrying β-lactamase genes, we screened 176 clinical isolates of Enterobacter cloacae with cefotaxime MICs of ≥16 μg/mL for qnr genes. The qnrA, qnrB, and qnrS genes were detected in 18 (10.2%), 11 (6.2%), and 1 (0.56%) of the isolates, respectively. The genetic environments of the plasmids encoding these qnr genes were analyzed.

  6. Calcium requirement of phytochrome-mediated fern-spore germination: no direct phytochrome-calcium interaction in the phytochrome-initiated transduction chain

    Science.gov (United States)

    Scheuerlein, R.; Wayne, R.; Roux, S. J.

    1989-01-01

    Phytochrome-mediated germination of fern spores of Dryopteris paleacea Sw. was initiated by a saturating red-light (R) irradiation after 20 h of imbibition. For its realization external Ca2+ was required, with a threshold at a submicromolar concentration, and an optimum was reached around 10(-4) M. At concentrations > or = 10(-1) M only a reduced response was obtained, based probably on an unspecific osmotic or ionic effect. The germination response was inhibited by La3+, an antagonist of Ca2+. From these results it is concluded that Ca2+ influx from the medium into the spores may be an important event in phytochrome-mediated germination. In the absence of Ca2+ the R-stimulated system remained capable of responding to Ca2+, added as late as 40 h after R. Moreover, Ca2+ was effective even if added after the active form of phytochrome, Pfr, had been abolished by far-red (FR) 24 h after R. Thus, the primary effect of Pfr, that initiates the transduction chain, does not require calcium. "Coupling" of Pfr to subsequent dark reactions has been investigated by R-FR irradiations with various dark intervals. The resulting "escape kinetics" were characterized by a lag phase (6 h) and half-maximal escape from FR reversibility (19 h). These kinetics were not significantly changed by the presence or absence of calcium. Thus, direct interaction of Pfr and calcium is not a step in the transduction chain initiated by the active form of phytochrome.

  7. Plasmid interference for curing antibiotic resistance plasmids in vivo

    Science.gov (United States)

    Kamruzzaman, Muhammad; Shoma, Shereen; Thomas, Christopher M.; Partridge, Sally R.

    2017-01-01

    Antibiotic resistance increases the likelihood of death from infection by common pathogens such as Escherichia coli and Klebsiella pneumoniae in developed and developing countries alike. Most important modern antibiotic resistance genes spread between such species on self-transmissible (conjugative) plasmids. These plasmids are traditionally grouped on the basis of replicon incompatibility (Inc), which prevents coexistence of related plasmids in the same cell. These plasmids also use post-segregational killing (‘addiction’) systems, which poison any bacterial cells that lose the addictive plasmid, to guarantee their own survival. This study demonstrates that plasmid incompatibilities and addiction systems can be exploited to achieve the safe and complete eradication of antibiotic resistance from bacteria in vitro and in the mouse gut. Conjugative ‘interference plasmids’ were constructed by specifically deleting toxin and antibiotic resistance genes from target plasmids. These interference plasmids efficiently cured the corresponding antibiotic resistant target plasmid from different Enterobacteriaceae in vitro and restored antibiotic susceptibility in vivo to all bacterial populations into which plasmid-mediated resistance had spread. This approach might allow eradication of emergent or established populations of resistance plasmids in individuals at risk of severe sepsis, enabling subsequent use of less toxic and/or more effective antibiotics than would otherwise be possible, if sepsis develops. The generalisability of this approach and its potential applications in bioremediation of animal and environmental microbiomes should now be systematically explored. PMID:28245276

  8. Detection of plasmid mediated colistin resistance (MCR-1) in Escherichia coli and Salmonella enterica isolated from poultry and swine in Spain.

    Science.gov (United States)

    Quesada, Alberto; Ugarte-Ruiz, María; Iglesias, M Rocío; Porrero, M Concepción; Martínez, Remigio; Florez-Cuadrado, Diego; Campos, María J; García, María; Píriz, Segundo; Sáez, José Luis; Domínguez, Lucas

    2016-04-01

    Recent findings suggest that use of colistin as a last resort antibiotic is seriously threatened by the rise of a new plasmid mediated mechanism of resistance (MCR-1). This work identifies, for the first time in Southern Europe, the gene mcr-1 in nine strains from farm animals (poultry and swine) corresponding to five Escherichia coli and four Salmonella enterica, among which three belong to serovar Typhimurium and one to Rissen. The MCR-1 was found encoded by a plasmid highly mobilizable by conjugation to the E. coli J53 strain. Two E. coli strains carried two determinants, mcr-1 plus pmrA or pmrB mutations, known to confer colistin resistance.

  9. Extended-Spectrum-Beta-Lactamases, AmpC Beta-Lactamases and Plasmid Mediated Quinolone Resistance in Klebsiella spp. from Companion Animals in Italy

    DEFF Research Database (Denmark)

    Donati, Valentina; Feltrin, Fabiola; Hendriksen, Rene S.

    2014-01-01

    We report the genetic characterization of 15 Klebsiella pneumoniae (KP) and 4 isolates of K. oxytoca (KO) from clinical cases in dogs and cats and showing extended-spectrum cephalosporin (ESC) resistance. Extended spectrum beta-lactamase (ESBL) and AmpC genes, plasmid-mediated quinolone resistance...... patterns observed, including two clusters of two (ST340) and four (ST101) indistinguishable isolates, respectively. All isolates harbored at least one ESBL or AmpC gene, all carried on transferable plasmids (IncR, IncFII, IncI1, IncN), and 16/19 were positive for PMQR genes (qnr family or aac(6')-Ib...... of multidrug-resistant Klebsiella with ESBL, AmpC and PMQR determinants, poses further and serious challenges in companion animal therapy and raise concerns for possible bidirectional transmission between pets and humans, especially at household level....

  10. Small-Molecule Inhibitors of Cytokine-Mediated STAT1 Signal Transduction In ß-Cells With Improved Aqueous Solubility

    DEFF Research Database (Denmark)

    Scully, Stephen Shane; Tang, Alicia J; Lundh, Morten;

    2013-01-01

    We previously reported the discovery of BRD0476 (1), a small molecule generated by diversity-oriented synthesis that suppresses cytokine-induced ß-cell apoptosis. Herein, we report the synthesis and biological evaluation of 1 and analogs with improved aqueous solubility. By replacing naphthyl wit...... with quinoline moieties, we prepared active analogs with up to a 1400-fold increase in solubility from 1. In addition, we demonstrated that compound 1 and analogs inhibit STAT1 signal transduction induced by IFN-¿....

  11. Plasmid-Mediated Colistin Resistance Gene mcr-1 in an Escherichia coli ST10 Bloodstream Isolate in the Sultanate of Oman.

    Science.gov (United States)

    Mohsin, Jalila; Pál, Tibor; Petersen, Jorgen Eskild; Darwish, Dania; Ghazawi, Akela; Ashraf, Tanveer; Sonnevend, Agnes

    2017-08-11

    To identify plasmid-mediated colistin resistance in clinical Enterobacteriaceae isolates in Oman, where this resistance mechanism has not been encountered yet. Twenty-two colistin-resistant Enterobacteriaceae clinical isolates collected between July 2014 and June 2016 in a tertiary care hospital in Muscat were screened by PCR for the mcr-1 and mcr-2 genes. The strain identified as mcr-1 positive was genotyped and its antibiotic susceptibility was established. The mcr-1 containing plasmid was mobilized into Escherichia coli K-12 and its sequence was determined. A single E. coli isolate (OM97) carrying mcr-1 gene was identified, while no strains carrying the mcr-2 gene was found. E. coli OM97 was isolated in June 2016 from blood culture of a male patient with multiple comorbidities. It belonged to ST10. Beyond colistin, it was resistant to amoxicillin-clavulanic acid, piperacillin-tazobactam, amikacin, ciprofloxacin, tetracycline, and cotrimoxazole. The mcr-1 gene was located on a conjugative IncI2-type plasmid of 63722 bp size, which did not harbor any further resistance genes. The genetic surrounding of the mcr-1 gene lacked the ISApl1 element. Although colistin resistance caused by the mcr-1 gene is not common in our collection of clinical isolates, the occurrence of the plasmid-mediated colistin resistance in an E. coli ST10 strain is of concern as this clonal group was already shown to spread ESBL genes and quinolone resistance worldwide. It is especially worrisome that as the mcr-1 gene occurred in a non-ESBL, carbapenem-susceptible E. coli strain, current susceptibility testing algorithms may not detect its presence.

  12. Plasmid Biopharmaceuticals.

    Science.gov (United States)

    Prazeres, Duarte Miguel F; Monteiro, Gabriel A

    2014-12-01

    Plasmids are currently an indispensable molecular tool in life science research and a central asset for the modern biotechnology industry, supporting its mission to produce pharmaceutical proteins, antibodies, vaccines, industrial enzymes, and molecular diagnostics, to name a few key products. Furthermore, plasmids have gradually stepped up in the past 20 years as useful biopharmaceuticals in the context of gene therapy and DNA vaccination interventions. This review provides a concise coverage of the scientific progress that has been made since the emergence of what are called today plasmid biopharmaceuticals. The most relevant topics are discussed to provide researchers with an updated overview of the field. A brief outline of the initial breakthroughs and innovations is followed by a discussion of the motivation behind the medical uses of plasmids in the context of therapeutic and prophylactic interventions. The molecular characteristics and rationale underlying the design of plasmid vectors as gene transfer agents are described and a description of the most important methods used to deliver plasmid biopharmaceuticals in vivo (gene gun, electroporation, cationic lipids and polymers, and micro- and nanoparticles) is provided. The major safety issues (integration and autoimmunity) surrounding the use of plasmid biopharmaceuticals is discussed next. Aspects related to the large-scale manufacturing are also covered, and reference is made to the plasmid products that have received marketing authorization as of today.

  13. Impact of Lentiviral Vector-Mediated Transduction on the Tightness of a Polarized Model of Airway Epithelium and Effect of Cationic Polymer Polyethylenimine

    Directory of Open Access Journals (Sweden)

    Stefano Castellani

    2010-01-01

    Full Text Available Lentiviral (LV vectors are promising agents for efficient and long-lasting gene transfer into the lung and for gene therapy of genetically determined pulmonary diseases, such as cystic fibrosis, however, they have not been evaluated for cytotoxicity and impact on the tightness of the airway epithelium. In this study, we evaluated the transduction efficiency of a last-generation LV vector bearing Green Fluorescent Protein (GFP gene as well as cytotoxicity and tight junction (TJ integrity in a polarized model of airway epithelial cells. High multiplicities of infection (MOI showed to be cytotoxic, as assessed by increase in propidium iodide staining and decrease in cell viability, and harmful for the epithelial tightness, as demonstrated by the decrease of transepithelial resistance (TER and delocalization of occludin from the TJs. To increase LV efficiency at low LV:cell ratio, we employed noncovalent association with the polycation branched 25ߙkDa polyethylenimine (PEI. Transduction of cells with PEI/LV particles resulted in 2.5–3.6-fold increase of percentage of GFP-positive cells only at the highest PEI:LV ratios (1×107 PEI molecules/transducing units with 50 MOI LV as compared to plain LV. At this dose PEI/LV transduction resulted in 6.5±2.4% of propidium iodide-positive cells. On the other hand, PEI/LV particles did not determine any alteration of TER and occludin localization. We conclude that PEI may be useful for improving the efficiency of gene transfer mediated by LV vectors in airway epithelial cells, in the absence of high acute cytotoxicity and alteration in epithelial tightness.

  14. The anaphase-promoting complex protein 5 (AnapC5 associates with A20 and inhibits IL-17-mediated signal transduction.

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    Allen W Ho

    Full Text Available IL-17 is the founding member of a family of cytokines and receptors with unique structures and signaling properties. IL-17 is the signature cytokine of Th17 cells, a relatively new T cell population that promotes inflammation in settings of infection and autoimmunity. Despite advances in understanding Th17 cells, mechanisms of IL-17-mediated signal transduction are less well defined. IL-17 signaling requires contributions from two receptor subunits, IL-17RA and IL-17RC. Mutants of IL-17RC lacking the cytoplasmic domain are nonfunctional, indicating that IL-17RC provides essential but poorly understood signaling contributions to IL-17-mediated signaling. To better understand the role of IL-17RC in signaling, we performed a yeast 2-hybrid screen to identify novel proteins associated with the IL-17RC cytoplasmic tail. One of the most frequent candidates was the anaphase promoting complex protein 7 (APC7 or AnapC7, which interacted with both IL-17RC and IL-17RA. Knockdown of AnapC7 by siRNA silencing exerted no detectable impact on IL-17 signaling. However, AnapC5, which associates with AnapC7, was also able to bind IL-17RA and IL-17RC. Moreover, AnapC5 silencing enhanced IL-17-induced gene expression, suggesting an inhibitory activity. Strikingly, AnapC5 also associated with A20 (TNFAIP3, a recently-identified negative feedback regulator of IL-17 signal transduction. IL-17 signaling was not impacted by knockdown of Itch or TAXBP1, scaffolding proteins that mediate A20 inhibition in the TNFα and IL-1 signaling pathways. These data suggest a model in which AnapC5, rather than TAX1BP1 and Itch, is a novel adaptor and negative regulator of IL-17 signaling pathways.

  15. Generation of CRISPR/Cas9-mediated lactoferrin-targeted mice by pronuclear injection of plasmid pX330

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    Mengxu GE,Fei LIU,Fei CHANG,Zhaolin SUN,Jing FEI,Ying GUO,Yunping DAI,Zhengquan YU,Yaofeng ZHAO,Ning LI,Qingyong MENG

    2015-09-01

    Full Text Available Lactoferrin is a member of the transferrin family of multifunctional iron binding glycoproteins. While numerous physiological functions have been described for lactoferrin, the mechanisms underlying these functions are not clear. To further study the functions and mechanisms of lactoferrin, we modified the lactoferrin promoter of mice using the CRISPR/Cas9 system to reduce or eliminate lactoferrin expression. Seven mice with lactoferrin promoter mutations were obtained with an efficiency of 24% (7/29 by injecting the plasmid pX330, expressing a small guide RNA and human codon-optimized SpCas9, into fertilized eggs of mice. Plasmid integration and off-targeting of pX330 were not detected. These results confirmed that pronuclear injection of a circular plasmid is a feasible and efficient method for targeted mutagenesis in mice.

  16. Prevalence of plasmid-mediated multidrug resistance determinants in fluoroquinolone-resistant bacteria isolated from sewage and surface water.

    Science.gov (United States)

    Osińska, Adriana; Harnisz, Monika; Korzeniewska, Ewa

    2016-06-01

    Fluoroquinolones (FQs) are fully synthetic broad-spectrum antibacterial agents that are becoming increasingly popular in the treatment of clinical and veterinary infections. Being excreted during treatment, mostly as active compounds, their biological action is not limited to the therapeutic site, but it is moved further as resistance selection pressure into the environment. Water environment is an ideal medium for the aggregation and dissemination of antibiotics, antibiotic-resistant bacteria (ARB), and antibiotic resistance genes (ARGs), which can pose a serious threat to human health. Because of this, the aim of this study was to determine the number of fluoroquinolone-resistant bacteria (FQRB) and their share in total heterotrophic plate counts (HPC) in treated wastewater (TWW), and upstream and downstream river water (URW, DRW) samples where TWW is discharged. The spread of plasmid-mediated quinolone resistance (PMQR) determinants and the presence/absence of resistance genes to other most popular antibiotic groups (against tetracyclines and beta-lactams) in selected 116 multiresistant isolates were investigated. The share of FQRB in total HPC in all samples was rather small and ranged from 0.7 % in URW samples to 7.5 % in TWW. Bacteria from Escherichia (25.0 %), Acinetobacter (25.0 %), and Aeromonas (6.9 %) genera were predominant in the FQRB group. Fluoroquinolone resistance was mostly caused by the presence of the gene aac(6')-1b-cr (91.4 %). More rarely reported was the occurrence of qnrS, qnrD, as well as oqxA, but qnrA, qnrB, qepA, and oqxB were extremely rarely or never noted in FQRB. The most prevalent bacterial genes connected with beta-lactams' resistance in FQRB were bla TEM, bla OXA, and bla CTX-M. The bla SHV was less common in the community of FQRB. The occurrence of bla genes was reported in almost 29.3 % of FQRB. The most abundant tet genes in FQRB were tet(A), tet(L), tet(K), and tet(S). The prevalence of tet genes was observed in 41.4

  17. Baculoviral transduction facilitates TALEN-mediated targeted transgene integration and Cre/LoxP cassette exchange in human-induced pluripotent stem cells.

    Science.gov (United States)

    Zhu, Haibao; Lau, Cia-Hin; Goh, Sal-Lee; Liang, Qingle; Chen, Can; Du, Shouhui; Phang, Rui-Zhe; Tay, Felix Chang; Tan, Wee-Kiat; Li, Zhendong; Tay, Johan Chin-Kang; Fan, Weimin; Wang, Shu

    2013-10-01

    Safety and reliability of transgene integration in human genome continue to pose challenges for stem cell-based gene therapy. Here, we report a baculovirus-transcription activator-like effector nuclease system for AAVS1 locus-directed homologous recombination in human induced pluripotent stem cells (iPSCs). This viral system, when optimized in human U87 cells, provided a targeted integration efficiency of 95.21% in incorporating a Neo-eGFP cassette and was able to mediate integration of DNA insert up to 13.5 kb. In iPSCs, targeted integration with persistent transgene expression was achieved without compromising genomic stability. The modified iPSCs continued to express stem cell pluripotency markers and maintained the ability to differentiate into three germ lineages in derived embryoid bodies. Using a baculovirus-Cre/LoxP system in the iPSCs, the Neo-eGFP cassette at the AAVS1 locus could be replaced by a Hygro-mCherry cassette, demonstrating the feasibility of cassette exchange. Moreover, as assessed by measuring γ-H2AX expression levels, genome toxicity associated with chromosomal double-strand breaks was not detectable after transduction with moderate doses of baculoviral vectors expressing transcription activator-like effector nucleases. Given high targeted integration efficiency, flexibility in transgene exchange and low genome toxicity, our baculoviral transduction-based approach offers great potential and attractive option for precise genetic manipulation in human pluripotent stem cells.

  18. Prevalence of Plasmid-Mediated Quinolone Resistance Genes among Extended-Spectrum β-Lactamase-Producing Klebsiella pneumoniae Human Isolates in Iran

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    Ehsaneh Shams

    2015-01-01

    Full Text Available The purpose of this study was to determine the prevalence and molecular characterization of plasmid-mediated quinolone resistance (PMQR genes (qnrA, qnrB, qnrS, aac(6′-Ib-cr, and qepA among ESBL-producing Klebsiella pneumoniae isolates in Kashan, Iran. A total of 185 K. pneumoniae isolates were tested for quinolone resistance and ESBL-producing using the disk diffusion method and double disk synergy (DDST confirmatory test. ESBL-producing strains were further evaluated for the blaCTX-M genes. The PCR method was used to show presence of plasmid-mediated quinolone resistance genes and the purified PCR products were sequenced. Eighty-seven ESBL-producing strains were identified by DDST confirmatory test and majority (70, 80.5% of which carried blaCTX-M genes including CTX-M-1 (60%, CTX-M-2 (42.9%, and CTX-M-9 (34.3%. Seventy-seven ESBL-producing K. pneumoniae isolates harbored PMQR genes, which mostly consisted of aac(6′-Ib-cr (70.1% and qnrB (46.0%, followed by qnrS (5.7%. Among the 77 PMQR-positive isolates, 27 (35.1% and 1 (1.3% carried 2 and 3 different PMQR genes, respectively. However, qnrA and qepA were not found in any isolate. Our results highlight high ESBL occurrence with CTX-M type and high frequency of plasmid-mediated quinolone resistance genes among ESBL-producing K. pneumoniae isolates in Kashan.

  19. Detection and occurrence of plasmid-mediated AmpC in highly resistant gram-negative Rods

    NARCIS (Netherlands)

    E.A. Reuland (E. Ascelijn); J.P. Hays (John); D.M.C. de Jongh (Denise); E. Abdelrehim (Eman); I. Willemsen (Ina); J.A.J.W. Kluytmans (Jan); P.H.M. Savelkoul (Paul); C.M.J.E. Vandenbroucke-Grauls (Christina); N.A. Naiemi (Nashwan Al)

    2014-01-01

    textabstractObjectives: The aim of this study was to compare the current screening methods and to evaluate confirmation tests for phenotypic plasmidal AmpC (pAmpC) detection. Methods: For this evaluation we used 503 Enterobacteriaceae from 18 Dutch hospitals and 21 isolates previously confirmed to b

  20. Detection of mcr-1 encoding plasmid-mediated colistin-resistant Escherichia coli isolates from human bloodstream infection and imported chicken meat, Denmark 2015.

    Science.gov (United States)

    Hasman, Henrik; Hammerum, Anette M; Hansen, Frank; Hendriksen, Rene S; Olesen, Bente; Agersø, Yvonne; Zankari, Ea; Leekitcharoenphon, Pimlapas; Stegger, Marc; Kaas, Rolf S; Cavaco, Lina M; Hansen, Dennis S; Aarestrup, Frank M; Skov, Robert L

    2015-01-01

    The plasmid-mediated colistin resistance gene, mcr-1, was detected in an Escherichia coli isolate from a Danish patient with bloodstream infection and in five E. coli isolates from imported chicken meat. One isolate from chicken meat belonged to the epidemic spreading sequence type ST131. In addition to IncI2, an incX4 replicon was found to be linked to mcr-1. This report follows a recent detection of mcr-1 in E. coli from animals, food and humans in China.

  1. Plasmid-mediated mcr-1 colistin resistance in Escherichia coli and Klebsiella spp. clinical isolates from the Western Cape region of South Africa.

    Science.gov (United States)

    Newton-Foot, Mae; Snyman, Yolandi; Maloba, Motlatji Reratilwe Bonnie; Whitelaw, Andrew Christopher

    2017-01-01

    Colistin is a last resort antibiotic for the treatment of carbapenem-resistant Gram negative infections. Until recently, mechanisms of colistin resistance were limited to chromosomal mutations which confer a high fitness cost and cannot be transferred between organisms. However, a novel plasmid-mediated colistin resistance mechanism, encoded by the mcr-1 gene, has been identified, and has since been detected worldwide. The mcr-1 colistin resistance mechanism is a major threat due to its lack of fitness cost and ability to be transferred between strains and species. Surveillance of colistin resistance mechanisms is critical to monitor the development and spread of resistance.This study aimed to determine the prevalence of the plasmid-mediated colistin resistance gene, mcr-1, in colistin-resistant E. coli and Klebsiella spp. isolates in the Western Cape of South Africa; and whether colistin resistance is spread through clonal expansion or by acquisition of resistance by diverse strains. Colistin resistant E. coli and Klebsiella spp. isolates were collected from the NHLS microbiology laboratory at Tygerberg Hospital. Species identification and antibiotic susceptibility testing was done using the API® 20 E system and the Vitek® 2 Advanced Expert System™. PCR was used to detect the plasmid-mediated mcr-1 colistin resistance gene and REP-PCR was used for strain typing of the isolates. Nineteen colistin resistant isolates, including 12 E. coli, six K. pneumoniae and one K. oxytoca isolate, were detected over 7 months from eight different hospitals in the Western Cape region. The mcr-1 gene was detected in 83% of isolates which were shown to be predominantly unrelated strains. The plasmid-mediated mcr-1 colistin resistance gene is responsible for the majority of colistin resistance in clinical isolates of E. coli and Klebsiella spp. from the Western Cape of South Africa. Colistin resistance is not clonally disseminated; the mcr-1 gene has been acquired by several

  2. IncA/C plasmid-mediated spread of CMY-2 in multidrug-resistant Escherichia coli from food animals in China.

    Science.gov (United States)

    Guo, Yu-Fang; Zhang, Wen-Hui; Ren, Si-Qi; Yang, Lin; Lü, Dian-Hong; Zeng, Zhen-Ling; Liu, Ya-Hong; Jiang, Hong-Xia

    2014-01-01

    To obtain a broad molecular epidemiological characterization of plasmid-mediated AmpC β-lactamase CMY-2 in Escherichia coli isolates from food animals in China. A total of 1083 E. coli isolates from feces, viscera, blood, drinking water, and sub-surface soil were examined for the presence of CMY-2 β-lactamases. CMY-2-producing isolates were characterized as follows: the blaCMY-2 genotype was determined using PCR and sequencing, characterization of the blaCMY-2 genetic environment, plasmid sizing using S1 nuclease pulsed-field gel electrophoresis (PFGE), PCR-based replicon typing, phylogenetic grouping, XbaI-PFGE, and multi-locus sequence typing (MLST). All 31 CMY-2 producers were only detected in feces, and presented with multidrug resistant phenotypes. All CMY-2 strains also co-harbored genes conferring resistance to other antimicrobials, including extended spectrum β-lactamases genes (blaCTX-M-14 or blaCTX-M-55), plasmid-mediated quinolone resistance determinants (qnr, oqxA, and aac-(6')-Ib-cr), floR and rmtB. The co-transferring of blaCMY-2 with qnrS1 and floR (alone and together) was mainly driven by the Inc A/C type plasmid, with sizes of 160 or 200 kb. Gene cassette arrays inserted in the class 1 or class 2 integron were amplified among 12 CMY-2 producers. CMY-2 producers belonged to avirulent groups B1 (n = 12) and A (n = 11), and virulent group D (n = 8). There was a good correlation between phylogenetic groups and sequence types (ST). Twenty-four STs were identified, of which the ST complexes (STC) 101/B1 (n = 6), STC10/A (n = 5), and STC155/B1 (n = 3) were dominant. CMY-2 is the dominant AmpC β-lactamase in food animals and is associated with a transferable replicon IncA/C plasmid in the STC101, STC10, and STC155 strains.

  3. IncA/C plasmid-mediated spread of CMY-2 in multidrug-resistant Escherichia coli from food animals in China.

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    Yu-Fang Guo

    Full Text Available OBJECTIVES: To obtain a broad molecular epidemiological characterization of plasmid-mediated AmpC β-lactamase CMY-2 in Escherichia coli isolates from food animals in China. METHODS: A total of 1083 E. coli isolates from feces, viscera, blood, drinking water, and sub-surface soil were examined for the presence of CMY-2 β-lactamases. CMY-2-producing isolates were characterized as follows: the blaCMY-2 genotype was determined using PCR and sequencing, characterization of the blaCMY-2 genetic environment, plasmid sizing using S1 nuclease pulsed-field gel electrophoresis (PFGE, PCR-based replicon typing, phylogenetic grouping, XbaI-PFGE, and multi-locus sequence typing (MLST. RESULTS: All 31 CMY-2 producers were only detected in feces, and presented with multidrug resistant phenotypes. All CMY-2 strains also co-harbored genes conferring resistance to other antimicrobials, including extended spectrum β-lactamases genes (blaCTX-M-14 or blaCTX-M-55, plasmid-mediated quinolone resistance determinants (qnr, oqxA, and aac-(6'-Ib-cr, floR and rmtB. The co-transferring of blaCMY-2 with qnrS1 and floR (alone and together was mainly driven by the Inc A/C type plasmid, with sizes of 160 or 200 kb. Gene cassette arrays inserted in the class 1 or class 2 integron were amplified among 12 CMY-2 producers. CMY-2 producers belonged to avirulent groups B1 (n = 12 and A (n = 11, and virulent group D (n = 8. There was a good correlation between phylogenetic groups and sequence types (ST. Twenty-four STs were identified, of which the ST complexes (STC 101/B1 (n = 6, STC10/A (n = 5, and STC155/B1 (n = 3 were dominant. CONCLUSIONS: CMY-2 is the dominant AmpC β-lactamase in food animals and is associated with a transferable replicon IncA/C plasmid in the STC101, STC10, and STC155 strains.

  4. Sugar-induced cephalic-phase insulin release is mediated by a T1r2+T1r3-independent taste transduction pathway in mice.

    Science.gov (United States)

    Glendinning, John I; Stano, Sarah; Holter, Marlena; Azenkot, Tali; Goldman, Olivia; Margolskee, Robert F; Vasselli, Joseph R; Sclafani, Anthony

    2015-09-01

    Sensory stimulation from foods elicits cephalic phase responses, which facilitate digestion and nutrient assimilation. One such response, cephalic-phase insulin release (CPIR), enhances glucose tolerance. Little is known about the chemosensory mechanisms that activate CPIR. We studied the contribution of the sweet taste receptor (T1r2+T1r3) to sugar-induced CPIR in C57BL/6 (B6) and T1r3 knockout (KO) mice. First, we measured insulin release and glucose tolerance following oral (i.e., normal ingestion) or intragastric (IG) administration of 2.8 M glucose. Both groups of mice exhibited a CPIR following oral but not IG administration, and this CPIR improved glucose tolerance. Second, we examined the specificity of CPIR. Both mouse groups exhibited a CPIR following oral administration of 1 M glucose and 1 M sucrose but not 1 M fructose or water alone. Third, we studied behavioral attraction to the same three sugar solutions in short-term acceptability tests. B6 mice licked more avidly for the sugar solutions than for water, whereas T1r3 KO mice licked no more for the sugar solutions than for water. Finally, we examined chorda tympani (CT) nerve responses to each of the sugars. Both mouse groups exhibited CT nerve responses to the sugars, although those of B6 mice were stronger. We propose that mice possess two taste transduction pathways for sugars. One mediates behavioral attraction to sugars and requires an intact T1r2+T1r3. The other mediates CPIR but does not require an intact T1r2+T1r3. If the latter taste transduction pathway exists in humans, it should provide opportunities for the development of new treatments for controlling blood sugar. Copyright © 2015 the American Physiological Society.

  5. Detection of the plasmid-mediated mcr-1 gene conferring colistin resistance in human and food isolates of Salmonella enterica and Escherichia coli in England and Wales.

    Science.gov (United States)

    Doumith, Michel; Godbole, Gauri; Ashton, Philip; Larkin, Lesley; Dallman, Tim; Day, Martin; Day, Michaela; Muller-Pebody, Berit; Ellington, Matthew J; de Pinna, Elizabeth; Johnson, Alan P; Hopkins, Katie L; Woodford, Neil

    2016-08-01

    In response to the first report of transmissible colistin resistance mediated by the mcr-1 gene in Escherichia coli and Klebsiella spp. from animals and humans in China, we sought to determine its presence in Enterobacteriaceae isolated in the UK. The PHE archive of whole-genome sequences of isolates from surveillance collections, submissions to reference services and research projects was retrospectively analysed for the presence of mcr-1 using Genefinder. The genetic environment of the gene was also analysed. Rapid screening of the genomes of ∼24 000 Salmonella enterica, E. coli, Klebsiella spp., Enterobacter spp., Campylobacter spp. and Shigella spp. isolated from food or humans identified 15 mcr-1-positive isolates. These comprised: 10 human S. enterica isolates submitted between 2012 and 2015 (8 Salmonella Typhimurium, 1 Salmonella Paratyphi B var Java and 1 Salmonella Virchow) from 10 patients; 3 isolates of E. coli from 2 patients; and 2 isolates of Salmonella Paratyphi B var Java from poultry meat imported from the EU. The mcr-1 gene was located on diverse plasmids belonging to the IncHI2, IncI2 and IncX4 replicon types and its association with ISApl1 varied. Six mcr-1-positive S. enterica isolates were from patients who had recently travelled to Asia. Analysis of WGS data allowed rapid confirmation of the presence of the plasmid-mediated colistin resistance gene mcr-1 in diverse genetic environments and plasmids. It has been present in E. coli and Salmonella spp. harboured by humans in England and Wales since at least 2012. © Crown copyright 2016.

  6. Photochemical Modulation of Ras-Mediated Signal Transduction using Caged Farnesyltransferase Inhibitors: Activation via One- and Two-Photon Excitation

    Science.gov (United States)

    Abate-Pella, Daniel; Zeliadt, Nicholette A.; Ochocki, Joshua D.; Warmka, Janel K.; Dore, Timothy M.; Blank, David A.; Wattenberg, Elizabeth V.; Distefano, Mark D.

    2012-01-01

    The creation of caged molecules involves the attachment of protecting groups to biologically active compounds such as ligands, substrates, and drugs that can be removed under specific conditions. Photoremovable caging groups are the most common due to their ability to be removed with high spatial and temporal resolution. Here, the synthesis and photochemistry of a caged inhibitor of protein farnesyltransferase, Bhc-FTI, is described. The inhibitor was caged by alkylation of a critical thiol functional group with a Bhc moiety; while Bhc is well established as a protecting group for carboxylates and phosphates, it has not been extensively used to cage sulfhydryls. The resulting caged molecule, Bhc-FTI, can be photolyzed with UV light to release the inhibitor (FTI) that prevents Ras farnesylation, Ras membrane localization and downstream signaling. Finally, it is shown that Bhc-FTI can be uncaged by two-photon excitation to produce FTI at levels sufficient to inhibit Ras localization and alter cell morphology. Given the widespread involvement of Ras proteins in signal transduction pathways, this caged inhibitor should be useful in a plethora of studies. PMID:22492666

  7. Photochemical modulation of Ras-mediated signal transduction using caged farnesyltransferase inhibitors: activation by one- and two-photon excitation.

    Science.gov (United States)

    Abate-Pella, Daniel; Zeliadt, Nicholette A; Ochocki, Joshua D; Warmka, Janel K; Dore, Timothy M; Blank, David A; Wattenberg, Elizabeth V; Distefano, Mark D

    2012-05-07

    The creation of caged molecules involves the attachment of protecting groups to biologically active compounds such as ligands, substrates and drugs that can be removed under specific conditions. Photoremovable caging groups are the most common due to their ability to be removed with high spatial and temporal resolution. Here, the synthesis and photochemistry of a caged inhibitor of protein farnesyltransferase is described. The inhibitor, FTI, was caged by alkylation of a critical thiol group with a bromohydroxycoumarin (Bhc) moiety. While Bhc is well established as a protecting group for carboxylates and phosphates, it has not been extensively used to cage sulfhydryl groups. The resulting caged molecule, Bhc-FTI, can be photolyzed with UV light to release the inhibitor that prevents Ras farnesylation, Ras membrane localization and downstream signaling. Finally, it is shown that Bhc-FTI can be uncaged by two-photon excitation to produce FTI at levels sufficient to inhibit Ras localization and alter cell morphology. Given the widespread involvement of Ras proteins in signal transduction pathways, this caged inhibitor should be useful in a plethora of studies.

  8. CNS-restricted Transduction and CRISPR/Cas9-mediated Gene Deletion with an Engineered AAV Vector

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    Giridhar Murlidharan

    2016-01-01

    Full Text Available Gene therapy using recombinant adeno-associated viral (AAV vectors is emerging as a promising approach to treat central nervous system disorders such as Spinal muscular atrophy, Batten, Parkinson and Alzheimer disease amongst others. A critical remaining challenge for central nervous system-targeted gene therapy, silencing or gene editing is to limit potential vector dose-related toxicity in off-target cells and organs. Here, we characterize a lab-derived AAV chimeric (AAV2g9, which displays favorable central nervous system attributes derived from both parental counterparts, AAV2 and AAV9. This synthetic AAV strain displays preferential, robust, and widespread neuronal transduction within the brain and decreased glial tropism. Importantly, we observed minimal systemic leakage, decreased sequestration and gene transfer in off-target organs with AAV2g9, when administered into the cerebrospinal fluid. A single intracranial injection of AAV2g9 vectors encoding guide RNAs targeting the schizophrenia risk gene MIR137 (encoding MIR137 in CRISPR/Cas9 knockin mice resulted in brain-specific gene deletion with no detectable events in the liver. This engineered AAV vector is a promising platform for treating neurological disorders through gene therapy, silencing or editing modalities.

  9. Transduction for pheromones in the main olfactory epithelium is mediated by the Ca2+ -activated channel TRPM5.

    Science.gov (United States)

    López, Fabián; Delgado, Ricardo; López, Roberto; Bacigalupo, Juan; Restrepo, Diego

    2014-02-26

    Growing evidence suggests that the main olfactory epithelium contains a subset of olfactory sensory neurons (OSNs) responding to pheromones. One candidate subpopulation expresses the calcium activated cation channel TRPM5 (transient receptor potential channel M5). Using GFP driven by the TRPM5 promoter in mice, we show that this subpopulation responds to putative pheromones, urine, and major histocompatibility complex peptides, but not to regular odors or a pheromone detected by other species. In addition, this subpopulation of TRPM5-GFP+ OSNs uses novel transduction. In regular OSNs, odorants elicit activation of the cyclic nucleotide-gated (CNG) channel, leading to Ca2+ gating of Cl- channels; in TRPM5-GFP+ OSNs, the Ca2+ -activated Cl- ANO2 (anoctamin 2) channel is not expressed, and pheromones elicit activation of the CNG channel leading to Ca2+ gating of TRPM5. In conclusion, we show that OSNs expressing TRPM5 respond to pheromones, but not to regular odors through the opening of CNG channels leading to Ca2+ gating of TRPM5.

  10. Ca2+ is involved in muscarine-acetylcholine-receptor-mediated acetylcholine signal transduction in guard cells of Vicia faba L.

    Institute of Scientific and Technical Information of China (English)

    MENG Fanxia; MIAO Long; ZHANG Shuqiu; LOU Chenghou

    2004-01-01

    Acetylcholine (ACh) is an important neurochemical transmitter in animals; it also exists in plants and plays a significant role in various kinds of physiological functions in plants. ACh has been known to induce the stomatal opening. By monitoring the changes of cytosolic Ca2+ with fluorescent probe Fluo-3 AM under the confocal microscopy,we found that exogenous ACh increased cytosolic Ca2+ concentration of guard cells of Vicia faba L. Muscarine, an agonist of muscarine acetylcholine receptor (mAChR), could do so as well. In contrast, atropine, the antagonist of mAChR abolished the ability of ACh to increase Ca2+ in guard cells.This mechanism is similar to mAChR in animals. When EGTA was used to chelate Ca2+ or ruthenium red to block Ca2+ released from vacuole respectively, the results showed that the increased cytosolic Ca2+ mainly come from intracellular Ca2+ store. The evidence supports that Ca2+ is involved in guard-cell response to ACh and that Ca2+ signal is coupled to mAChRs in ACh signal transduction in guard cells.

  11. Plasmid mediated multiple antibiotic resistance in Escherichia coli isolated from community acquired infection of urinary tract in Aligarh Hospital

    Institute of Scientific and Technical Information of China (English)

    Asad U Khan; Saeedut Zafar Ali; Mohammed S Zaman

    2008-01-01

    This study was to investigate the current trends of multiple drug resistance in bacteria against antibiotics for the proper empirical treatmen.Clinical isolates were collected from community-acquired infection of urinary tract patients in Aligarh India from March 1999 to August 1999.Antibiotic susceptibility test was performed,using the disc diffusion method followed by plasmid isolation by the method of Kado and Liu.Transfer experiments were performed by the method of Lederberg and Cohen.Clinical study revealed that this infection was more common in young women.Various strains of E.coli isolated during the course of study were found to show multiple antibiotic resistance which was further characterized as plasmid-borne drug resistance.This study shows that E.coli may be one of the important causative agents of urinary tract infection (UTI )in young women.

  12. Multiple Antibiotic Resistance Plasmids Allow Scalable, PCR-Mediated DNA Manipulation and Near-Zero Background Cloning

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    Remigiusz Arnak

    2016-01-01

    Full Text Available We have constructed two plasmids that can be used for cloning as templates for PCR-based gene disruption, mutagenesis and the construction of DNA chromosome translocation cassettes. To our knowledge, these plasmids are the first vectors that confer resistance to ampicillin, kanamycin and hygromycin B in bacteria, and to geneticin (G418 and hygromycin B in Saccharomyces cerevisiae simultaneously. The option of simultaneously using up to three resistance markers provides a highly stringent control of recombinant selection and the almost complete elimination of background resistance, while unique restriction sites allow easy cloning of chosen genetic material. Moreover, we successfully used these new vectors as PCR templates for the induction of chromosome translocation in budding yeast by the bridge-induced translocation system. Cells in which translocation was induced carried chromosomal rearrangements as expected and exhibited resistance to both, G418 and hygromycin B. These features make our constructs very handy tools for many molecular biology applications.

  13. The presence of plasmid-mediated resistance genes among uropathogenes isolated from diabetic and non-diabetic patients with chronic pyelonephritis

    Directory of Open Access Journals (Sweden)

    O.I. Chub

    2016-08-01

    Full Text Available Increased multidrug resistance of extended-spectrum beta-lactamases (ESBLs compromises the efficacy of treatment of urinary tract infections. The objective of this study is to determine the prevalence of ESBL-producing uropathogens from patients with chronic pyelonephritis (CP and to evaluate the risk factors of these types of infections. Screening for the presence of plasmid-mediated ESBL was performed by polymerase chain reaction. Out of 105 patients, 22 (20.9% revealed strains with resistance genes: 11 (36.7%, 11 (36.7% and 8 (26.7% were identified to carry bla(TEM, bla(SHV and bla(CTX-M beta-lactamase genes, respectively. We have demonstrated that prevalence of the resistance among patients with CP combined with type 2 DM was 31.3%, while among patients with CP without type 2 DM was 27.4%; however the difference between these groups was not significant. The main factors related with appearance of plasmid-mediated resistance genes were age range above 55 years, Chronic Kidney Disease stage ІІІ and ІV, in-patient treatment history, history of using antibiotics last year. Isolation and detection of ESBL-producing strains are essential fоr the sеlection оf the mоst effеctive antibiоtic for the empiric trеatment.

  14. Extended-spectrum-beta-lactamases, AmpC beta-lactamases and plasmid mediated quinolone resistance in klebsiella spp. from companion animals in Italy.

    Directory of Open Access Journals (Sweden)

    Valentina Donati

    Full Text Available We report the genetic characterization of 15 Klebsiella pneumoniae (KP and 4 isolates of K. oxytoca (KO from clinical cases in dogs and cats and showing extended-spectrum cephalosporin (ESC resistance. Extended spectrum beta-lactamase (ESBL and AmpC genes, plasmid-mediated quinolone resistance (PMQR and co-resistances were investigated. Among KP isolates, ST101 clone was predominant (8/15, 53%, followed by ST15 (4/15, 27%. ST11 and ST340, belonging to Clonal Complex (CC11, were detected in 2012 (3/15, 20%. MLST on KP isolates corresponded well with PFGE results, with 11 different PFGE patterns observed, including two clusters of two (ST340 and four (ST101 indistinguishable isolates, respectively. All isolates harbored at least one ESBL or AmpC gene, all carried on transferable plasmids (IncR, IncFII, IncI1, IncN, and 16/19 were positive for PMQR genes (qnr family or aac(6'-Ib-cr. The most frequent ESBL was CTX-M-15 (11/19, 58%, detected in all KP ST101, in one KP ST15 and in both KP ST340. blaCTX-M-15 was carried on IncR plasmids in all but one KP isolate. All KP ST15 isolates harbored different ESC resistance genes and different plasmids, and presented the non-transferable blaSHV-28 gene, in association with blaCTX-M-15, blaCTX-M-1 (on IncR, or on IncN, blaSHV-2a (on IncR or blaCMY-2 genes (on IncI1. KO isolates were positive for blaCTX-M-9 gene (on IncHI2, or for the blaSHV-12 and blaDHA-1 genes (on IncL/M. They were all positive for qnr genes, and one also for the aac(6'-Ib-cr gene. All Klebsiella isolates showed multiresistance towards aminoglycosides, sulfonamides, tetracyclines, trimethoprim and amphenicols, mediated by strA/B, aadA2, aadB, ant (2"-Ia, aac(6'-Ib, sul, tet, dfr and cat genes in various combinations. The emergence in pets of multidrug-resistant Klebsiella with ESBL, AmpC and PMQR determinants, poses further and serious challenges in companion animal therapy and raise concerns for possible bi-directional transmission between

  15. Toxin Plasmids of Clostridium perfringens

    Science.gov (United States)

    Li, Jihong; Adams, Vicki; Bannam, Trudi L.; Miyamoto, Kazuaki; Garcia, Jorge P.; Uzal, Francisco A.; Rood, Julian I.

    2013-01-01

    SUMMARY In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch's postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract. PMID:23699255

  16. Mechanism of macrophage injury following traumatic hemorrhagic shock: through PTX-sensitive G-protein-mediated signal transduction pathway

    Institute of Scientific and Technical Information of China (English)

    刘靖华; 刘良明; 陈惠孙; 胡德耀; 刘怀琼

    2002-01-01

    Objective: To study the mechanism of macrophage injury after trauma-hemorrhagic shock.   Methods: Wistar male rats underwent trauma (closed bone fracture) and hemorrhage (mean arterial blood pressure of 35 mm Hg±5 mm Hg for 60 minutes, following fluid resuscitation). Rats without trauma, hemorrhage or fluid resuscitation served as controls. Peritoneal macrophages were harvested at 6 hours and 1, 2, 3, 7 days after traumatic hemorrhagic shock to determine the effects of pertussis toxin (PTX, as a specific inhibitor to Giα) and cholera toxin (CTX, as a stimulant to Gsα) on macrophage-Ia expression and TNF-α production and levels of Giα and Gsα.   Results: The macrophages from the injured rats revealed a significant decrease of Ia positive number and TNF-α release in response to LPS. With pretreatment with PTX 10-100 ng/ml Ia positive cells and LPS-induced TNFα production in both control and impaired macrophages populations were dose-dependently increased. Both macrophages populations were not responding to CTX treatment (10-100 ng/ml). Western blot analyses showed that the levels of Giα protein expression increased as much as 116.5%-148.8% of the control level from 6 hours through 7 days after traumatic hemorrhage. The levels of Gsα protein expression were reduced at 6 hours and decreased to the lowest degree; 36% of the control at day 1, began to return at day 2 and returned to the normal level at day 7, following traumatic hemorrhagic shock.   Conclusions: PTX-sensitive G-protein may participate in the modulation of macrophage-Ia expression and TNF-α release following traumatic hemorrhagic shock. Analyses of the alteration of Giα and Gsα protein expressions further supports the concept that G-protein is involved in trauma-induced macrophage signal transduction pathways.

  17. Association of iss and iucA, but not tsh, with plasmid-mediated virulence of avian pathogenic Escherichia coli.

    Science.gov (United States)

    Tivendale, Kelly A; Allen, Joanne L; Ginns, Carol A; Crabb, Brendan S; Browning, Glenn F

    2004-11-01

    Avian pathogenic Escherichia coli (APEC) is an economically important respiratory pathogen of chickens worldwide. Factors previously associated with the virulence of APEC include adhesins, iron-scavenging mechanisms, the production of colicin V (ColV), serum resistance, and temperature-sensitive hemagglutination, but virulence has generally been assessed by parenteral inoculation, which does not replicate the normal respiratory route of infection. A large plasmid, pVM01, is essential for virulence in APEC strain E3 in chickens after aerosol exposure. Here we establish the size of pVM01 to be approximately 160 kb and show that the putative virulence genes iss (increased serum survival) and tsh (temperature-sensitive hemagglutinin) and the aerobactin operon are on the plasmid. These genes were not clustered on pVM01 but, rather, were each located in quite distinct regions. Examination of APEC strains with defined levels of respiratory pathogenicity after aerosol exposure showed that both the aerobactin operon and iss were associated with high levels of virulence in APEC but that the possession of either gene was sufficient for intermediate levels of virulence. In contrast, the presence of tsh was not necessary for high levels of virulence. Thus, both the aerobactin operon and iss are associated with virulence in APEC after exposure by the natural route of infection. The similarities between APEC and extraintestinal E. coli infection in other species suggests that they may be useful models for definition of the role of these virulence genes and of other novel virulence genes that may be located on their virulence plasmids.

  18. Emergence of co-production of plasmid-mediated AmpC beta-lactamase and ESBL in cefoxitin-resistant uropathogenic Escherichia coli.

    Science.gov (United States)

    Ghosh, B; Mukherjee, M

    2016-09-01

    Plasmid-mediated AmpC (pAmpC) and ESBL co-production was detected in Escherichia coli a major etiologic agent of urinary tract infection. Isolates resistant to cefoxitin by CLSI methodology were tested for pAmpC beta-lactamase using phenylboronic acid and ESBLs by combined disk diffusion method. pAmpC/ESBL genes were characterized by PCR and sequencing. Transconjugation experiments were done to study the transfer of pAmpC and ESBL production from clinical isolates as donor to E. coli J53 AziR as recipient. Incompatibility groups of transmissible plasmids were classified by PCR-based replicon typing (PBRT). Among 148 urine culture positive isolates, E. coli was reported in 39.86 % (59/148), with 93.22 % (55/59) of cefoxitin resistance. pAmpC production was detected in 25, with varied distribution of blaCMY-2 and blaDHA-1type genes alone (n = 13 and 7 respectively) or in combination (n = 5). ESBL co-production was observed in 88 % (22/25) of pAmpC producing isolates with predominance of blaTEM (n = 20). Twenty-three transconjugants showed transmission of pAmpC-and ESBL-resistant genes with co-carriage of blaCMY-2 and blaTEM (n = 15) in plasmids of IncF type (n = 9) being predominant, followed by IncI1 (n = 4) and IncH1 (n = 2) in combination. All clinical isolates were clonally diverse. Resistance against different beta-lactams in uropathogenic E. coli has been an emerging concern in resource- poor countries such as India. Knowledge on the occurrence of AmpC beta-lactamases and ESBL amongst this pathogen and its transmission dynamics may aid in hospital infection control.

  19. Gene Flow Across Genus Barriers - Conjugation of Dinoroseobacter shibae's 191-kb Killer Plasmid into Phaeobacter inhibens and AHL-mediated Expression of Type IV Secretion Systems.

    Science.gov (United States)

    Patzelt, Diana; Michael, Victoria; Päuker, Orsola; Ebert, Matthias; Tielen, Petra; Jahn, Dieter; Tomasch, Jürgen; Petersen, Jörn; Wagner-Döbler, Irene

    2016-01-01

    Rhodobacteraceae harbor a conspicuous wealth of extrachromosomal replicons (ECRs) and therefore the exchange of genetic material via horizontal transfer has been supposed to be a major evolutionary driving force. Many plasmids in this group encode type IV secretion systems (T4SS) that are expected to mediate transfer of proteins and/or DNA into host cells, but no experimental evidence of either has yet been provided. Dinoroseobacter shibae, a species of the Roseobacter group within the Rhodobacteraceae family, contains five ECRs that are crucial for anaerobic growth, survival under starvation and the pathogenicity of this model organism. Here we tagged two syntenous but compatible RepABC-type plasmids of 191 and 126-kb size, each encoding a T4SS, with antibiotic resistance genes and demonstrated their conjugational transfer into a distantly related Roseobacter species, namely Phaeobacter inhibens. Pulsed field gel electrophoresis showed transfer of those replicons into the recipient both individually but also together documenting the efficiency of conjugation. We then studied the influence of externally added quorum sensing (QS) signals on the expression of the T4SS located on the sister plasmids. A QS deficient D. shibae null mutant (ΔluxI1 ) lacking synthesis of N-acyl-homoserine lactones (AHLs) was cultivated with a wide spectrum of chemically diverse long-chain AHLs. All AHLs with lengths of the acid side-chain ≥14 reverted the ΔluxI1 phenotype to wild-type. Expression of the T4SS was induced up to log2 ∼3fold above wild-type level. We hypothesize that conjugation in roseobacters is QS-controlled and that the QS system may detect a wide array of long-chain AHLs at the cell surface.

  20. ClpP/ClpX-mediated degradation of the bacteriophage lambda O protein and regulation of lambda phage and lambda plasmid replication.

    Science.gov (United States)

    Wegrzyn, A; Czyz, A; Gabig, M; Wegrzyn, G

    2000-01-01

    The O protein is a replication initiator that binds to the orilambda region and promotes assembly of the bacteriophage lambda replication complex. This protein, although protected from proteases by other elements of the replication complex, in a free form is rapidly degraded in the host, Escherichia coli, by the ClpP/ClpX protease. Nevertheless, the physiological role of this rapid degradation remains unclear. Here we demonstrate that the copy number of plasmids derived from bacteriophage lambda is significantly higher in wild-type cells growing in rich media than in slowly growing bacteria. However, lambda plasmid copy number in bacteria devoid of the ClpP/ClpX protease was not dependent on the bacterial growth rate and in all minimal media tested was comparable to that observed in wildtype cells growing in a rich medium. Contrary to lambda plasmid replication, the efficiency of lytic growth of bacteriophage lambda was found to be dependent on the host growth rate in both wild-type bacteria and clpP and clpX mutants. The activities of two major lambda promoters operating during the lytic development, p(R) and p(L), were found to be slightly dependent on the host growth rate. However, when p(R) activity was significantly decreased in the dnaA mutant, production of phage progeny was completely abolished at low growth rates. These results indicate that the O protein (whose level in E. coli cells depends on the activity of ClpP/ClpX protease) is a major limiting factor in the regulation of lambda plasmid replication at low bacterial growth rates. However, this protein seems to be only one of the limiting factors in the bacteriophage lambda lytic development under poor growth conditions of host cells. Therefore, it seems that the role of the rapid ClpP/ClpX-mediated proteolysis of the O protein is to decrease the efficiency of early DNA replication of the phage in slowly growing host cells.

  1. The application of polymer-mediated plasmid DNA transit systems in bone tissue engineering%聚合物介导质粒DNA转运系统在骨组织工程中的应用

    Institute of Scientific and Technical Information of China (English)

    夏伦果; 蒋欣泉; 张志愿

    2009-01-01

    Gene therapy can further promote osteogenesis in bone tissue engineering. By protecting DNA from degradation and maintaining the concentration of DNA effectively, polymer-mediated plasmid DNA transit systems could extend its endocytosis opportunities and enhance the efficiency of gene transfer. At present, polymer-mediated plasmid DNA transit systems used for bone tissue engineering mainly include plasmid DNA and collagen protein composite transit system, plasmid DNA and polyethylene glycol hyaluronic acid hydroge composite transit system, plasmid DNA and liposome composite transit system, plasmid DNA and cationic polymer composite transit systems. This review focuses on the present status of application of polymer-mediated plasmid DNA transit systems.%基因治疗技术应用于骨组织工程,可以进一步促进成骨.聚合物介导的质粒DNA转运系统通过保护DNA免受降解并维持DNA在效应浓度,延长其内吞的机会,从而提高基因转染效率.目前用于骨组织工程研究的聚合物介导的质粒DNA转运系统主要有质粒DNA与胶原蛋白复合转运系统、质粒DNA与聚乙二醇-透明质酸水凝胶复合转运系统、质粒DNA与脂质体复合转运系统、质粒DNA与阳离子聚合物复合转运系统等.本文对近年来聚合物介导的质粒DNA复合转运系统在骨组织工程中的应用进展做一综述.

  2. Prevalence and characterisation of plasmid-mediated quinolone resistance and mutations in the gyrase and topoisomerase IV genes among Shigella isolates from Henan, China, between 2001 and 2008.

    Science.gov (United States)

    Yang, Haiyan; Duan, Guangcai; Zhu, Jingyuan; Zhang, Weidong; Xi, Yuanlin; Fan, Qingtang

    2013-08-01

    A total of 293 Shigella isolates were isolated from patients with diarrhoea in four villages of Henan, China. This study investigated the prevalence of the plasmid-mediated quinolone resistance (PMQR) genes qnrA, qnrB, qnrS, qepA and aac(6')-Ib-cr and compared the polymorphic quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC and parE. Of the isolates, 292 were found to be resistant to nalidixic acid and pipemidic acid, whereas 77 were resistant to ciprofloxacin (resistance rate of 26.3%). Resistance of the Shigella isolates to ciprofloxacin significantly increased from 2001 to 2008 (PShigella isolates are common in China. This study found that there was a significant increase in mutation rates of the QRDR and the resistant rates to ciprofloxacin. Other mechanisms may be present in the isolates that also contribute to their resistance to ciprofloxacin.

  3. Real-time quantitative PCR assay with Taqman® probe for rapid detection of MCR-1 plasmid-mediated colistin resistance

    Directory of Open Access Journals (Sweden)

    S. Chabou

    2016-09-01

    Full Text Available Here we report the development of two rapid real-time quantitative PCR assays with TaqMan® probes to detect the MCR-1 plasmid-mediated colistin resistance gene from bacterial isolates and faecal samples from chickens. Specificity and sensitivity of the assay were 100% on bacterial isolates including 18 colistin-resistant isolates carrying the mcr-1 gene (six Klebsiella pneumoniae and 12 Escherichia coli with a calibration curve that was linear from 101 to 108 DNA copies. Five out of 833 faecal samples from chickens from Algeria were positive, from which three E. coli strains were isolated and confirmed to harbour the mcr-1 gene by standard PCR and sequencing.

  4. Occurrence of Extended-Spectrum β-Lactamases, Plasmid-Mediated Quinolone Resistance, and Disinfectant Resistance Genes in Escherichia coli Isolated from Ready-To-Eat Meat Products

    DEFF Research Database (Denmark)

    Li, Lili; Ye, Lei; Kromann, Sofie

    2017-01-01

    There are growing concerns about the coselection of resistance against antibiotics and disinfectants in bacterial pathogens. The aim of this study was to characterize the antimicrobial susceptibility profiles, the prevalence of extended-spectrum β-lactamases (ESBLs), plasmid-mediated quinolone...... resistance genes (PMQRs), and quaternary ammonium compound resistance genes (QACs) in Escherichia coli isolated from ready-to-eat (RTE) meat products obtained in Guangzhou, China, and to determine whether these genes were colocalized in the isolates. A total of 64 E. coli isolates were obtained from 720 RTE...... meat samples. Multidrug resistance was observed in 70.3% of the isolates. A 100% of the isolates were resistant to benzalkonium chloride. Four types of β-lactamase genes were identified in the 16 ESBL-producing E. coli isolates: blaSHV (9.4%), blaTEM (7.8%), blaCTX-M-15 (1.6%), and blaCTX-M-9 (1...

  5. Plasmid-mediated extended-spectrum beta-lactamase-producing strains of Enterobacteriaceae isolated from diabetes foot infections in a Brazilian diabetic center

    Directory of Open Access Journals (Sweden)

    R.N. Motta

    2003-04-01

    Full Text Available We bacteriologically analyzed 156 species of Enterobacteriaceae, isolated from 138 patients with community-acquired diabetic foot ulcers, in a prospective study made at a diabetic center and at the Federal University of Ceará, Brazil, from March, 2000, to November, 2001.The samples were cultured using selective media, and identification, susceptibility tests and detection of plasmid-mediated-extended-spectrum-beta-lactamase (ESBL producing strains were made with conventional and automated methods. The most frequently occurring pathogens were K. pneumoniae (21.2%, Morganella morganii (19.9% and E. coli (15.4%. High resistance rates were noted for ampicillin, first generation cephalosporin, trimethoprim/sulfamethoxazole, tetracycline, amoxicillin-clavulanic acid and chloramphenicol. ESBL-producing strains were detected in 6% of the patients. Resistance among gram-negative bacteria has become increasingly common, even in community-acquired infections.

  6. Travelers Can Import Colistin-Resistant Enterobacteriaceae, Including Those Possessing the Plasmid-Mediated mcr-1 Gene.

    Science.gov (United States)

    Bernasconi, Odette J; Kuenzli, Esther; Pires, João; Tinguely, Regula; Carattoli, Alessandra; Hatz, Christoph; Perreten, Vincent; Endimiani, Andrea

    2016-08-01

    Stool samples from 38 travelers returning from India were screened for extended-spectrum cephalosporin- and carbapenem-resistant Enterobacteriaceae implementing standard selective plates. Twenty-six (76.3%) people were colonized with CTX-M or DHA producers, but none of the strains was colistin resistant and/or mcr-1 positive. Nevertheless, using overnight enrichment and CHROMagar Orientation plates supplemented with colistin, four people (10.5%) were found to be colonized with colistin-resistant Escherichia coli One cephalosporin-susceptible sequence type 10 (ST10) strain carried a 4,211-bp ISApl1-mcr-1-ISApl1 element in an IncHI2 plasmid backbone. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  7. Phenotypic plasticity in bacterial plasmids.

    Science.gov (United States)

    Turner, Paul E

    2004-01-01

    Plasmid pB15 was previously shown to evolve increased horizontal (infectious) transfer at the expense of reduced vertical (intergenerational) transfer and vice versa, a key trade-off assumed in theories of parasite virulence. Whereas the models predict that susceptible host abundance should determine which mode of transfer is selectively favored, host density failed to mediate the trade-off in pB15. One possibility is that the plasmid's transfer deviates from the assumption that horizontal spread (conjugation) occurs in direct proportion to cell density. I tested this hypothesis using Escherichia coli/pB15 associations in laboratory serial culture. Contrary to most models of plasmid transfer kinetics, my data show that pB15 invades static (nonshaking) bacterial cultures only at intermediate densities. The results can be explained by phenotypic plasticity in traits governing plasmid transfer. As cells become more numerous, the plasmid's conjugative transfer unexpectedly declines, while the trade-off between transmission routes causes vertical transfer to increase. Thus, at intermediate densities the plasmid's horizontal transfer can offset selection against plasmid-bearing cells, but at high densities pB15 conjugates so poorly that it cannot invade. I discuss adaptive vs. nonadaptive causes for the phenotypic plasticity, as well as potential mechanisms that may lead to complex transfer dynamics of plasmids in liquid environments. PMID:15166133

  8. Boldo prevents UV light and nitric oxide-mediated plasmid DNA damage and reduces the expression of Hsp70 protein in melanoma cancer cells.

    Science.gov (United States)

    Russo, Alessandra; Cardile, Venera; Caggia, Silvia; Gunther, Germán; Troncoso, Nicolas; Garbarino, Juan

    2011-09-01

    This study was designed to investigate the potential protective effect of a methanolic extract of Peumus boldus leaves on UV light and nitric oxide (NO)-mediated DNA damage. In addition, we investigated the growth inhibitory activity of this natural product against human melanoma cells (M14). Boldine, catechin, quercetin and rutin were identified using a HPLC method. The extract was incubated with plasmid DNA and, before irradiating the samples with UV-R, H(2) O(2) was added. For analysis of DNA single-strand breaks induced by NO, the experiments were performed by incubating the extract with Angeli's salt. In the study on M14 cell line, cell viability was measured using MTT assay. Release of lactate dehydrogenase, a marker of membrane breakdown, was also measured. For the detection of apoptosis, the evaluation of DNA fragmentation (COMET assay) and caspase-3 activity assay were employed. The expression of heat shock protein 70 (Hsp70) was detected by Western blot analysis. Generation of reactive oxygen species was measured by using a fluorescent probe. The extract (demonstrating the synergistic effect of the constituents boldine and flavonoids), showed a protective effect on plasmid DNA and selectively inhibited the growth of melanoma cells. But a novel finding was that apoptosis evoked by this natural product in M14 cells, appears to be mediated, at least in part, via the inhibition of Hsp70 expression, which may be correlated with a modulation of redox-sensitive mechanisms. These results confirm the promising biological properties of Peumus boldus and encourage in-vivo investigations into its potential anti-cancer activity. © 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.

  9. Gαi2-protein-mediated signal transduction: central nervous system molecular mechanism countering the development of sodium-dependent hypertension.

    Science.gov (United States)

    Wainford, Richard D; Carmichael, Casey Y; Pascale, Crissey L; Kuwabara, Jill T

    2015-01-01

    Excess dietary salt intake is an established cause of hypertension. At present, our understanding of the neuropathophysiology of salt-sensitive hypertension is limited by a lack of identification of the central nervous system mechanisms that modulate sympathetic outflow and blood pressure in response to dietary salt intake. We hypothesized that impairment of brain Gαi2-protein-gated signal transduction pathways would result in increased sympathetically mediated renal sodium retention, thus promoting the development of salt-sensitive hypertension. To test this hypothesis, naive or renal denervated Dahl salt-resistant and Dahl salt-sensitive (DSS) rats were assigned to receive a continuous intracerebroventricular control scrambled or a targeted Gαi2-oligodeoxynucleotide infusion, and naive Brown Norway and 8-congenic DSS rats were fed a 21-day normal or high-salt diet. High salt intake did not alter blood pressure, suppressed plasma norepinephrine, and evoked a site-specific increase in hypothalamic paraventricular nucleus Gαi2-protein levels in naive Brown Norway, Dahl salt-resistant, and scrambled oligodeoxynucleotide-infused Dahl salt-resistant but not DSS rats. In Dahl salt-resistant rats, Gαi2 downregulation evoked rapid renal nerve-dependent hypertension, sodium retention, and sympathoexcitation. In DSS rats, Gαi2 downregulation exacerbated salt-sensitive hypertension via a renal nerve-dependent mechanism. Congenic-8 DSS rats exhibited sodium-evoked paraventricular nucleus-specific Gαi2-protein upregulation and attenuated hypertension, sodium retention, and global sympathoexcitation compared with DSS rats. These data demonstrate that paraventricular nucleus Gαi2-protein-gated pathways represent a conserved central molecular pathway mediating sympathoinhibitory renal nerve-dependent responses evoked to maintain sodium homeostasis and a salt-resistant phenotype. Impairment of this mechanism contributes to the development of salt-sensitive hypertension.

  10. Signal Transduction Mechanisms Underlying Group I mGluR-mediated Increase in Frequency and Amplitude of Spontaneous EPSCs in the Spinal Trigeminal Subnucleus Oralis of the Rat

    Directory of Open Access Journals (Sweden)

    Ahn Dong-Kuk

    2009-09-01

    Full Text Available Abstract Group I mGluRs (mGluR1 and 5 pre- and/or postsynaptically regulate synaptic transmission at glutamatergic synapses. By recording spontaneous EPSCs (sEPSCs in the spinal trigeminal subnucleus oralis (Vo, we here investigated the regulation of glutamatergic transmission through the activation of group I mGluRs. Bath-applied DHPG (10 μM/5 min, activating the group I mGluRs, increased sEPSCs both in frequency and amplitude; particularly, the increased amplitude was long-lasting. The DHPG-induced increases of sEPSC frequency and amplitude were not NMDA receptor-dependent. The DHPG-induced increase in the frequency of sEPSCs, the presynaptic effect being further confirmed by the DHPG effect on paired-pulse ratio of trigeminal tract-evoked EPSCs, an index of presynaptic modulation, was significantly but partially reduced by blockades of voltage-dependent sodium channel, mGluR1 or mGluR5. Interestingly, PKC inhibition markedly enhanced the DHPG-induced increase of sEPSC frequency, which was mainly accomplished through mGluR1, indicating an inhibitory role of PKC. In contrast, the DHPG-induced increase of sEPSC amplitude was not affected by mGluR1 or mGluR5 antagonists although the long-lasting property of the increase was disappeared; however, the increase was completely inhibited by blocking both mGluR1 and mGluR5. Further study of signal transduction mechanisms revealed that PLC and CaMKII mediated the increases of sEPSC in both frequency and amplitude by DHPG, while IP3 receptor, NO and ERK only that of amplitude during DHPG application. Altogether, these results indicate that the activation of group I mGluRs and their signal transduction pathways differentially regulate glutamate release and synaptic responses in Vo, thereby contributing to the processing of somatosensory signals from orofacial region.

  11. International collaborative study on the occurrence of plasmid mediated quinolone resisitance in Salmonella enterica en Escherichia coli isolated from animals, humans, food and the environment in 13 European countries.

    NARCIS (Netherlands)

    Veldman, K.T.; Cavaco, L.M.; Mevius, D.J.; Battisti, A.; Botteldoorn, N.; Bruneau, M.; Cerny, T.; Franco, A.; Frutos Escobar, De C.; Guerra, B.; Gutierrez, M.; Hopkins, K.; Myllyniemi, A.L.; Perrin-Guyomard, A.; Schroeter, A.; Sunde, M.; Wasyl, D.; Aarestrup, F.M.

    2011-01-01

    Objectives This study was initiated to collect retrospective information on the occurrence of plasmid-mediated quinolone resistance (PMQR) in Salmonella enterica and Escherichia coli isolates in Europe and to identify the responsible genes. Methods Databases of national reference laboratories contai

  12. Lichen metabolites prevent UV light and nitric oxide-mediated plasmid DNA damage and induce apoptosis in human melanoma cells.

    Science.gov (United States)

    Russo, A; Piovano, M; Lombardo, L; Garbarino, J; Cardile, V

    2008-09-26

    In humans both UV-A and UV-B can cause gene mutations and suppress immunity, which leads to skin cancer, including melanoma. Inhibition of reactive oxygen species (ROS) and reactive nitrogen species (RNS) appears particularly promising as ROS and RNS production by both UV-A and UV-B contributes to inflammation, immunosuppression, gene mutation and carcinogenesis. We evaluated the effect of two lichen compounds, sphaerophorin (depside) and pannarin (depsidone) on pBR322 DNA cleavage induced by hydroxyl radicals (()OH), and by nitric oxide (NO), and their superoxide anion (O(2)(-)) scavenging capacity. In addition, we investigated the growth inhibitory activity of these compounds against human melanoma cells (M14 cell line). Sphaerophorin and pannarin showed a protective effect on plasmid DNA and exhibited a superoxide dismutase like effect. The data obtained in cell culture show that these lichen metabolites inhibit the growth of melanoma cells, inducing an apoptotic cell death, demonstrated by the fragmentation of genomic DNA (COMET and TUNEL Assays) and by a significant increase of caspase-3 activity, and correlated, at least in part, to the increase of ROS generation, These results confirm the promising biological properties of sphaerophorin and pannarin and encourage further investigations on their molecular mechanisms.

  13. High prevalence of plasmid-mediated 16S rRNA methylase gene rmtB among Escherichia coli clinical isolates from a Chinese teaching hospital

    Directory of Open Access Journals (Sweden)

    Zhang Xue-qing

    2010-06-01

    transposon, Tn3, was located upstream of the rmtB. Nineteen clonal patterns were obtained by PFGE, with type H representing the prevailing pattern. Conclusion A high prevalence of plasmid-mediated rmtB gene was found among clinical E. coli isolates from a Chinese teaching hospital. Both horizontal gene transfer and clonal spread were responsible for the dissemination of the rmtB gene.

  14. Identification of DHA-23, a Novel Plasmid-mediated and Inducible AmpC beta-Lactamase from Enterobacteriaceae in Northern Taiwan

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    Wen-Shyang eHsieh

    2015-05-01

    Full Text Available Objectives: AmpC β-lactamases are classified as Amber Class C and Bush Group 1. AmpC β-lactamases can hydrolyze broad and extended-spectrum cephalosporins, and are not inhibited by β-lactamase inhibitors such as clavulanic acid. This study was conducted to identify DHA-23, a novel plasmid-mediated and inducible AmpC β-lactamase obtained from Enterobacteriaceae. Methods: A total of 210 carbapenem-resistant Enterobacteriaceae isolates were collected from a medical center (comprising 2 branches in Northern Taiwan during 2009–2012. AmpC β-lactamase genes were analyzed through a polymerase chain reaction using plasmid DNA templates and gene sequencing. The genetic relationships of the isolates were typed using pulsed-field gel electrophoresis following the digestion of intact genomic DNA by using XbaI. Results: Three enterobacterial isolates (one Escherichia coli and 2 Klebsiella pneumoniae were obtained from 3 hospitalized patients. All 3 isolates were resistant or intermediately susceptible to all β-lactams, and exhibited reduced susceptibility to carbapenems. These 3 isolates expressed a novel AmpC β-lactamase, designated DHA-23, approved by the curators of the Lahey website. DHA-23 differs from DHA-1 and DHA-6 by one amino acid substitution (Ser245Ala, exhibiting 2 amino acid changes compared with DHA-7 and DHA-Morganella morganii; 3 amino acid changes compared with DHA-3; 4 amino acid changes compared with DHA-5; and 8 amino acid changes compared with DHA-2 (> 97% identity. This AmpC β-lactamase is inducible using a system involving ampR. Conclusion: This is the first report to address DHA-23, a novel AmpC β-lactamase. DHA-type β-lactamases are continuous threat in Taiwan.

  15. Melatonin Signal Transduction Pathways Require E-Box-Mediated Transcription of Per1 and Per2 to Reset the SCN Clock at Dusk

    Science.gov (United States)

    Kandalepas, Patty C.; Mitchell, Jennifer W.; Gillette, Martha U.

    2016-01-01

    Melatonin is released from the pineal gland into the circulatory system at night in the absence of light, acting as “hormone of darkness” to the brain and body. Melatonin also can regulate circadian phasing of the suprachiasmatic nucleus (SCN). During the day-to-night transition, melatonin exposure advances intrinsic SCN neural activity rhythms via the melatonin type-2 (MT2) receptor and downstream activation of protein kinase C (PKC). The effects of melatonin on SCN phasing have not been linked to daily changes in the expression of core genes that constitute the molecular framework of the circadian clock. Using real-time RT-PCR, we found that melatonin induces an increase in the expression of two clock genes, Period 1 (Per1) and Period 2 (Per2). This effect occurs at CT 10, when melatonin advances SCN phase, but not at CT 6, when it does not. Using anti-sense oligodeoxynucleotides (α ODNs) to Per 1 and Per 2, as well as to E-box enhancer sequences in the promoters of these genes, we show that their specific induction is necessary for the phase-altering effects of melatonin on SCN neural activity rhythms in the rat. These effects of melatonin on Per1 and Per2 were mediated by PKC. This is unlike day-active non-photic signals that reset the SCN clock by non-PCK signal transduction mechanisms and by decreasing Per1 expression. Rather, this finding extends roles for Per1 and Per2, which are critical to photic phase-resetting, to a nonphotic zeitgeber, melatonin, and suggest that the regulation of these clock gene transcripts is required for clock resetting by diverse regulatory cues. PMID:27362940

  16. Melatonin Signal Transduction Pathways Require E-Box-Mediated Transcription of Per1 and Per2 to Reset the SCN Clock at Dusk.

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    Patty C Kandalepas

    Full Text Available Melatonin is released from the pineal gland into the circulatory system at night in the absence of light, acting as "hormone of darkness" to the brain and body. Melatonin also can regulate circadian phasing of the suprachiasmatic nucleus (SCN. During the day-to-night transition, melatonin exposure advances intrinsic SCN neural activity rhythms via the melatonin type-2 (MT2 receptor and downstream activation of protein kinase C (PKC. The effects of melatonin on SCN phasing have not been linked to daily changes in the expression of core genes that constitute the molecular framework of the circadian clock. Using real-time RT-PCR, we found that melatonin induces an increase in the expression of two clock genes, Period 1 (Per1 and Period 2 (Per2. This effect occurs at CT 10, when melatonin advances SCN phase, but not at CT 6, when it does not. Using anti-sense oligodeoxynucleotides (α ODNs to Per 1 and Per 2, as well as to E-box enhancer sequences in the promoters of these genes, we show that their specific induction is necessary for the phase-altering effects of melatonin on SCN neural activity rhythms in the rat. These effects of melatonin on Per1 and Per2 were mediated by PKC. This is unlike day-active non-photic signals that reset the SCN clock by non-PCK signal transduction mechanisms and by decreasing Per1 expression. Rather, this finding extends roles for Per1 and Per2, which are critical to photic phase-resetting, to a nonphotic zeitgeber, melatonin, and suggest that the regulation of these clock gene transcripts is required for clock resetting by diverse regulatory cues.

  17. Mucin-like glycoprotein secretion is mediated by cyclic-AMP and protein kinase C signal transduction pathways in rat corneal epithelium.

    Science.gov (United States)

    Nakamura, M; Endo, K; Nakata, K

    1998-05-01

    Ocular surface mucin is secreted from both goblet cells in the conjunctival epithelium and corneal epithelial cells. To clarify its mechanism of secretion in corneal epithelial cells, a rat cornea organ culture system was used to evaluate the second messenger roles of cyclic-AMP (cAMP), cyclic-GMP (cGMP) and protein kinase C (PKC) in modulating mucin-like glycoprotein secretion. Rat cornea sections (3 mm diameter) were cultured in TC-199 medium, and radiolabeled with sodium sulfate for 18 hr. After washing, the corneas were treated with various second messenger modulating agents for 30 min. The culture media were reacted with Dolichos biflorus (DBA)-lectin, and mucin-like glycoprotein was isolated. Then the radioactivity of DBA-binding mucin-like glycoprotein was isolated. Then the radioactivity of DBA-binding mucin-like glycoprotein was measured. There was a time-dependent increase in mucin-like glycoprotein was measured. There was a time-dependent increase in mucin-like glycoprotein secretion, whereas after corneal epithelial debridement the secretion was markedly inhibited by 81%. Mucin-like glycoprotein secretion was stimulated in a dose-dependent manner following elevation of cAMP levels by exposure to either forskolin, dibutyryl cAMP or 3-isobutyl-1-methylxanthine. Concomitant exposure to the cAMP dependent protein kinase inhibitor, KT5720 completely inhibited their stimulatory effects. Neither exposure to dibutyryl cGMP nor nitroprusside affected mucin-like glycoprotein secretion. Stimulation by PKC, phorbol 12, 13-dibutyrate (PDBu) also increased mucin-like glycoprotein secretion in a dose-dependent fashion. The PKC inhibitor, calphostin C completely inhibited the stimulation by PDBu of mucine-like glycoprotein secretion. These results demonstrate that corneal epithelial cells secrete mucin-like glycoprotein, which is mediated by cAMP and PKC signal transduction pathways.

  18. Adeno-associated virus and lentivirus vectors mediate efficient and sustained transduction of cultured mouse and human dorsal root ganglia sensory neurons.

    Science.gov (United States)

    Fleming, J; Ginn, S L; Weinberger, R P; Trahair, T N; Smythe, J A; Alexander, I E

    2001-01-01

    Peripheral nervous system (PNS) sensory neurons are directly involved in the pathophysiology of numerous inherited and acquired neurological conditions. Therefore, efficient and stable gene delivery to these postmitotic cells has significant therapeutic potential. Among contemporary vector systems capable of neuronal transduction, only those based on herpes simplex virus have been extensively evaluated in PNS neurons. We therefore investigated the transduction performance of recombinant adeno-associated virus type 2 (AAV) and VSV-G-pseudotyped lentivirus vectors derived from human immunodeficiency virus (HIV-1) in newborn mouse and fetal human dorsal root ganglia (DRG) sensory neurons. In dissociated mouse DRG cultures both vectors achieved efficient transduction of sensory neurons at low multiplicities of infection (MOIs) and sustained transgene expression within a 28-day culture period. Interestingly, the lentivirus vector selectively transduced neurons in murine cultures, in contrast to human cultures, in which Schwann and fibroblast-like cells were also transduced. Recombinant AAV transduced all three cell types in both mouse and human cultures. After direct microinjection of murine DRG explants, maximal transduction efficiencies of 20 and 200 transducing units per neuronal transductant were achieved with AAV and lentivirus vectors, respectively. Most importantly, both vectors achieved efficient and sustained transduction of human sensory neurons in dissociated cultures, thereby directly demonstrating the exciting potential of these vectors for gene therapy applications in the PNS.

  19. Clostridium perfringens type A–E toxin plasmids

    Science.gov (United States)

    Freedman, John C.; Theoret, James R.; Wisniewski, Jessica A.; Uzal, Francisco A.; Rood, Julian I.; McClane, Bruce A.

    2014-01-01

    Clostridium perfringens relies upon plasmid-encoded toxin genes to cause intestinal infections. These toxin genes are associated with insertion sequences that may facilitate their mobilization and transfer, giving rise to new toxin plasmids with common backbones. Most toxin plasmids carry a transfer of clostridial plasmids locus mediating conjugation, which likely explains the presence of similar toxin plasmids in otherwise unrelated C. perfringens strains. The association of many toxin genes with insertion sequences and conjugative plasmids provides virulence flexibility when causing intestinal infections. However, incompatibility issues apparently limit the number of toxin plasmids maintained by a single cell. PMID:25283728

  20. Importin β1 mediates nuclear factor-κB signal transduction into the nuclei of myeloma cells and affects their proliferation and apoptosis.

    Science.gov (United States)

    Yan, Wenqing; Li, Rong; He, Jie; Du, Juan; Hou, Jian

    2015-04-01

    Multiple myeloma (MM) is a plasma cell neoplasm that is currently incurable. The activation of nuclear factor-κB (NF-κB) signalling plays a crucial role in the immortalisation of MM cells. As the most important transcription factor of the canonical NF-κB pathway, the p50/p65 heterodimer requires transportation into the nucleus for its successful signal transduction. Importin β1 is the key transport receptor that mediates p50/p65 nuclear import. Currently, it remains unclear whether the regulation of importin β1 function affects the biological behaviour of MM cells. In the present study, we investigated the changes in p65 translocation and the proliferation and apoptosis of MM cells after treatment with small interfering RNA (siRNA) or an importin β1 inhibitor. The underlying mechanisms were also investigated. We found importin β1 over-expression and the excessive nuclear transport of p65 in myeloma cells. Confocal laser scanning microscopy and Western blot analysis results indicated that p65 nuclear transport was blocked after inhibiting importin β1 expression with siRNA and the importin β1-specific inhibitor importazole (IPZ). Importantly, electronic mobility shift assay results also verified that p65 nuclear transport was dramatically reduced. Moreover, the expression of the NF-κB signalling target genes involved in MM cell apoptosis, such as BCL-2, c-IAP1 and XIAP, were markedly reduced, as demonstrated by the RT-PCR results. Furthermore, the proliferation of MM cells was inhibited, as demonstrated by MTT assay results, and the MM cell apoptosis rate was higher, as demonstrated by the annexin V/propidium iodide (PI) double-staining assay results. Additionally, the percentage of S phase cells in the myeloma cell lines treated with IPZ was dramatically reduced. In conclusion, our results clearly show that importin β1 mediates the translocation of NF-κB into the nuclei of myeloma cells, thereby regulating proliferation and blocking apoptosis, which

  1. High Prevalence of β-lactamase and Plasmid-Mediated Quinolone Resistance Genes in Extended-Spectrum Cephalosporin-Resistant Escherichia coli from Dogs in Shaanxi, China

    Science.gov (United States)

    Liu, Xiaoqiang; Liu, Haixia; Li, Yinqian; Hao, Caiju

    2016-01-01

    Objective: The aim of this study was to investigate the occurrence and molecular characterization of extended-spectrum β-lactamases (ESBL), plasmid-mediated AmpC β-lactamase (pAmpC) and carbapenemases as well as plasmid-mediated quinolone-resistant (PMQR) among extended-spectrum cephalosporin-resistant (ESC-R) Escherichia coli from dogs in Shaanxi province in China. Methods: A total of 40 ESC-R Escherichia coli selected from 165 Extraintestinal pathogenic E. coli (ExPEC) isolated from dogs were screened and characterized for the genes encoding for the ESBLs, pAmpC, carbapenemases and PMQR genes by PCR and sequencing. Phylogenetic groups, virulence gene profiles and multilocus sequence typing (MLST) were used to investigate the genetic background of the ESC-R E. coli isolates. Results: Among 40 ESC-R E. coli, the predominant β-lactamase gene was blaCTX−Ms (n = 35), and followed by blaTEM−1 (n = 31), blaSHV−12 (n = 14), blaOXA−48 (n = 8), blaTEM−30 (n = 4), blaCMY−2 (n = 3) and blaDHA−1 (n = 2). The most common specific blaCTX−M gene subtype was blaCTX−M−15 (n = 31), and followed by blaCTX−M−123 (n = 14), blaCTX−M−1 (n = 10), blaCTX−M−14 (n = 10) and blaCTX−M−9 (n = 7). PMQR genes were detected in 32 (80%) isolates, and the predominant PMQR gene was aac(6′)-Ib-cr (n = 26), followed by qnrS (n = 12), qnrD (n = 9), qnrB (n = 8), qepA (n = 4), and all PMQR genes were detected in co-existence with β-lactamase genes. traT (n = 34) and fimH (n = 32) were the most prevalent virulence genes, and virulence genes fimH, iutA, fyuA, malX, iha, and sat were more prevalent in phylogenetic group B2. The 40 ESC-R isolates analyzed were assigned to 22 sequence types (STs), and the clonal lineages ST131 (n = 10) and ST10 (n = 9) were the predominant STs. Conclusion: High prevalence of β-lantamases and PMQR genes were detected among ESC-R E. coli from companion animals. This is also the first description of the co-existence of six

  2. Prevalence and characteristics of extended-spectrum β-lactamase and plasmid-mediated fluoroquinolone resistance genes in Escherichia coli isolated from chickens in Anhui province, China.

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    Lin Li

    Full Text Available The aim of this study was to characterize the prevalence of extended-spectrum β-lactamase (ESBL genes and plasmid-mediated fluoroquinolone resistance (PMQR determinants in 202 Escherichia coli isolates from chickens in Anhui Province, China, and to determine whether ESBL and PMQR genes co-localized in the isolates. Antimicrobial susceptibility for 12 antimicrobials was determined by broth microdilution. Polymerase chain reactions (PCRs, DNA sequencing, and pulsed field gel electrophoresis (PFGE were employed to characterize the molecular basis for β-lactam and fluoroquinolone resistance. High rates of antimicrobial resistance were observed, 147 out of the 202 (72.8% isolates were resistant to at least 6 antimicrobial agents and 28 (13.9% of the isolates were resistant to at least 10 antimicrobials. The prevalence of blaCTX-M, blaTEM-1 and blaTEM-206 genes was 19.8%, 24.3% and 11.9%, respectively. Seventy-five out of the 202 (37.1% isolates possessed a plasmid-mediated quinolone resistance determinant in the form of qnrS (n = 21; this determinant occurred occasionally in combination with aac(6'-1b-cr (n = 65. Coexistence of ESBL and/or PMQR genes was identified in 31 of the isolates. Two E. coli isolates carried blaTEM-1, blaCTX-M and qnrS, while two others carried blaCTX-M, qnrS and aac(6'-1b-cr. In addition, blaTEM-1, qnrS and aac(6'-1b-cr were co-located in two other E. coli isolates. PFGE analysis showed that these isolates were not clonally related and were genetically diverse. To the best of our knowledge, this study is the first to describe detection of TEM-206-producing E. coli in farmed chickens, and the presence of blaTEM-206, qnrS and aac(6'-1b-cr in one of the isolates.

  3. High Prevalence of β-lactamase and Plasmid-mediated Quinolone Resistance Genes in Extended-spectrum Cephalosporin-resistant Escherichia coli from Dogs in Shaanxi, China

    Directory of Open Access Journals (Sweden)

    Xiaoqiang Liu

    2016-11-01

    Full Text Available Objective: The aim of this study was to investigate the occurrence and molecular characterization of extended-spectrum β-lactamases (ESBL, plasmid-mediated AmpC β-lactamase (pAmpC and carbapenemases as well as plasmid-mediated quinolone-resistant (PMQR among extended-spectrum cephalosporin-resistant (ESC-R Escherichia coli from dogs in Shaanxi province in China.Methods: A total of 40 ESC-R Escherichia coli selected from 165 Extraintestinal pathogenic E. coli (ExPEC isolated from dogs were screened and characterized for the genes encoding for the ESBLs, pAmpC, carbapenemases and PMQR genes by PCR and sequencing. Phylogenetic groups, virulence gene profiles and multilocus sequence typing (MLST were used to investigate the genetic background of the ESC-R E. coli isolates. Results: Among 40 ESC-R E. coli, the predominant β-lactamase gene was blaCTX-Ms (n=35, and followed by blaTEM-1 (n=31, blaSHV-12 (n=14, blaOXA-48 (n=8, blaTEM-30 (n=4, blaCMY-2 (n=3 and blaDHA-1 (n=2. The most common specific blaCTX-M gene subtype was blaCTX-M-15 (n=31, and followed by blaCTX-M-123 (n=14, blaCTX-M-1 (n=10, blaCTX-M-14 (n=10 and blaCTX-M-9 (n=7. PMQR genes were detected in 32 (80% isolates, and the predominant PMQR gene was aac(6'-Ib-cr (n=26, followed by qnrS (n=12, qnrD (n=9, qnrB (n=8, qepA (n=4, and all PMQR genes were detected in co-existence with β-lactamase genes. traT (n=34 and fimH (n=32 were the most prevalent virulence genes, and virulence genes fimH, iutA, fyuA, malX, iha and sat were more prevalent in phylogenetic group B2. The 40 ESC-R isolates analyzed were assigned to 22 sequence types (STs, and the clonal lineages ST131 (n=10 and ST10 (n=9 were the predominant STs. Conclusion: High prevalence of β-lantamases and PMQR genes were detected among ESC-R E. coli from companion animals. This is also the first description of the co-existence of six β-lantamase genes and five PMQR genes in one E. coli isolate. Moreover, ten ST131 clones harboring CTX

  4. Identification of plasmid-mediated quinolone resistance genes qnrA1, qnrB1 and aac(6′-1b-cr in a multiple drug-resistant isolate of Klebsiella pneumoniae from Chennai

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    H Magesh

    2011-01-01

    Full Text Available Purpose: Resistance to fluoroquinolones, a commonly prescribed antimicrobial for Gram-negative and Gram-positive microorganisms, is of importance in therapy. The purpose of this study was to screen for the presence of Plasmid-Mediated Quinolone Resistance (PMQR determinants in clinical isolates of Klebsiella pneumoniae. Materials and Methods: Extended-Spectrum Beta-Lactamase (ESBL isolates of K. pneumoniae collected during October 2009 were screened by the antimicrobial susceptibility test. The plasmids from these isolates were analysed by specific Polymerase chain Reaction (PCR for qnrA, qnrB and aac(6′-1b. The amplified products were sequenced to confirm the allele. Results: Our analysis showed that 61% out of the 23 ESBL K. pneumoniae isolates were resistant to ciprofloxacin and 56% to levofloxacin. The PMQR was demonstrated by transforming the plasmids from two isolates P12 and P13 into E. coli JM109. The PMQR gene qnrA was found in 16 isolates and qnrB in 11 isolates. The plasmid pKNMGR13 which conferred an minimum inhibitory concentration (MIC of more than 240 ΅g/ml in sensitive E. coli was found to harbour the qnrA1 and qnrB1 allele. Furthermore, the gene aac(6′-1b-cr encoding a variant aminoglycoside 6′-N Acetyl transferase which confers resistance to fluoroquinolones was found in the same plasmid. Conclusions: Our report shows the prevalence of PMQR mediated by qnrA and qnrB in multidrug-resistant K. pneumoniae isolates from Chennai. A multidrug-resistant plasmid conferring high resistance to ciprofloxacin was found to harbour another PMQR gene, aac(6′-1b-cr mutant gene. This is the first report screening for PMQR in K. pneumoniae isolates from India.

  5. Complete nucleotide sequence of pGA45, a 140,698-bp incFIIY plasmid encoding blaIMI-3-mediated carbapenem resistance, from river sediment

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    Bingjun eDang

    2016-02-01

    Full Text Available Plasmid pGA45 was isolated from the sediment of Haihe River using E. coli CV601 (gfp-tagged as recipients and indigenous bacteria from sediment as donors. This plasmid confers reduced susceptibility to imipenem which belongs to carbapenem group. Plasmid pGA45 was fully sequenced on an Illumina HiSeq 2000 sequencing system. The complete sequence of plasmid pGA45 was 140,698 bp in length with an average G+C content of 52.03%. Sequence analysis shows that pGA45 belongs to incFIIY group and harbors a backbone region shares high homology and gene synteny to several other incF plasmids including pNDM1_EC14653, pYDC644, pNDM-Ec1GN574, pRJF866, pKOX_NDM1 and pP10164-NDM. In addition to the backbone region, plasmid pGA45 harbors two notable features including one blaIMI-3-containing region and one type VI secretion system region. The blaIMI-3-containing region is responsible for bacteria carbapenem resistance and the type VI secretion system region is probably involved in bacteria virulence, respectively. Plasmid pGA45 represents the first complete nucleotide sequence of the blaIMI-harboring plasmid from environment sample and the sequencing of this plasmid provided insight into the architecture used for the dissemination of blaIMI carbapenemase genes.

  6. Vpx mediated degradation of SAMHD1 has only a very limited effect on lentiviral transduction rate in ex vivo cultured HSPCs

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    Duo Li

    2015-09-01

    Full Text Available Understanding how to achieve efficient transduction of hematopoietic stem and progenitor cells (HSPCs, while preserving their long-term ability to self-reproduce, is key for applying lentiviral-based gene engineering methods. SAMHD1 is an HIV-1 restriction factor in myeloid and resting CD4+ T cells that interferes with reverse transcription by decreasing the nucleotide pools or by its RNase activity. Here we show that SAMHD1 is expressed at high levels in HSPCs cultured in a medium enriched with cytokines. Thus, we hypothesized that degrading SAMHD1 in HSPCs would result in more efficient lentiviral transduction rates. We used viral like particles (VLPs containing Vpx, shRNA against SAMHD1, or provided an excess of dNTPs or dNs to study this question. Regardless of the method applied, we saw no increase in the lentiviral transduction rate. The result was different when we used viruses (HR-GFP-Vpx+ which carry Vpx and encode GFP. These viruses allow assessment of the effects of Vpx specifically in the transduced cells. Using HR-GFP-Vpx+ viruses, we observed a modest but significant increase in the transduction efficiency. These data suggest that SAMHD1 has some limited efficacy in blocking reverse transcription but the major barrier for efficient lentiviral transduction occurs before reverse transcription.

  7. Insights into the Mechanistic Basis of Plasmid-Mediated Colistin Resistance from Crystal Structures of the Catalytic Domain of MCR-1

    Science.gov (United States)

    Hinchliffe, Philip; Yang, Qiu E.; Portal, Edward; Young, Tom; Li, Hui; Tooke, Catherine L.; Carvalho, Maria J.; Paterson, Neil G.; Brem, Jürgen; Niumsup, Pannika R.; Tansawai, Uttapoln; Lei, Lei; Li, Mei; Shen, Zhangqi; Wang, Yang; Schofield, Christopher J.; Mulholland, Adrian J; Shen, Jianzhong; Fey, Natalie; Walsh, Timothy R.; Spencer, James

    2017-01-01

    The polymixin colistin is a “last line” antibiotic against extensively-resistant Gram-negative bacteria. Recently, the mcr-1 gene was identified as a plasmid-mediated resistance mechanism in human and animal Enterobacteriaceae, with a wide geographical distribution and many producer strains resistant to multiple other antibiotics. mcr-1 encodes a membrane-bound enzyme catalysing phosphoethanolamine transfer onto bacterial lipid A. Here we present crystal structures revealing the MCR-1 periplasmic, catalytic domain to be a zinc metalloprotein with an alkaline phosphatase/sulphatase fold containing three disulphide bonds. One structure captures a phosphorylated form representing the first intermediate in the transfer reaction. Mutation of residues implicated in zinc or phosphoethanolamine binding, or catalytic activity, restores colistin susceptibility of recombinant E. coli. Zinc deprivation reduces colistin MICs in MCR-1-producing laboratory, environmental, animal and human E. coli. Conversely, over-expression of the disulphide isomerase DsbA increases the colistin MIC of laboratory E. coli. Preliminary density functional theory calculations on cluster models suggest a single zinc ion may be sufficient to support phosphoethanolamine transfer. These data demonstrate the importance of zinc and disulphide bonds to MCR-1 activity, suggest that assays under zinc-limiting conditions represent a route to phenotypic identification of MCR-1 producing E. coli, and identify key features of the likely catalytic mechanism. PMID:28059088

  8. Comparison of two DNA microarrays for detection of plasmid-mediated antimicrobial resistance and virulence factor genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae.

    LENUS (Irish Health Repository)

    Walsh, Fiona

    2010-06-01

    A DNA microarray was developed to detect plasmid-mediated antimicrobial resistance (AR) and virulence factor (VF) genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae. The array was validated with the following bacterial species: Escherichiacoli (n=17); Klebsiellapneumoniae (n=3); Enterobacter spp. (n=6); Acinetobacter genospecies 3 (n=1); Acinetobacterbaumannii (n=1); Pseudomonasaeruginosa (n=2); and Stenotrophomonasmaltophilia (n=2). The AR gene profiles of these isolates were identified by polymerase chain reaction (PCR). The DNA microarray consisted of 155 and 133 AR and VF gene probes, respectively. Results were compared with the commercially available Identibac AMR-ve Array Tube. Hybridisation results indicated that there was excellent correlation between PCR and array results for AR and VF genes. Genes conferring resistance to each antibiotic class were identified by the DNA array. Unusual resistance genes were also identified, such as bla(SHV-5) in a bla(OXA-23)-positive carbapenem-resistant A. baumannii. The phylogenetic group of each E. coli isolate was verified by the array. These data demonstrate that it is possible to screen simultaneously for all important classes of mobile AR and VF genes in Enterobacteriaceae and non-Enterobacteriaceae whilst also assigning a correct phylogenetic group to E. coli isolates. Therefore, it is feasible to test clinical Gram-negative bacteria for all known AR genes and to provide important information regarding pathogenicity simultaneously.

  9. A Model for Direction Sensing in Dictyostelium discoideum: Ras Activity and Symmetry Breaking Driven by a Gβγ-Mediated, Gα2-Ric8 -- Dependent Signal Transduction Network

    Science.gov (United States)

    Cheng, Yougan; Othmer, Hans

    2016-01-01

    Chemotaxis is a dynamic cellular process, comprised of direction sensing, polarization and locomotion, that leads to the directed movement of eukaryotic cells along extracellular gradients. As a primary step in the response of an individual cell to a spatial stimulus, direction sensing has attracted numerous theoretical treatments aimed at explaining experimental observations in a variety of cell types. Here we propose a new model of direction sensing based on experiments using Dictyostelium discoideum (Dicty). The model is built around a reaction-diffusion-translocation system that involves three main component processes: a signal detection step based on G-protein-coupled receptors (GPCR) for cyclic AMP (cAMP), a transduction step based on a heterotrimetic G protein Gα2βγ, and an activation step of a monomeric G-protein Ras. The model can predict the experimentally-observed response of cells treated with latrunculin A, which removes feedback from downstream processes, under a variety of stimulus protocols. We show that Gα2βγ cycling modulated by Ric8, a nonreceptor guanine exchange factor for Gα2 in Dicty, drives multiple phases of Ras activation and leads to direction sensing and signal amplification in cAMP gradients. The model predicts that both Gα2 and Gβγ are essential for direction sensing, in that membrane-localized Gα2*, the activated GTP-bearing form of Gα2, leads to asymmetrical recruitment of RasGEF and Ric8, while globally-diffusing Gβγ mediates their activation. We show that the predicted response at the level of Ras activation encodes sufficient ‘memory’ to eliminate the ‘back-of-the wave’ problem, and the effects of diffusion and cell shape on direction sensing are also investigated. In contrast with existing LEGI models of chemotaxis, the results do not require a disparity between the diffusion coefficients of the Ras activator GEF and the Ras inhibitor GAP. Since the signal pathways we study are highly conserved between Dicty

  10. A Model for Direction Sensing in Dictyostelium discoideum: Ras Activity and Symmetry Breaking Driven by a Gβγ-Mediated, Gα2-Ric8 -- Dependent Signal Transduction Network.

    Science.gov (United States)

    Cheng, Yougan; Othmer, Hans

    2016-05-01

    Chemotaxis is a dynamic cellular process, comprised of direction sensing, polarization and locomotion, that leads to the directed movement of eukaryotic cells along extracellular gradients. As a primary step in the response of an individual cell to a spatial stimulus, direction sensing has attracted numerous theoretical treatments aimed at explaining experimental observations in a variety of cell types. Here we propose a new model of direction sensing based on experiments using Dictyostelium discoideum (Dicty). The model is built around a reaction-diffusion-translocation system that involves three main component processes: a signal detection step based on G-protein-coupled receptors (GPCR) for cyclic AMP (cAMP), a transduction step based on a heterotrimetic G protein Gα2βγ, and an activation step of a monomeric G-protein Ras. The model can predict the experimentally-observed response of cells treated with latrunculin A, which removes feedback from downstream processes, under a variety of stimulus protocols. We show that [Formula: see text] cycling modulated by Ric8, a nonreceptor guanine exchange factor for [Formula: see text] in Dicty, drives multiple phases of Ras activation and leads to direction sensing and signal amplification in cAMP gradients. The model predicts that both [Formula: see text] and Gβγ are essential for direction sensing, in that membrane-localized [Formula: see text], the activated GTP-bearing form of [Formula: see text], leads to asymmetrical recruitment of RasGEF and Ric8, while globally-diffusing Gβγ mediates their activation. We show that the predicted response at the level of Ras activation encodes sufficient 'memory' to eliminate the 'back-of-the wave' problem, and the effects of diffusion and cell shape on direction sensing are also investigated. In contrast with existing LEGI models of chemotaxis, the results do not require a disparity between the diffusion coefficients of the Ras activator GEF and the Ras inhibitor GAP. Since

  11. Phase IIb trial of in vivo electroporation mediated dual-plasmid hepatitis B virus DNA vaccine in chronic hepatitis B patients under lamivudine therapy

    Science.gov (United States)

    Yang, Fu-Qiang; Rao, Gui-Rong; Wang, Gui-Qiang; Li, Yue-Qi; Xie, Yao; Zhang, Zhan-Qing; Deng, Cun-Liang; Mao, Qing; Li, Jun; Zhao, Wei; Wang, Mao-Rong; Han, Tao; Chen, Shi-Jun; Pan, Chen; Tan, De-Ming; Shang, Jia; Zhang, Ming-Xiang; Zhang, Yue-Xin; Yang, Ji-Ming; Chen, Guang-Ming

    2017-01-01

    AIM To assess the efficacy and safety of in vivo electroporation (EP)-mediated dual-plasmid hepatitis B virus (HBV) DNA vaccine vs placebo for sequential combination therapy with lamivudine (LAM) in patients with chronic hepatitis B. METHODS Two hundred and twenty-five patients were randomized to receive either LAM + vaccine (vaccine group, n = 109) or LAM + placebo (control group, n = 116). LAM treatment lasted 72 wk. Patients received the DNA vaccine or placebo by intramuscular injection mediated by EP at weeks 12 (start of treatment with vaccine or placebo, SOT), 16, 24, and 36 (end of treatment with vaccine or placebo, EOT). RESULTS In the modified intent-to-treat population, more patients had a decrease in HBV DNA > 2 log10 IU/mL in the vaccine group at week 12 after EOT compared with the control group. A trend toward a difference in the number of patients with undetectable HBV DNA at week 28 after EOT was obtained. Adverse events were similar. In the dynamic per-protocol set, which excluded adefovir (ADV) add-on cases at each time point instantly after ADV administration due to LAM antiviral failure, more patients had a decrease in HBV DNA > 2 log10 IU/mL in the vaccine group at week 12 and 28 after EOT compared with the control group. More patients with undetectable HBV DNA at week 28 after EOT in the vaccine group were also observed. Among patients with a viral load < 1000 copies/mL at week 12, more patients achieved HBeAg seroconversion in the vaccine group than among controls at week 36 after EOT, as well as less virological breakthrough and YMDD mutations. CONCLUSION The primary endpoint was not achieved using the HBV DNA vaccine. The HBV DNA vaccine could only be beneficial in subjects that have achieved initial virological response under LAM chemotherapy. PMID:28127204

  12. Plasmid-Mediated Sulfamethoxazole Resistance Encoded by the sul2 Gene in the Multidrug-Resistant Shigella flexneri 2a Isolated from Patients with Acute Diarrhea in Dhaka, Bangladesh

    Science.gov (United States)

    Iqbal, Mohd S.; Rahman, Mostafizur; Islam, Rafiad; Banik, Atanu; Amin, M. Badrul; Akter, Fatema; Talukder, Kaisar Ali

    2014-01-01

    In this study, mechanisms of plasmid-mediated sulfamethoxazole resistances in the clinical strains of multi-drug resistant (MDR) Shigella flexneri 2a were elucidated for the first time in Bangladesh. From 2006 to 2011, a total of 200 S. flexneri 2a strains were randomly selected from the stock of the Enteric and Food Microbiology Laboratory of icddr,b. Antimicrobial susceptibility of the strains showed 73%, 98%, 93%, 58%, 98%, 64% and 4% resistance to trimethoprim-sulfamethoxazole, nalidixic acid, ampicillin, erythromycin, tetracycline, ciprofloxacin and ceftriaxone respectively. Plasmid profiling revealed heterogeneous patterns and interestingly, all the trimethoprim-sulfamethoxazole resistant (SXTR) strains yielded a distinct 4.3 MDa plasmid compared to that of the trimethoprim-sulfamethoxazole susceptible (SXTS) strains. Curing of this 4.3 MDa plasmid resulted in the susceptibility to sulfamethoxazole alone suggesting the involvement of this plasmid in the resistance of sulfamethoxazole. Moreover, PCR analysis showed the presence of sul2 gene in SXTR strains which is absent in SXTS strains as well as in the 4.3 MDa plasmid-cured derivatives, confirming the involvement of sul2 in the resistance of sulfamethoxazole. Furthermore, pulsed-field gel electrophoresis (PFGE) analysis revealed that both the SXTR and SXTS strains were clonal. This study will significantly contributes to the knowledge on acquired drug resistance of the mostly prevalent S. flexneri 2a and further warrants continuous monitoring of the prevalence and correlation of this resistance determinants amongst the clinical isolates of Shigella and other enteric pathogens around the world to provide effective clinical management of the disease. PMID:24416393

  13. Plasmid-mediated sulfamethoxazole resistance encoded by the sul2 gene in the multidrug-resistant Shigella flexneri 2a isolated from patients with acute diarrhea in Dhaka, Bangladesh.

    Directory of Open Access Journals (Sweden)

    Mohd S Iqbal

    Full Text Available In this study, mechanisms of plasmid-mediated sulfamethoxazole resistances in the clinical strains of multi-drug resistant (MDR Shigella flexneri 2a were elucidated for the first time in Bangladesh. From 2006 to 2011, a total of 200 S. flexneri 2a strains were randomly selected from the stock of the Enteric and Food Microbiology Laboratory of icddr,b. Antimicrobial susceptibility of the strains showed 73%, 98%, 93%, 58%, 98%, 64% and 4% resistance to trimethoprim-sulfamethoxazole, nalidixic acid, ampicillin, erythromycin, tetracycline, ciprofloxacin and ceftriaxone respectively. Plasmid profiling revealed heterogeneous patterns and interestingly, all the trimethoprim-sulfamethoxazole resistant (SXT(R strains yielded a distinct 4.3 MDa plasmid compared to that of the trimethoprim-sulfamethoxazole susceptible (SXT(S strains. Curing of this 4.3 MDa plasmid resulted in the susceptibility to sulfamethoxazole alone suggesting the involvement of this plasmid in the resistance of sulfamethoxazole. Moreover, PCR analysis showed the presence of sul2 gene in SXT(R strains which is absent in SXT(S strains as well as in the 4.3 MDa plasmid-cured derivatives, confirming the involvement of sul2 in the resistance of sulfamethoxazole. Furthermore, pulsed-field gel electrophoresis (PFGE analysis revealed that both the SXT(R and SXT(S strains were clonal. This study will significantly contributes to the knowledge on acquired drug resistance of the mostly prevalent S. flexneri 2a and further warrants continuous monitoring of the prevalence and correlation of this resistance determinants amongst the clinical isolates of Shigella and other enteric pathogens around the world to provide effective clinical management of the disease.

  14. Signal transduction by HLA-DR is mediated by tyrosine kinase(s) and regulated by CD45 in activated T cells

    DEFF Research Database (Denmark)

    Odum, Niels; Martin, P J; Schieven, G L;

    1991-01-01

    , but the inhibitory effect of CD45 dominated over the enhancing effect of CD4. These data indicate that PTK activation is obligatory for DR-induced (Ca2+)i responses, suggesting a linkage between these pathways in class II signal transduction. This conclusion is consistent with our observation that in activated human...

  15. Progress in research of plasmid-mediated quinolone resistance gene of enterobacteria%肠杆菌科细菌质粒介导的喹诺酮耐药基因研究进展

    Institute of Scientific and Technical Information of China (English)

    符浩; 夏兴; 陈代杰

    2011-01-01

    继首个质粒介导的喹诺酮耐药基因qnrAl之后,qnrB,qnrS,qnrC和qnrD等其他一些类似基因也相继被发现.另 外,两种质粒介导的喹诺酮耐药机制,即外排泵QepA和OqxAB以及氨基糖苷甲基转移酶Aac(6’)-Ib-cr陆续被报道.本文综述肠杆菌科细菌质粒介导的喹诺酮耐药基因研究进展.%Since the first plasmid-mediated quinolone antibiotics resistance gene (PMQR, currently named qnrAl) was reported, some other genes such as qnrB, qnrS, qnrC and qnrD have also been characterized. In addition, two other plasmid-mediated resistance mechanisms: the modification of quinolones with a piperazinyl substituent by the acetyltransferase, Aac (6') -Ib-cr, and active efflux by QepA and OqxAB have also been reported. This review describes the progress in research of plasmid-mediated quinolone resistance gene of enterobacteria.

  16. The tra locus of streptomycete plasmid pIJ101 mediates efficient transfer of a circular but not a linear version of the same replicon.

    Science.gov (United States)

    Wang, Jing; Pettis, Gregg S

    2010-09-01

    Conjugal transfer of circular plasmids in Streptomyces involves a unique mechanism employing few plasmid-encoded loci and the transfer of double-stranded DNA by an as yet uncharacterized intercellular route. Efficient transfer of the circular streptomycete plasmid pIJ101 requires only two plasmid loci: the pIJ101 tra gene, and as a cis-acting function known as clt. Here, we compared the ability of the pIJ101 transfer apparatus to promote conjugal transfer of circular versus linear versions of the same replicon. While the pIJ101 tra locus readily transferred the circular form of the replicon, the linear version was transferred orders of magnitude less efficiently and all plasmids isolated from the transconjugants were circular, regardless of their original configuration in the donor. Additionally, relatively rare circularization of linear plasmids was detectable in the donor cells, which is consistent with the notion that this event was a prerequisite for transfer by TraB(pIJ101). Linear versions of this same replicon did transfer efficiently, in that configuration, from strains containing the conjugative linear plasmid SLP2. Our data indicate that functions necessary and sufficient for transfer of circular DNA were insufficient for transfer of a related linear DNA molecule. The results here suggest that the conjugation mechanisms of linear versus circular DNA in Streptomyces spp. are inherently different and/or that efficient transfer of linear DNA requires additional components.

  17. Identification of plasmid-mediated quinolone resistance qnr genes in multidrug-resistant Gram-negative bacteria from hospital wastewaters and receiving waters in the Jinan area, China.

    Science.gov (United States)

    Xia, Ruirui; Ren, Ye; Xu, Hai

    2013-12-01

    We investigated the prevalence of plasmid-mediated quinolone resistance (PMQR) qnr genes by the polymerase chain reaction (PCR) in antibiotic-resistant bacteria isolates collected from aquatic environments in Jinan during 2 years (2008.3-2009.11). Genes were identified to variant level by PCR restriction fragment length polymorphism analysis or sequencing. qnrA1, qnrB2, qnrB4, qnrB6, qnrB9, qnrS1, and the new qnrB variant qnrB26 were detected in 31 strains from six genera (Klebsiella spp., Escherichia coli, Enterobacter spp., Proteus spp., Shigella spp., and Citrobacter spp.), four of which contained double qnr genes. Other PMQR genes, aac(6')-Ib-cr and qepA, were found in 12 (38.7%) and 5 (16.1%) of 31 isolates, respectively; while qepA was found in Shigella spp. for the first time. Eight types of β-lactamase genes and eight other types of resistance genes were also present in the 31 qnr-positive isolates. The detection rate for five β-lactamase genes (blaTEM, blaCTX, ampR, blaDHA, and blaSHV) was >45%. Class 1 integrons and complex class 1 integrons were prevalent in these strains, which contained 15 different gene cassette arrays and 5 different insertion sequence common region 1 (ISCR1)-mediated downstream structures. qnrA1, qnrB2, and qnrB6 were present in three ISCR1-mediated downstream structures: qnrA1-ampR, sapA-like-qnrB2, and sdr-qnrB6. We also analyzed the horizontal transferability of PMQR genes and other resistance determinants. The qnr genes and some integrons and resistance genes from 18 (58.1%) of the 31 qnr-positive strains could be transferred to E. coli J53 Azi(R) or E. coli DH5α recipient strains using conjugation or transformation methods. The results showed that a high number of qnr genes were associated with other resistance genes in aquatic environments in Jinan. This suggests that we should avoid over-using antibiotics and monitor aquatic environments to control the spread of antibiotic resistance genes.

  18. Seawater is a reservoir of multi-resistant Escherichia coli, including strains hosting plasmid-mediated quinolones resistance and extended-spectrum beta-lactamases genes.

    Science.gov (United States)

    Alves, Marta S; Pereira, Anabela; Araújo, Susana M; Castro, Bruno B; Correia, António C M; Henriques, Isabel

    2014-01-01

    The aim of this study was to examine antibiotic resistance (AR) dissemination in coastal water, considering the contribution of different sources of fecal contamination. Samples were collected in Berlenga, an uninhabited island classified as Natural Reserve and visited by tourists for aquatic recreational activities. To achieve our aim, AR in Escherichia coli isolates from coastal water was compared to AR in isolates from two sources of fecal contamination: human-derived sewage and seagull feces. Isolation of E. coli was done on Chromocult agar. Based on genetic typing 414 strains were established. Distribution of E. coli phylogenetic groups was similar among isolates of all sources. Resistances to streptomycin, tetracycline, cephalothin, and amoxicillin were the most frequent. Higher rates of AR were found among seawater and feces isolates, except for last-line antibiotics used in human medicine. Multi-resistance rates in isolates from sewage and seagull feces (29 and 32%) were lower than in isolates from seawater (39%). Seawater AR profiles were similar to those from seagull feces and differed significantly from sewage AR profiles. Nucleotide sequences matching resistance genes bla TEM, sul1, sul2, tet(A), and tet(B), were present in isolates of all sources. Genes conferring resistance to 3rd generation cephalosporins were detected in seawater (bla CTX-M-1 and bla SHV-12) and seagull feces (bla CMY-2). Plasmid-mediated determinants of resistance to quinolones were found: qnrS1 in all sources and qnrB19 in seawater and seagull feces. Our results show that seawater is a relevant reservoir of AR and that seagulls are an efficient vehicle to spread human-associated bacteria and resistance genes. The E. coli resistome recaptured from Berlenga coastal water was mainly modulated by seagulls-derived fecal pollution. The repertoire of resistance genes covers antibiotics critically important for humans, a potential risk for human health.

  19. Extended spectrum β-lactamase and plasmid mediated quinolone resistance in Escherichia coli fecal isolates from healthy companion animals in Algeria.

    Science.gov (United States)

    Yousfi, Massilia; Mairi, Assia; Touati, Abdelaziz; Hassissene, Lila; Brasme, Lucien; Guillard, Thomas; De Champs, Christophe

    2016-07-01

    The aim of this study was to evaluate the rate of fecal carriage of Escherichia coli strains producing Extended-spectrum β-lactamases (ESBLs) and plasmid-mediated quinolone resistance (PMQR) isolated from healthy pets (dogs and cats) in Algeria. Fecal samples from 171 healthy pets (102 dogs and 69 cats) in one veterinary practice and private owners were included. After isolates identification, antibiotic susceptibility was determined by disk diffusion procedure. ESBL were detected by combination disk tests. PCR and sequencing were used to characterize genes encoding ESBLs and PMQR. Transfer of ESBL and PMQR genes was assessed by conjugation experiments. Phylogenetic groups of E. coli were determined by PCR. Of the 171 animals, 20 carried an ESBL producing E. coli giving a prevalence of ESBL fecal carriage of 11.7%. All isolates were susceptible to carbapenems, cefoxitin, piperacillin-tazobactam, amikacin and fosfomycine. For the rest of the tested β-lactams, susceptibility rates ranged from 35% to 70% for cefepime and amoxicillin-clavulanic acid respectively. Concerning the non-beta-lactams antibiotics, the rates of susceptibility ranged between 5% to trimethoprim and 95% for chloramphenicol. The beta-lactamase genes identified in E. coli isolates were blaCTX-M-15, blaCTX-M-1, blaSHV-12 and blaTEM-1. The PMQR determinants aac(6')-Ib-cr, qnrS1 and qnrB5 genes were identified in 15 isolates. Transconjugants were obtained for two isolates. Phylogenetic analysis showed that E. coli isolates belong to commensal phylogroups of A and B1. We reported here for the first time in Algeria ESBL and PMQR-producing E. coli in healthy cats and dogs.

  20. An E3-14.7K peptide that promotes microtubules-mediated transport of plasmid DNA increases polyplexes transfection efficiency.

    Science.gov (United States)

    Pigeon, Lucie; Gonçalves, Cristine; Gosset, David; Pichon, Chantal; Midoux, Patrick

    2013-11-25

    Chemical vectors as cationic polymers and cationic lipids are promising alternatives to viral vectors for gene therapy. Beside endosome escape and nuclear import, plasmid DNA (pDNA) migration in the cytosol toward the nuclear envelope is also regarded as a limiting step for efficient DNA transfection with non-viral vectors. Here, the interaction between E3-14.7K and FIP-1 to favor migration of pDNA along microtubules is exploited. E3-14.7K is an early protein of human adenoviruses that interacts via FIP-1 (Fourteen.7K Interacting Protein 1) protein with the light-chain components of the human microtubule motor protein dynein (TCTEL1). This peptide is conjugated with pDNA and mediates interaction of pDNA in vitro with isolated microtubules as well as with microtubules in cellulo. Videomicroscopy and tracking treatment of images clearly demonstrate that P79-98/pDNA conjugate exhibits a linear transport with large amplitude along microtubules upon 2 h transfection with polyplexes whereas control pDNA conjugate exhibits small non-directional movements in the cytoplasm. Remarkably, P79-98/peGFP polyplexes enhance by a factor 2.5 (up to 76%) the number of transfected cells. The results demonstrate, for the first time, that the transfection efficiency of polyplexes can be drastically increased when the microtubules migration of pDNA is facilitated by a peptide allowing pDNA docking to TCTEL1. This is a real breakthrough in the non viral gene delivery field that opens hope to build artificial viruses.

  1. Seawater is a reservoir of multi-resistant Escherichia coli, including strains hosting plasmid-mediated quinolones resistance and extended-spectrum beta-lactamases genes

    Directory of Open Access Journals (Sweden)

    Marta S. Alves

    2014-08-01

    Full Text Available The aim of this study was to examine antibiotic resistance (AR dissemination in coastal water, considering the contribution of different sources of faecal contamination. Samples were collected in Berlenga, an uninhabited island classified as Natural Reserve and visited by tourists for aquatic recreational activities. To achieve our aim, AR in Escherichia coli isolates from coastal water was compared to AR in isolates from two sources of faecal contamination: human-derived sewage and seagull faeces. Isolation of E. coli was done on Chromocult agar. Based on genetic typing 414 strains were established. Distribution of E. coli phylogenetic groups was similar among isolates of all sources. Resistances to streptomycin, tetracycline, cephalothin and amoxicillin were the most frequent. Higher rates of AR were found among seawater and faeces isolates, except for last-line antibiotics used in human medicine. Multi-resistance rates in isolates from sewage and seagull faeces (29% and 32% were lower than in isolates from seawater (39%. Seawater AR profiles were similar to those from seagull faeces and differed significantly from sewage AR profiles. Nucleotide sequences matching resistance genes blaTEM, sul1, sul2, tet(A and tet(B, were present in isolates of all sources. Genes conferring resistance to 3rd generation cephalosporins were detected in seawater (blaCTX-M-1 and blaSHV-12 and seagull faeces (blaCMY-2. Plasmid-mediated determinants of resistance to quinolones were found: qnrS1 in all sources and qnrB19 in seawater and seagull faeces. Our results show that seawater is a relevant reservoir of AR and that seagulls are an efficient vehicle to spread human-associated bacteria and resistance genes. The E. coli resistome recaptured from Berlenga coastal water was mainly modulated by seagulls-derived faecal pollution. The repertoire of resistance genes covers antibiotics critically important for humans, a potential risk for human health.

  2. Novel genetic environment of the plasmid-mediated KPC-3 gene detected in Escherichia coli and Citrobacter freundii isolates from China

    OpenAIRE

    Li, G.; Wei, Q; Wang, Y.; Du, X.; Zhao, Y.; Jiang, X.

    2010-01-01

    The imipenem and meropenem-resistant strains Citrobacter freundii HS70 and Escherichia coli HS510 were isolated from patients in Shanghai, China. By isoelectric focusing, PCR amplification and sequencing, these strains were each found to produce four β-lactamases: TEM-1, KPC-3, SHV-7 and CTX-M-14. A conjugation experiment and plasmid restriction digestion revealed that the bla KPC-3 gene was located on the same plasmid in both isolates. Bidirectional primer walking sequencing showed that the ...

  3. Enhanced cardioprotective effects mediated by plasmid containing the short-hairpin RNA of angiotensin converting enzyme with a biodegradable hydrogel after myocardial infarction.

    Science.gov (United States)

    Wan, Wei-Guo; Jiang, Xue-Jun; Li, Xiao-Yan; Zhang, Cui; Yi, Xin; Ren, Shan; Zhang, Xian-Zheng

    2014-10-01

    The expression of foreign gene was enhanced and prolonged by sustained releasing a target gene to cells from biodegradable dextran-poly(e-caprolactone)-2-hydroxylethylmethacrylate-poly(N-isopropylacrylamide) (Dex-PCL-HEMA/PNIPAAm) hydrogel in vitro. Moreover, we have demonstrated that injection of the same hydrogel improved post-infarct ventricular remodeling. Therefore, we hypothesized that intramyocardial injection of plasmid containing the short-hairpin RNA (shRNA) of angiotensin converting enzyme (ACE) with the same hydrogel enhances the cardioprotective effects superior to either alone or after rat myocardial infarction (MI). In this study, equal volume of phosphate-buffered solution (PBS), 10 μg ACE-shRNA plasmids, hydrogel containing 10 μg negative control ACE-shRNA plasmids and hydrogel containing 10 μg ACE-shRNA plasmids were shortly injected into the infarct area of rats after MI, respectively. We found that ACE-shRNA plasmid-loaded hydrogel extended the duration of gene expression in vivo. Moreover, it was shown that direct intramyocardial injection of ACE-shRNA plasmid-loaded hydrogel significantly decreased the expression of local ACE expression, inhibited cell apoptosis, reduced infarct size, and improved cardiac function compared with the injection of either alone 30 days after MI in rats. These results suggest that injection of ACE-shRNA plasmid-loaded hydrogel into impaired myocardium obtains more cardioprotective effects than either alone in rat with MI by prolonging the gene silencing of ACE. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 3452-3458, 2014.

  4. Transfection of bone marrow mesenchymal stem cells using green fluorescence protein labeled hVEGF165 recombinant plasmid mediated by liposome

    Institute of Scientific and Technical Information of China (English)

    Tao Wang; Tian-An Liao; Shao-Bo Zhong

    2013-01-01

    Objective:To study the role of bone marrow mesenchymal stem cells (BMSCs) in construction of vascularized engineered tissue. Methods: hVEGF165 was amplified via RT-PCR before recombinant with pShuttle-green fluorescence protein;green fluorescent protein (GFP)-CMV. Then the recombinant shuttle plasmid was transfected into BMSCs with LipofectamineTM 2000 for packaging and amplifying. hVEGF165 mRNA expression in BMSCs cells was tested. Results:The sequence of hVEGF165 in pShuttle-GFP-hVEGF165 plasmid was confirmed by double-enzyme cleavage method and sequencing. hVEGF165 was highly expressed in BMSCs. Conclusions:The GFP/hVEGF165 recombinant plasmid vector was constructed successfully and expressed effectively in host cells, which may be helpful for discussing the possibility of the application of VEGF165-BMSCs in tissue engineering and ischemic disease cure.

  5. Characterization of plasmid-mediated quinolone resistance (PMQR genes in extended-spectrum β-lactamase-producing Enterobacteriaceae pediatric clinical isolates in Mexico.

    Directory of Open Access Journals (Sweden)

    Jesus Silva-Sánchez

    Full Text Available This work describes the characterization of plasmid-mediated quinolone-resistance (PMQR genes from a multicenter study of ESBL-producing Enterobacteriaceae pediatric clinical isolates in Mexico. The PMQR gene-positive isolates were characterized with respect to ESBLs, and mutations in the GyrA and ParC proteins were determined. The phylogenetic relationship was established by PFGE and the transfer of PMQR genes was determined by mating assays. The prevalence of the PMQR genes was 32.1%, and the rate of qnr-positive isolates was 15.1%; 93.3% of the latter were qnrB and 6.4% were qnrA1. The distribution of isolates in terms of bacterial species was as follows: 23.5% (4/17 corresponded to E. cloacae, 13.7% (7/51 to K. pneumoniae, and 13.6% (6/44 to E. coli. In addition, the prevalence of aac(6'-Ib-cr and qepA was 15.1% and 1.7%, respectively. The molecular characteristics of qnr- and qepA-positive isolates pointed to extended-spectrum β-lactamase (ESBL CTX-M-15 as the most prevalent one (70.5%, and to SHV-12 in the case of aac(6'-Ib-cr-positive isolates. GyrA mutations at codons Ser-83 and Asp-87, and ParC mutations at codons Ser-80 were observed in 41.1% and 35.2% of the qnr-positive isolates, respectively. The analysis of the transconjugants revealed a co-transmission of bla(CTX-M-15 with the qnrB alleles. In general, the prevalence of PMQR genes (qnr and aac(6'-Ib-cr presented in this work was much lower in the pediatric isolates, in comparison to the adult isolates in Mexico. Also, ESBL CTX-M-15 was the main ESBL identified in the pediatric isolates, whereas in the adult ones, ESBLs corresponded to the CTX-M and the SHV families. In comparison with other studies, among the PMQR-genes identified in this study, the qnrB-alleles and the aac(6'-Ib-cr gene were the most prevalent, whereas the qnrS1, qnrA1 and qnrB-like alleles were the most prevalent in China and Uruguay.

  6. Plasmid-mediated transfer of CTX-M-55 extended-spectrum beta-lactamase among different strains of Salmonella and Shigella spp. in the Republic of Korea.

    Science.gov (United States)

    Kim, Jin Seok; Kim, Soojin; Park, Jungsun; Shin, Eunkyung; Yun, Young-Sun; Lee, Deog-Yong; Kwak, Hyo-Sun; Seong, Won Keun; Chung, Gyung Tae; Kim, Junyoung

    2017-09-01

    We screened 10 CTX-M-55-producing Shigella and Salmonella isolates from a national surveillance in Korea. The blaCTX-M-55 was located on the IncI1 (n=5), IncA/C (n=4) and IncZ (n=1) plasmids, downstream of ISEcp1, IS26-ISEcp1 and ISEcp1-IS5 sequences, respectively. These results indicate that CTX-M-55 has disseminated to other bacteria by lateral plasmid transfer. Copyright © 2017. Published by Elsevier Inc.

  7. Chemotherapy of Bacterial Plasmids

    Science.gov (United States)

    1979-01-29

    render them non-susceptible to K: z plasmid-encoded enzymes. (3) Development of drugs which are selective inhibitor! 1 4, of plasmid DNA replication. (4... Development of drugs which inhibit phenotypic as expression of plasmid genes, and (5) Development of drugs which are inhibitors o, drug-inactivating...Barnes [2] them non-susceptible to plasmid-encoded enzymes, tabulated data on the incidence of Gram-negative 3) development of drugs which are

  8. Cell-penetrating peptides (CPPs): From delivery of nucleic acids and antigens to transduction of engineered nucleases for application in transgenesis.

    Science.gov (United States)

    Rádis-Baptista, Gandhi; Campelo, Iana S; Morlighem, Jean-Étienne R L; Melo, Luciana M; Freitas, Vicente J F

    2017-06-20

    Cell-penetrating peptides (CPPs) have been studied for their capacity to translocate across the lipid membrane of several cell types. In membrane translocation, these peptides can remarkably transport biologically active hydrophilic molecules, such as pharmaceuticals, nucleic acids (DNA and RNA) and even high-molecular-weight proteins, Fig. 3 into the cell cytoplasm and organelles. The development of CPPs as transduction agents includes the modification of gene and protein expression, the reprogramming and differentiation of induced pluripotent stem cells and the preparation of cellular vaccines. A relatively recent field of CPP application is the transduction of plasmid DNA vectors and CPP-fusion proteins to modify genomes and introduce new traits in cells and organisms. CPP-mediated transduction of components for genome editing is an advantageous alternative to viral DNA vectors. Engineered site-specific nucleases, such as Cre recombinase, ZFN, TALENs and CRISPR associated protein (Cas), have been coupled to CPPs, and the fused proteins have been used to permeate targeted cells and tissues. The functionally active fusion CPP-nucleases subsequently home to the nucleus, incise genomic DNA at specific sites and induce repair and recombination. This review has the objective of discussing CPPs and elucidating the prospective use of CPP-mediated transduction technology, particularly in genome modification and transgenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Plasmids spread very fast in heterogeneous bacterial communities.

    Science.gov (United States)

    Dionisio, Francisco; Matic, Ivan; Radman, Miroslav; Rodrigues, Olivia R; Taddei, François

    2002-01-01

    Conjugative plasmids can mediate gene transfer between bacterial taxa in diverse environments. The ability to donate the F-type conjugative plasmid R1 greatly varies among enteric bacteria due to the interaction of the system that represses sex-pili formations (products of finOP) of plasmids already harbored by a bacterial strain with those of the R1 plasmid. The presence of efficient donors in heterogeneous bacterial populations can accelerate plasmid transfer and can spread by several orders of magnitude. Such donors allow millions of other bacteria to acquire the plasmid in a matter of days whereas, in the absence of such strains, plasmid dissemination would take years. This "amplification effect" could have an impact on the evolution of bacterial pathogens that exist in heterogeneous bacterial communities because conjugative plasmids can carry virulence or antibiotic-resistance genes. PMID:12524329

  10. Plasmid segregation mechanisms

    DEFF Research Database (Denmark)

    Ebersbach, Gitte; Gerdes, Kenn; Charbon, Gitte Ebersbach

    2005-01-01

    Bacterial plasmids encode partitioning (par) loci that ensure ordered plasmid segregation prior to cell division. par loci come in two types: those that encode actin-like ATPases and those that encode deviant Walker-type ATPases. ParM, the actin-like ATPase of plasmid R1, forms dynamic filaments ...

  11. Accumulation of plasmid-mediated fluoroquinolone resistance genes, qepA and qnrS1, in Enterobacter aerogenes co-producing RmtB and class A beta-lactamase LAP-1.

    Science.gov (United States)

    Park, Yeon-Joon; Yu, Jin Kyung; Kim, Sang-Il; Lee, Kyungwon; Arakawa, Yoshichika

    2009-01-01

    A new plasmid-mediated fluoroquinolone efflux pump gene, qepA, is known to be associated with the rmtB gene, which confers high-level resistance to aminoglycosides. We investigated the qepA gene in 573 AmpC-producing Enterobacteriaceae including one Citrobacter freundii known to harbor rmtB. Of them, two clonally unrelated E. aerogenes harbored qepA. Both isolates co-harbored rmtB, qnrS1, qepA, and bla(LAP-1) on an IncFI type plasmid. The qepA was flanked by two copies of IS26 containing ISCR3C, tnpA, tnpR, bla(TEM), and rmtB. The qnrS1 and bla(LAP-1) were located upstream of qepA. All the resistance determinants (qepA, qnrS1, rmtB, and bla(LAP-1)) were co-transferred to E. coli J53 by filter mating from both isolates. Although the prevalence of qepA is currently low, considering the presence of ISCR3C and the possibility of co-selection and co-transferability of plasmids, more active surveillance for these multi-drug resistant bacteria and prudent use of antimicrobials are needed.

  12. KPC-mediated resistance in Klebsiella pneumoniae in two hospitals in Padua, Italy, June 2009-December 2011: massive spreading of a KPC-3-encoding plasmid and involvement of non-intensive care units

    Directory of Open Access Journals (Sweden)

    Richter Sara N

    2012-07-01

    Full Text Available Abstract Background Klebsiella pneumoniae carbapenemases (KPCs producing bacteria have emerged as a cause of multidrug-resistant nosocomial infections worldwide. KPCs are plasmid-encoded enzymes capable of hydrolysing a broad spectrum of beta-lactams, including carbapenems and monobactams, therefore worryingly limiting antimicrobial treatment options. Analysis of circulating bacterial strains and KPC alleles may help understanding the route of KPC dissemination and therefore help containing the infection. Methods KPC-producing Klebsiella pneumoniae dissemination in two 1580- and 300- bed hospitals in Padua, Italy, from initial outbreak in 2009 to late 2011 was analysed. Molecular and clinical epidemiology, including bacterial strains, KPC-encoding plasmid sequences and associated resistance genes, involved hospital wards and relocation of patients were described. Routine antimicrobial susceptibility testing and MIC of carbapenems on clinical isolates were performed. Detection of resistance genes was obtained by PCR and sequencing. MLST, PFGE and ERIC were used for molecular genotyping. Plasmid analysis was obtained by digestion with restriction enzymes and deep sequencing. Results KPC-positive clinical samples were isolated from nearly 200 patients. In the initial outbreak intensive care units were almost exclusively involved, while medical, surgical and long-term wards were successively massively concerned. Analysis of KPC alleles, plasmids and bacterial sequence types (STs indicated that during the initial outbreak KPC-3 in ST258 and KPC-2 in ST147 were each confined in one of the two surveilled hospitals. While KPC-2 dissemination was effectively contained, KPC-3 in ST258 cross-spreading was observed. The simultaneous presence of two carbapenemases, VIM-1 and KPC-2, in the same isolate was also observed in three patients. Total sequencing of plasmid content of two KPC-3 strains showed novel association of resistance plasmids. Conclusions The

  13. Cross-sectional study on prevalence and molecular characteristics of plasmid mediated ESBL/AmpC-producing Escherichia coli isolated from veal calves at slaughter.

    Directory of Open Access Journals (Sweden)

    Joost Hordijk

    Full Text Available OBJECTIVES: The presence of ESBL/AmpC-producing E. coli in cattle has been reported previously, however information on veal calves is limited. This study describes the prevalence and molecular characteristics of E. coli with non-wild type susceptibility to cefotaxime in veal calves at slaughter. METHODS: Faecal samples from 100 herds, 10 individual animals per herd, were screened for E. coli with non-wild type susceptibility for cefotaxime. Molecular characterization of ESBL/AmpC genes and plasmids was performed on one isolate per herd by microarray, PCR and sequence analysis. RESULTS: 66% of the herds were positive for E. coli with non-wild type susceptibility for cefotaxime. Within-herd prevalence varied from zero to 90%. 83% of E. coli producing ESBL/AmpC carried bla(CTX-M genes, of which bla(CTX-M-1, bla(CTX-M-14 and bla(CTX-M-15 were most prevalent. The dominant plasmids were IncI1 and IncF-type plasmids. CONCLUSIONS: A relatively high prevalence of various bla(CTX-M producing E. coli was found in veal calves at slaughter. The genes were mainly located on IncI1 and IncF plasmids.

  14. Prevalence of plasmid-mediated AmpC β-lactamase-producing Escherichia coli and spread of the ST131 clone among extended-spectrum β-lactamase-producing E. coli in Japan.

    OpenAIRE

    2012-01-01

    In 2010, a total of 1327 clinical Escherichia coli isolates from five hospitals in the Kyoto and Shiga regions of Japan were analysed by PCR. The prevalences of plasmid-mediated AmpC β-lactamase (pAmpC)-producers, extended-spectrum β-lactamase (ESBL)-producers and co-producers of pAmpC and ESBL were 1.7%, 9.7% and 0.3%, respectively. Less than one-half of the pAmpC-producers were reported to be resistant to third-generation cephalosporins, cephamycins and β-lactam/β-lactam inhibitors using th...

  15. Protein tyrosine kinases p53/56lyn and p72syk in MHC class I-mediated signal transduction in B lymphoma cells

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Bregenholt, S; Skov, S

    1998-01-01

    Crosslinking of major histocompatibility complex class I (MHC-I) molecules on the surface of human B lymphoma cells was shown to induce protein tyrosine phosphorylation and mobilization of intracellular free calcium. Immunoprecipitations indicated that the protein tyrosine kinases p53/56lyn and p72...... and the results indicate that these two kinases have different substrate specificity and regulate intracellular free calcium differently in response to MHC-I crosslinking. In addition MHC-I crosslinking of a sIgM-negative DT40 chicken B cell variant results in less activity of tyrosine kinases and less...... mobilization of intracellular free calcium compared with MHC-I crosslinking of wild-type DT40 cells. Thus, expression of BCR at the cell surface is likely to be important for the signal cascade initiated by MHC-I crosslinking. Our data suggest that signal transduction initiated through ligation of the MHC...

  16. [Investigation of plasmid-mediated quinolone resistance genes in quinolone-resistant Escherichia coli and Klebsiella spp. isolates from bloodstream infections].

    Science.gov (United States)

    Buruk, Celal Kurtuluş; Öztel Ocak, Hikmet; Bayramoğlu, Gülçin; Aydın, Faruk

    2016-04-01

    One of the treatment options of Escherichia coli and Klebsiella spp. infections which are the most common opportunistic pathogens of gram-negative sepsis is quinolones. Resistance to quinolones which act by disrupting DNA synthesis has been increasing. Horizontal transfer of plasmid-mediated quinolone resistance (PMQR) genes play an important role in the spread of resistance. The data about the prevalence of PMQR genes in our country is quite limited. The aim of this study was to investigate the presence of known PMQR genes namely qnrA, qnrB, qnrC, qnrS, qnrD, aac(6')-Ib-cr, qepA and oqxAB amongst quinolone-resistant E. coli and Klebsiella spp. strains isolated from blood cultures. One hundred twenty seven E.coli and 66 Klebsiella isolates detected as nalidixic acid- and/or ciprofloxacin-resistant by phenotypical methods, from 193 blood samples of 187 patients admitted to Karadeniz Technical University, Faculty of Medicine, Department of Medical Microbiology, Bacteriology Unit of Patient Service Laboratory between January 2012 to August 2013 were included in the study. The presence of PMQR genes were investigated by polymerase chain reaction (PCR) and for the detection of aac(6')-Ib-cr variants PCR-restriction fragment length polymorphism (PCR-RFLP) method was used. The positive bands were sequenced using the same primers, and aligned with formerly defined resistance gene sequences, and confirmed. In the study, 56.7% (72/127) of E.coli and 19.7% (13/66) of Klebsiella spp. isolates, with a total of 44% (85/193) of all the isolates were found to be phenotypically resistant to quinolones. Of the 13 resistant Klebsiella isolates, 11 were K.pneumoniae, and two were K.oxytoca. Extended-spectrum beta-lactamase (ESBL)-producing isolates showed higher resistance (50/80, 62.5%) to quinolones than the negative ones (35/113, 30.9%). The prevalence of quinolone resistance genes among resistant E. coli and Klebsiella spp. isolates was determined as qnrA, 1.4% and 15.4%; qnrB, 4

  17. Plasmid-mediated AmpC beta-lactamase-producing Escherichia coli causing urinary tract infection in the Auckland community likely to be resistant to commonly prescribed antimicrobials.

    Science.gov (United States)

    Drinkovic, Dragana; Morris, Arthur J; Dyet, Kristin; Bakker, Sarah; Heffernan, Helen

    2015-03-13

    To estimate the prevalence and characterise plasmid-mediated AmpC beta-lactamase (PMACBL)- producing Escherichia coli in the Auckland community. All cefoxitin non-susceptible (NS) E. coli identified at the two Auckland community laboratories between 1 January and 31 August 2011 were referred to ESR for boronic acid double-disc synergy testing, to detect the production of AmpC beta-lactamase, and polymerase chain reaction (PCR) to identify the presence of PMACBL genes. PMACBL-producing isolates were typed using pulsed-field gel electrophoresis (PFGE), and PCR was used to determine their phylogenetic group and to identify multilocus sequence type (ST)131. Antimicrobial susceptibility testing and detection of extended-spectrum beta-lactamases (ESBLs) were performed according to the Clinical and Laboratory Standards Institute recommendations. 101 (51%) and 74 (37%) of 200 non-duplicate cefoxitin-NS E. coli were PMACBL producers or assumed hyper-producers of chromosomal AmpC beta-lactamase, respectively. The prevalence of PMACBL-producing E. coli was 0.4%. PMACBL-producing E. coli were significantly less susceptible to norfloxacin, trimethoprim and nitrofurantoin than E. coli that produced neither a PMACBL nor an ESBL. Very few (4%) PMACBL-producing E. coli co-produced an ESBL. Most (88%) of the PMACBL-producing isolates had a CMY-2-like PMACBL. The PMACBL-producing E. coli isolates were diverse based on their PFGE profiles, 44% belonged to phylogenetic group D, and only four were ST131. 100 of the 101 PMACBL-producing E. coli were cultured from urine, and were causing urinary tract infection (UTI) in the majority of patients. The median patient age was 56 years and most (94%) of the patients were women. A greater proportion of patients with community-acquired UTI caused by PMACBL-producing E. coli received a beta-lactam antimicrobial than patients with community-acquired UTI caused by other non-AmpC, non-ESBL-producing E. coli. Thirty-six (43%) patients with community

  18. Quantitation of signal transduction.

    Science.gov (United States)

    Krauss, S; Brand, M D

    2000-12-01

    Conventional qualitative approaches to signal transduction provide powerful ways to explore the architecture and function of signaling pathways. However, at the level of the complete system, they do not fully depict the interactions between signaling and metabolic pathways and fail to give a manageable overview of the complexity that is often a feature of cellular signal transduction. Here, we introduce a quantitative experimental approach to signal transduction that helps to overcome these difficulties. We present a quantitative analysis of signal transduction during early mitogen stimulation of lymphocytes, with steady-state respiration rate as a convenient marker of metabolic stimulation. First, by inhibiting various key signaling pathways, we measure their relative importance in regulating respiration. About 80% of the input signal is conveyed via identifiable routes: 50% through pathways sensitive to inhibitors of protein kinase C and MAP kinase and 30% through pathways sensitive to an inhibitor of calcineurin. Second, we quantify how each of these pathways differentially stimulates functional units of reactions that produce and consume a key intermediate in respiration: the mitochondrial membrane potential. Both the PKC and calcineurin routes stimulate consumption more strongly than production, whereas the unidentified signaling routes stimulate production more than consumption, leading to no change in membrane potential despite increased respiration rate. The approach allows a quantitative description of the relative importance of signal transduction pathways and the routes by which they activate a specific cellular process. It should be widely applicable.

  19. Research of quinolones plasmid-mediated resistance mechanisms and countermeasure%喹诺酮类药物的质粒介导耐药机制及其对抗防御措施研究

    Institute of Scientific and Technical Information of China (English)

    李建华; 宋丰贵

    2008-01-01

    随着喹诺酮类抗菌药物在临床上的广泛应用,细菌对喹诺酮类药物的耐药性上升迅速.研究发现,细菌对喹诺酮类药物耐药的机制主要为靶位改变及主动外排,两者均为染色体介导.近年发现与两者完全不同的质粒介导耐药机制,且越来越多的临床菌株得以证实.本文主要对喹诺酮类药物的质粒介导的耐药机制及如何采取相应对抗防御措施进行综述.%Along with widespread application of quinolones antibiotics in clinic,quinolones resistanceof bacteria has rapidly risen. It is discovered that mechanisms of quinolones resistance of bacteria are mainlyinvolves change of target site and initiative excretion, which are both mediated by chromosome. In recentyears,plasmid-mediated drug resistance mechanism has been discovered,which is completely different fromthem. More and more clinical bacteria strains have been confirmed. The paper summarizes plasmid-mediatedquinolones resistance mechanisms and measures taken.

  20. Transductive Ordinal Regression

    CERN Document Server

    Seah, Chun-Wei; Ong, Yew-Soon

    2011-01-01

    Ordinal regression is commonly formulated as a multi-class problem with ordinal constraints. The challenge of designing accurate classifiers for ordinal regression generally increases with the number of classes involved, due to the large number of labeled patterns that are needed. The availability of ordinal class labels, however, are often costly to calibrate or difficult to obtain. Unlabeled patterns, on the other hand, often exist in much greater abundance and are freely available. To take benefits from the abundance of unlabeled patterns, we present a novel transductive learning paradigm for ordinal regression in this paper, namely Transductive Ordinal Regression (TOR). The key challenge of the present study lies in the precise estimation of both the ordinal class label of the unlabeled data and the decision functions of the ordinal classes, simultaneously. The core elements of the proposed TOR include an objective function that caters to several commonly used loss functions casted in transductive setting...

  1. TAT-mediated transduction of MafA protein in utero results in enhanced pancreatic insulin expression and changes in islet morphology.

    Directory of Open Access Journals (Sweden)

    Nancy Vargas

    Full Text Available Alongside Pdx1 and Beta2/NeuroD, the transcription factor MafA has been shown to be instrumental in the maintenance of the beta cell phenotype. Indeed, a combination of MafA, Pdx1 and Ngn3 (an upstream regulator of Beta2/NeuroD was recently reported to lead to the effective reprogramming of acinar cells into insulin-producing beta cells. These experiments set the stage for the development of new strategies to address the impairment of glycemic control in diabetic patients. However, the clinical applicability of reprogramming in this context is deemed to be poor due to the need to use viral vehicles for the delivery of the above factors. Here we describe a recombinant transducible version of the MafA protein (TAT-MafA that penetrates across cell membranes with an efficiency of 100% and binds to the insulin promoter in vitro. When injected in utero into living mouse embryos, TAT-MafA significantly up-regulates target genes and induces enhanced insulin production as well as cytoarchitectural changes consistent with faster islet maturation. As the latest addition to our armamentarium of transducible proteins (which already includes Pdx1 and Ngn3, the purification and characterization of a functional TAT-MafA protein opens the door to prospective therapeutic uses that circumvent the use of viral delivery. To our knowledge, this is also the first report on the use of protein transduction in utero.

  2. Sensory Transduction in Caenorhabditis elegans

    Science.gov (United States)

    Brown, Austin L.; Ramot, Daniel; Goodman, Miriam B.

    The roundworm Caenorhabditis elegans has a well-defined and comparatively simple repertoire of sensory-guided behaviors, all of which rely on its ability to detect chemical, mechanical or thermal stimuli. In this chapter, we review what is known about the ion channels that mediate sensation in this remarkable model organism. Genetic screens for mutants defective in sensory-guided behaviors have identified genes encoding channel proteins, which are likely transducers of chemical, thermal, and mechanical stimuli. Such classical genetic approaches are now being coupled with molecular genetics and in vivo cellular physiology to elucidate how these channels are activated in specific sensory neurons. The ion channel superfamilies implicated in sensory transduction in C. elegans - CNG, TRP, and DEG/ENaC - are conserved across phyla and also appear to contribute to sensory transduction in other organisms, including vertebrates. What we learn about the role of these ion channels in C. elegans sensation is likely to illuminate analogous processes in other animals, including humans.

  3. Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Q. [Department of Hyperbaric Medicine, No. 401 Hospital of PLA, Qingdao (China); Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Xiong, J. [Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Lu, J. [Office of Medical Education, Training Department, Second Military Medical University, Shanghai (China); Xu, S. [Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Li, Y. [State Food and Drug Administration of China,Huangdao Branch, Qingdao (China); Zhong, X.P.; Gao, G.K. [Department of Hyperbaric Medicine, No. 401 Hospital of PLA, Qingdao (China); Liu, H.Q. [2Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China)

    2012-06-22

    The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

  4. Anti-CD3 antibodies modulate anti-factor VIII immune responses in hemophilia A mice after factor VIII plasmid-mediated gene therapy.

    Science.gov (United States)

    Peng, Baowei; Ye, Peiqing; Rawlings, David J; Ochs, Hans D; Miao, Carol H

    2009-11-12

    One major obstacle in gene therapy is the generation of immune responses directed against transgene product. Five consecutive anti-CD3 treatments concomitant with factor VIII (FVIII) plasmid injection prevented the formation of inhibitory antibodies against FVIII and achieved persistent, therapeutic levels of FVIII gene expression in treated hemophilia A mice. Repeated plasmid gene transfer is applicable in tolerized mice without eliciting immune responses. Anti-CD3 treatment significantly depleted both CD4+ and CD8+ T cells, whereas increased transforming growth factor-beta levels in plasma and the frequency of both CD4+CD25+FoxP3+ and CD4+CD25-Foxp3+ regulatory T cells in the initial few weeks after treatment. Although prior depletion of CD4+CD25+ cells did not abrogate tolerance induction, adoptive transfer of CD4+ cells from tolerized mice at 6 weeks after treatment protected recipient mice from anti-FVIII immune responses. Anti-CD3-treated mice mounted immune responses against both T-dependent and T-independent neo-antigens, indicating that anti-CD3 did not hamper the immune systems in the long term. Concomitant FVIII plasmid + anti-CD3 treatment induced long-term tolerance specific to FVIII via a mechanism involving the increase in transforming growth factor-beta levels and the generation of adaptive FVIII-specific CD4+Foxp3+ regulatory T cells at the periphery. Furthermore, anti-CD3 can reduce the titers of preexisting anti-FVIII inhibitory antibodies in hemophilia A mice.

  5. Complete sequence of pOZ176, a 500-kilobase IncP-2 plasmid encoding IMP-9-mediated carbapenem resistance, from outbreak isolate Pseudomonas aeruginosa 96.

    Science.gov (United States)

    Xiong, Jianhui; Alexander, David C; Ma, Jennifer H; Déraspe, Maxime; Low, Donald E; Jamieson, Frances B; Roy, Paul H

    2013-08-01

    Pseudomonas aeruginosa 96 (PA96) was isolated during a multicenter surveillance study in Guangzhou, China, in 2000. Whole-genome sequencing of this outbreak strain facilitated analysis of its IncP-2 carbapenem-resistant plasmid, pOZ176. The plasmid had a length of 500,839 bp and an average percent G+C content of 57%. Of the 618 predicted open reading frames, 65% encode hypothetical proteins. The pOZ176 backbone is not closely related to any plasmids thus far sequenced, but some similarity to pQBR103 of Pseudomonas fluorescens SBW25 was observed. Two multiresistant class 1 integrons and several insertion sequences were identified. The blaIMP-9-carrying integron contained aacA4 → bla(IMP-9) → aacA4, flanked upstream by Tn21 tnpMRA and downstream by a complete tni operon of Tn402 and a mer module, named Tn6016. The second integron carried aacA4 → catB8a → bla(OXA-10) and was flanked by Tn1403-like tnpRA and a sul1-type 3' conserved sequence (3'-CS), named Tn6217. Other features include three resistance genes similar to those of Tn5, a tellurite resistance operon, and two pil operons. The replication and maintenance systems exhibit similarity to a genomic island of Ralstonia solanacearum GM1000. Codon usage analysis suggests the recent acquisition of bla(IMP-9). The origins of the integrons on pOZ176 indicated separate horizontal gene transfer events driven by antibiotic selection. The novel mosaic structure of pOZ176 suggests that it is derived from environmental bacteria.

  6. Heat Resistance Mediated by a New Plasmid Encoded Clp ATPase, ClpK, as a Possible Novel Mechanism for Nosocomial Persistence of Klebsiella pneumoniae

    DEFF Research Database (Denmark)

    Bojer, Martin Saxtorph; Struve, Carsten; Ingmer, Hanne;

    2010-01-01

    Klebsiella pneumoniae is an important opportunistic pathogen and a frequent cause of nosocomial infections. We have characterized a K. pneumoniae strain responsible for a series of critical infections in an intensive care unit over a two-year period. The strain was found to be remarkably...... resistant to lethal heat shock. Furthermore, one third of a collection of nosocomial K. pneumoniae isolates carry clpK and exhibit a heat resistant phenotype. The discovery of ClpK as a plasmid encoded factor and its profound impact on thermal stress survival sheds new light on the biological relevance...

  7. Heat resistance mediated by a new plasmid encoded Clp ATPase, ClpK, as a possible novel mechanism for nosocomial persistence of Klebsiella pneumoniae

    DEFF Research Database (Denmark)

    Bojer, Martin Saxtorph; Struve, Carsten; Ingmer, Hanne;

    2010-01-01

    Klebsiella pneumoniae is an important opportunistic pathogen and a frequent cause of nosocomial infections. We havecharacterized a K. pneumoniae strain responsible for a series of critical infections in an intensive care unit over a two-year period. The strain was found to be remarkably...... resistant to lethal heat shock. Furthermore, one third of a collection of nosocomial K. pneumoniae isolates carry clpK and exhibit a heat resistant phenotype. The discovery of ClpK as a plasmid encoded factor and its profound impact on thermal stress survival sheds new light on the biological relevance...

  8. Gene flow across genus barriers – Conjugation of Dinoroseobacter shibae´s 191-kb killer plasmid into Phaeobacter inhibens and AHL-mediated expression of type IV secretion systems

    Directory of Open Access Journals (Sweden)

    Diana ePatzelt

    2016-05-01

    Full Text Available Rhodobacteraceae harbor a conspicuous wealth of extrachromosomal replicons (ECRs and therefore the exchange of genetic material via horizontal transfer has been supposed to be a major evolutionary driving force. Many plasmids in the roseobacter group encode type IV secretion systems (T4SS that generally mediate transfer of proteins and/or DNA into host cells, but no experimental evidence of either has yet been provided. Dinoroseobacter shibae contains five ECRs that are crucial for anaerobic growth, survival under starvation and the pathogenicity of this model organism. Here we tagged two syntenous but compatible RepABC-type plasmids of 191 kb and 126-kb size, each harboring a T4SS, with antibiotic resistance genes and demonstrated their conjugational transfer into a distantly related roseo-bacter species, namely Phaeobacter inhibens. Pulsed field gel electrophoresis showed transfer of those replicons into the recipient both individually but also together documenting the efficiency of conjugation. We then studied the influence of externally added quorum sensing (QS signals on the expression of the T4SS located on the sister plasmids. A QS deficient D. shibae null mutant (ΔluxI1 lacking synthesis of N-acyl-homoserine lactones (AHLs was cultivated with a wide spectrum of chemically diverse long-chain AHLs. All AHLs with lengths of the acid side-chain >14 reverted the ΔluxI1 phenotype to wild-type. Expression of the T4SS was induced above wild-type level. We hypothesize that conjugation in roseobacters is QS-controlled and that the QS system may detect a wide array of long-chain AHLs at the cell surface.

  9. The combined transduction of copper, zinc-superoxide dismutase and catalase mediated by cell-penetrating peptide, PEP-1, to protect myocardium from ischemia-reperfusion injury

    Directory of Open Access Journals (Sweden)

    Guo Ling-Yun

    2011-05-01

    Full Text Available Abstract Background Our previous studies indicate that either PEP-1-superoxide dismutase 1 (SOD1 or PEP-1-catalase (CAT fusion proteins protects myocardium from ischemia-reperfusion-induced injury in rats. The aim of this study is to explore whether combined use of PEP-1-SOD1 and PEP-1-CAT enhances their protective effects. Methods SOD1, PEP-1-SOD1, CAT or PEP-1-CAT fusion proteins were prepared and purified by genetic engineering. In vitro and in vivo effects of these proteins on cell apoptosis and the protection of myocardium after ischemia-reperfusion injury were measured. Embryo cardiac myocyte H9c2 cells were used for the in vitro studies. In vitro cellular injury was determined by the expression of lactate dehydrogenase (LDH. Cell apoptosis was quantitatively assessed with Annexin V and PI double staining by Flow cytometry. In vivo, rat left anterior descending coronary artery (LAD was ligated for one hour followed by two hours of reperfusion. Hemodynamics was then measured. Myocardial infarct size was evaluated by TTC staining. Serum levels of myocardial markers, creatine kinase-MB (CK-MB and cTnT were quantified by ELISA. Bcl-2 and Bax expression in left ventricle myocardium were analyzed by western blot. Results In vitro, PEP-1-SOD1 or PEP-1-CAT inhibited LDH release and apoptosis rate of H9c2 cells. Combined transduction of PEP-1-SOD1 and PEP-1-CAT, however, further reduced the LDH level and apoptosis rate. In vivo, combined usage of PEP-1-SOD1 and PEP-1-CAT produced a greater effect than individual proteins on the reduction of CK-MB, cTnT, apoptosis rate, lipoxidation end product malondialdehyde, and the infarct size of myocardium. Functionally, the combination of these two proteins further increased left ventricle systolic pressure, but decreased left ventricle end-diastolic pressure. Conclusion This study provided a basis for the treatment or prevention of myocardial ischemia-reperfusion injury with the combined usage of PEP-1-SOD1

  10. Novel genetic environment of the plasmid-mediated KPC-3 gene detected in Escherichia coli and Citrobacter freundii isolates from China.

    Science.gov (United States)

    Li, G; Wei, Q; Wang, Y; Du, X; Zhao, Y; Jiang, X

    2011-04-01

    The imipenem and meropenem-resistant strains Citrobacter freundii HS70 and Escherichia coli HS510 were isolated from patients in Shanghai, China. By isoelectric focusing, PCR amplification and sequencing, these strains were each found to produce four β-lactamases: TEM-1, KPC-3, SHV-7 and CTX-M-14. A conjugation experiment and plasmid restriction digestion revealed that the bla (KPC-3) gene was located on the same plasmid in both isolates. Bidirectional primer walking sequencing showed that the nucleotide sequence surrounding the 3.8 kb bla(KPC-3) contained a 671-bp insertion similar to that previously characterized in China. The insertion was located between the promoter and the coding region of the bla(KPC-3) gene. Susceptibility testing performed on recombinant strains carrying the bla(KPC-3) gene with or without the insertion revealed that minimum inhibitory concentrations of imipenem, meropenem, cefepime, and cefotaxime for E. coli EMU-KPC3 (without insertion) were four times higher than that of E. coli EKPC3 (with insertion). The 671 bp insertion reduced bla(KPC-3) expression significantly. Taken together, these results suggest that KPC-3-producing C. freundii and E. coli have begun to emerge in our hospital.

  11. Efficient microbubble- and ultrasound-mediated plasmid DNA delivery into a specific rat liver lobe via a targeted injection and acoustic exposure using a novel ultrasound system.

    Science.gov (United States)

    Song, Shuxian; Noble, Misty; Sun, Samuel; Chen, Liping; Brayman, Andrew A; Miao, Carol H

    2012-08-06

    To develop efficient gene delivery in larger animals, based on a previous mouse study, we explored the luciferase reporter gene transfer in rats by establishing a novel unfocused ultrasound system with simultaneous targeted injection of a plasmid and microbubble mixture into a specific liver lobe through a portal vein branch. Luciferase expression was significantly enhanced over 0-30 vol % of the Definity microbubbles, with a plateau between 0.5 and 30 vol %. The increase of gene delivery efficiency also depended on the acoustic peak negative pressure, achieving over 100-fold enhancement at 2.5 MPa compared with plasmid only controls. Transient, modest liver damage following treatment was assessed by transaminase assays and histology, both of which correlated with gene expression induced by acoustic cavitation. In addition, pulse-train ultrasound exposures (i.e., with relatively long quiescent periods between groups of pulses to allow tissue refill with microbubbles) produced gene expression levels comparable to the standard US exposure but reduced the extent of liver damage. These results indicated that unfocused high intensity therapeutic ultrasound exposure with microbubbles is highly promising for safe and efficient gene delivery into the liver of rats or larger animals.

  12. Plasmid-Mediated Quinolone Resistance (PMQR) Genes and Class 1 Integrons in Quinolone-Resistant Marine Bacteria and Clinical Isolates of Escherichia coli from an Aquacultural Area.

    Science.gov (United States)

    Tomova, Alexandra; Ivanova, Larisa; Buschmann, Alejandro H; Godfrey, Henry P; Cabello, Felipe C

    2017-06-23

    Antimicrobial usage in aquaculture selects for antimicrobial-resistant microorganisms in the marine environment. The relevance of this selection to terrestrial animal and human health is unclear. Quinolone-resistance genes qnrA, qnrB, and qnrS were chromosomally located in four randomly chosen quinolone-resistant marine bacteria isolated from an aquacultural area with heavy quinolone usage. In quinolone-resistant uropathogenic clinical isolates of Escherichia coli from a coastal area bordering the same aquacultural region, qnrA was chromosomally located in two E. coli isolates, while qnrB and qnrS were located in small molecular weight plasmids in two other E. coli isolates. Three quinolone-resistant marine bacteria and three quinolone-resistant E. coli contained class 1 integrons but without physical association with PMQR genes. In both marine bacteria and uropathogenic E. coli, class 1 integrons had similar co-linear structures, identical gene cassettes, and similarities in their flanking regions. In a Marinobacter sp. marine isolate and in one E. coli clinical isolate, sequences immediately upstream of the qnrS gene were homologous to comparable sequences of numerous plasmid-located qnrS genes while downstream sequences were different. The observed commonality of quinolone resistance genes and integrons suggests that aquacultural use of antimicrobials might facilitate horizontal gene transfer between bacteria in diverse ecological locations.

  13. Emergence of CTX-M-3, TEM-1 and a new plasmid-mediated MOX-4 AmpC in a multiresistant Aeromonas caviae isolate from a patient with pneumonia.

    Science.gov (United States)

    Ye, Ying; Xu, Xi-Hai; Li, Jia-Bin

    2010-07-01

    Aeromonas species rarely cause pulmonary infection. We report, for what is believed to be the first time, a case of severe pneumonia in a cancer patient caused by Aeromonas caviae. Detailed microbiological investigation revealed that this isolate carried three beta-lactamase-encoding genes (encoding MOX-4, CTX-M-3 and TEM-1) conferring resistance to all beta-lactams but imipenem. The beta-lactamase with a pI of 9.0 was transferred by conjugation and associated with a 7.3 kb plasmid, as demonstrated by Southern blot hybridization. Analysis of the nucleotide and amino acid sequences showed a new ampC gene that was closely related to those encoding the MOX-1, MOX-2 and MOX-3 beta-lactamases. This new plasmid-mediated AmpC beta-lactamase from China was named MOX-4. This is believed to be the first report of MOX-4, CTX-M-3 and TEM-1 beta-lactamases in a multiresistant A. caviae.

  14. Phenotypic and Molecular Characterization of Plasmid Mediated AmpC β-Lactamases among Escherichia coli, Klebsiella spp., and Proteus mirabilis Isolated from Urinary Tract Infections in Egyptian Hospitals

    Directory of Open Access Journals (Sweden)

    Mai M. Helmy

    2014-01-01

    Full Text Available The incidence of resistance by Enterobacteriaceae to β-lactam/β-lactamase inhibitors combination is increasing in Egypt. Three phenotypic techniques, comprising AmpC disk diffusion and inhibition dependent methods using phenylboronic acid (PBA and cloxacillin, were compared to PCR based method for detection of plasmid mediated AmpC β-lactamase in common urinary tract isolates. A total of 143 isolates, including E. coli, Klebsiella pneumonia, and Proteus mirabilis, were collected from urinary tract infections cases in Egyptian hospitals. Plasmid encoded AmpC genes were detected by PCR in 88.46% of cefoxitin resistant isolates. The most prevalent AmpC gene family was CIT including CMY-2, CMY-4, and two CMY-2 variants. The second prevalent gene was DHA-1 which was detected in E. coli and Klebsiella pneumonia. The genes EBC, FOX, and MOX were also detected but in small percentage. Some isolates were identified as having more than one pAmpC gene. The overall sensitivity and specificity of phenotypic tests for detection of AmpC β-lactamase showed that AmpC disk diffusion and inhibition dependent method by cloxacillin were the most sensitive and the most specific disk tests. PCR remains the gold standard for detection of AmpC β-lactamases. This study represents the first report of CMY-2 variants of CMY-42 and CMY-102 β-lactamase-producing E. coli, Klebsiella pneumonia, and Proteus mirabilis isolates in Egypt.

  15. Antibiotic-Resistant Extended Spectrum ß-Lactamase- and Plasmid-Mediated AmpC-Producing Enterobacteriaceae Isolated from Retail Food Products and the Pearl River in Guangzhou, China

    Science.gov (United States)

    Ye, Qinghua; Wu, Qingping; Zhang, Shuhong; Zhang, Jumei; Yang, Guangzhu; Wang, Huixian; Huang, Jiahui; Chen, Mongtong; Xue, Liang; Wang, Juan

    2017-01-01

    We conducted a survey in 2015 to evaluate the presence of extended spectrum β-lactamase (ESBL)- and plasmid-mediated AmpC-producing Enterobacteriaceae in retail food and water of the Pearl River in Guangzhou, China, as well as their antibiotic resistance profiles. Samples (88 fresh food samples and 43 water samples) from eight different districts were analyzed by direct plating and after enrichment. Multidrug-resistant strains were found in 41.7 and 43.4% of food and water samples, respectively. ESBLs were found in 3.4 and 11.6% of food and water samples, respectively, and AmpC producers were found in 13.6 and 16.3% of food and water samples, respectively. Molecular characterization revealed the domination of blaCTX−Mgenes; plasmidic AmpC was of the type DHA-1 both in food and water samples. Thirteen of Fifty one β-lactamase-producing positive isolates were detected to be transconjugants, which readily received the β-lactamase genes conferring resistance to β-lactam antibiotics as well as some non-β-lactam antibiotics. These findings provide evidence that retail food and the river water may be considered as reservoirs for the dissemination of β-lactam antibiotics, and these resistance genes could readily be transmitted to humans through the food chain and water. PMID:28217112

  16. Survival and evolution of a large multidrug resistance plasmid in new clinical bacterial hosts

    DEFF Research Database (Denmark)

    Porse, Andreas; Schønning, Kristian; Munck, Christian

    2016-01-01

    of these plasmids within pathogenic hosts are poorly understood. Here we study plasmid-host adaptations following transfer of a 73 kb conjugative multidrug resistance plasmid to naïve clinical isolates of Klebsiella pneumoniae and Escherichia coli We use experimental evolution, mathematical modelling and population...... sequencing to show that the long-term persistence and molecular integrity of the plasmid is highly influenced by multiple factors within a 25 kb plasmid region constituting a host-dependent burden. In the E. coli hosts investigated here, improved plasmid stability readily evolves via IS26 mediated deletions...... of plasmid adaptation. While insertion sequences are well known to supply plasmids with adaptive traits, our findings suggest that they also play an important role in plasmid evolution by maintaining the plasticity necessary to alleviate plasmid-host constrains. Further, the observed evolutionary strategy...

  17. Proteasomal degradation of the metabotropic glutamate receptor 1α is mediated by Homer-3 via the proteasomal S8 ATPase: Signal transduction and synaptic transmission.

    Science.gov (United States)

    Rezvani, Khosrow; Baalman, Kelli; Teng, Yanfen; Mee, Maureen P; Dawson, Simon P; Wang, Hongmin; De Biasi, Mariella; Mayer, R John

    2012-07-01

    The metabotropic glutamate receptors (mGluRs) fine-tune the efficacy of synaptic transmission. This unique feature makes mGluRs potential targets for the treatment of various CNS disorders. There is ample evidence to show that the ubiquitin proteasome system mediates changes in synaptic strength leading to multiple forms of synaptic plasticity. The present study describes a novel interaction between post-synaptic adaptors, long Homer-3 proteins, and one of the 26S proteasome regulatory subunits, the S8 ATPase, that influences the degradation of the metabotropic glutamate receptor 1α (mGluR1α). We have shown that the two human long Homer-3 proteins specifically interact with human proteasomal S8 ATPase. We identified that mGluR1α and long Homer-3s immunoprecipitate with the 26S proteasome both in vitro and in vivo. We further found that the mGluR1α receptor can be ubiquitinated and degraded by the 26S proteasome and that Homer-3A facilitates this process. Furthermore, the siRNA mediated silencing of Homer-3 led to increased levels of total and plasma membrane-associated mGluR1α receptors. These results suggest that long Homer-3 proteins control the degradation of mGluR1α receptors by shuttling ubiquitinated mGluR-1α receptors to the 26S proteasome via the S8 ATPase which may modulate synaptic transmission.

  18. Prevalence and characterization of plasmid-mediated blaESBL with their genetic environment in Escherichia coli and Klebsiella pneumoniae in patients with pneumonia

    Institute of Scientific and Technical Information of China (English)

    WANG Xiao-rong; CHEN Ji-chao; KANG Yu; JIANG Ning; AN Shu-chang; GAO Zhan-cheng

    2012-01-01

    Background The extended spectrum β-lactamase (ESBL)-producing Escherichia coli (E.coli) and Klebsiella pneumoniae (K.pneumoniae) are the major pathogens causing pneumonia and have a significant impact on the clinical course.Limited data exist on molecular characterization of ESBL-producing E.coli and K.pneumoniae that cause pneumonia.The aim of this study was to investigate the comprehensive multilevel characteristics of E.coli and K.pneumoniae causing pneumonia in China for the first time.Methods E.coli (17) and K.pneumoniae (21) isolates responsible for pneumonia were isolated from 1270 specimens collected in a prospective multi-center study in eight teaching hospitals in China from June to December in 2007.The susceptibilities,ESBL confirmation,sequence typing,blaCTX-M and blaSHV genes,their genetic environment and plasmid Inc/rep types were determined.Results Sixteen E.coli (94.1%) and eleven K.pneumoniae (52.4%) isolates were ESBL producers.About 77.8% and 66.7% of them were resistance to ciprofloxacin and levofloxacin,and 100% were susceptible to imipenem.The most prevalent ESBL gene was CTX-M-14,followed by SHV-2,CTX-M-15,CTX-M-3,CTX-M-65,SHV-12,SHV-26 and SHV-28.SHV-1 and SHV-11 were also detected and coexisted with blaCTX-Ms in five strains,and three strains contained only SHV-1.All CTX-M-14 were detected ISEcp1 upstream and nine were found IS903 downstream and the majority of them (64.3%) were carried by IncF plasmids.All blasHv were flanked by recFand deoR,located on IncF,IncN,IncX and IncH plasmids.Two SHV-2,one SHV-1 and the only SHV-28 were further preceded by IS26.Genes lacYand lacZwere detected at further upstream of two blaSHv-1.The K.pneumoniae carrying SHV-28 was susceptible to β-lactams,and no mutations or deletions in gene or promoter sequences were identified to account for susceptibility.Multilocus sequence typing experiments showed the ESBL-producing strains were genetically diverse.Conclusions The rate of occurrence of bla

  19. Heat resistance mediated by a new plasmid encoded Clp ATPase, ClpK, as a possible novel mechanism for nosocomial persistence of Klebsiella pneumoniae

    DEFF Research Database (Denmark)

    Bojer, Martin Saxtorph; Struve, Carsten; Ingmer, Hanne;

    2010-01-01

    Klebsiella pneumoniae is an important opportunistic pathogen and a frequent cause of nosocomial infections. We have characterized a K. pneumoniae strain responsible for a series of critical infections in an intensive care unit over a two-year period. The strain was found to be remarkably...... of Clp ATPases in acquired environmental fitness and highlights the challenges of mobile genetic elements in fighting nosocomial infections....... resistant to lethal heat shock. Furthermore, one third of a collection of nosocomial K. pneumoniae isolates carry clpK and exhibit a heat resistant phenotype. The discovery of ClpK as a plasmid encoded factor and its profound impact on thermal stress survival sheds new light on the biological relevance...

  20. Emergence of serotype K1 Klebsiella pneumoniae ST23 strains co-producing the plasmid-mediated AmpC beta-lactamase DHA-1 and an extended-spectrum beta-lactamase in Korea

    Directory of Open Access Journals (Sweden)

    Hae Suk Cheong

    2016-11-01

    Full Text Available Abstract Background Serotype K1 Klebsiella pneumoniae has emerged as an important community pathogen causing various infections, including liver abscesses. Although serotype K1 K. pneumoniae community isolates have been reported as susceptible to most classes of antimicrobial agents, a few cases of infection caused by extended-spectrum beta-lactamase (ESBL-producing serotype K1 K. pneumoniae have recently been reported in Asian countries. We identified three ESBL-producing strains of serotype K1 K. pneumoniae and conducted a molecular characterization of their drug resistance. Methods Three ESBL-producing serotype K1 K. pneumoniae ST23 strains were identified from strains in the Asian Bacterial Bank. Antimicrobial susceptibility testing was performed using the broth microdilution method, and ESBL production was tested by the double-disk synergy test and a confirmatory test. PCR was performed to detect the genes for plasmid-mediated ESBL and AmpC beta-lactamases. Results All three strains were resistant to cefotaxime, ceftazidime, and piperacillin/tazobactam, and all were determined to be ESBL-producers. No known ESBL genes, including bla SHV, bla TEM, bla CTX-M, bla GES, bla PER, and bla VEB, were detected among the three strains. Of all plasmid-mediated AmpC beta-lactamase (PAB genes, including bla DHA-1, bla CMY, bla FOX, and bla MOX, the bla DHA-1 gene was detected in two of the strains. The PFGE patterns revealed that the two isolates carrying bla DHA-1 were closely related (84% similarity. Conclusions No ESBL genes were detected among three ESBL-producing serotype K1 K. pneumoniae ST23 strains. Two strains contained the PAB gene bla DHA-1. The emergence of resistant strains of community-origin serotype K1 K. pneumoniae has important implications for effective treatment and infection control practices.

  1. Signal transduction mediated by Bid, a pro-death Bcl-2 family proteins, connects the death receptor and mitochondria apoptosis pathways

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Two major apoptosis pathways have been defined in mammalian cells, the Fas/TNF-R1 death receptor pathway and the mitochondria pathway. The Bcl-2 family proteins consist of both anti-apoptosis and pro- apoptosis members that regulate apoptosis, mainly by controlling the release of cytochrome c and other mitochondrial apoptotic events. However, death signals mediated by Fas/TNF-R1 receptors can usually activate caspases directly, bypassing the need for mitochondria and escaping the regulation by Bcl-2 family proteins. Bid is a novel pro-apoptosis Bcl-2 family protein that is activated by caspase 8 in response to Fas/TNF-R1 death receptor signals. Activated Bid is translocated to mitochondria and induces cytochrome c release, which in turn activates downstream caspases. Such a connection between the two apoptosis pathways could be important for induction of apoptosis in certain types of cells and responsible for the pathogenesis of a number of human diseases.

  2. Protein diversity confers specificity in plasmid segregation.

    Science.gov (United States)

    Fothergill, Timothy J G; Barillà, Daniela; Hayes, Finbarr

    2005-04-01

    The ParG segregation protein (8.6 kDa) of multidrug resistance plasmid TP228 is a homodimeric DNA-binding factor. The ParG dimer consists of intertwined C-terminal domains that adopt a ribbon-helix-helix architecture and a pair of flexible, unstructured N-terminal tails. A variety of plasmids possess partition loci with similar organizations to that of TP228, but instead of ParG homologs, these plasmids specify a diversity of unrelated, but similarly sized, partition proteins. These include the proteobacterial pTAR, pVT745, and pB171 plasmids. The ParG analogs of these plasmids were characterized in parallel with the ParG homolog encoded by the pseudomonal plasmid pVS1. Like ParG, the four proteins are dimeric. No heterodimerization was detectable in vivo among the proteins nor with the prototypical ParG protein, suggesting that monomer-monomer interactions are specific among the five proteins. Nevertheless, as with ParG, the ParG analogs all possess significant amounts of unordered amino acid residues, potentially highlighting a common structural link among the proteins. Furthermore, the ParG analogs bind specifically to the DNA regions located upstream of their homologous parF-like genes. These nucleoprotein interactions are largely restricted to cognate protein-DNA pairs. The results reveal that the partition complexes of these and related plasmids have recruited disparate DNA-binding factors that provide a layer of specificity to the macromolecular interactions that mediate plasmid segregation.

  3. Molecular pathway profiling of T lymphocyte signal transduction pathways; Th1 and Th2 genomic fingerprints are defined by TCR and CD28-mediated signaling

    Directory of Open Access Journals (Sweden)

    Smeets Ruben L

    2012-03-01

    Full Text Available Abstract Background T lymphocytes are orchestrators of adaptive immunity. Naïve T cells may differentiate into Th1, Th2, Th17 or iTreg phenotypes, depending on environmental co-stimulatory signals. To identify genes and pathways involved in differentiation of Jurkat T cells towards Th1 and Th2 subtypes we performed comprehensive transcriptome analyses of Jurkat T cells stimulated with various stimuli and pathway inhibitors. Results from these experiments were validated in a human experimental setting using whole blood and purified CD4+ Tcells. Results Calcium-dependent activation of T cells using CD3/CD28 and PMA/CD3 stimulation induced a Th1 expression profile reflected by increased expression of T-bet, RUNX3, IL-2, and IFNγ, whereas calcium-independent activation via PMA/CD28 induced a Th2 expression profile which included GATA3, RXRA, CCL1 and Itk. Knock down with siRNA and gene expression profiling in the presence of selective kinase inhibitors showed that proximal kinases Lck and PKCθ are crucial signaling hubs during T helper cell activation, revealing a clear role for Lck in Th1 development and for PKCθ in both Th1 and Th2 development. Medial signaling via MAPkinases appeared to be less important in these pathways, since specific inhibitors of these kinases displayed a minor effect on gene expression. Translation towards a primary, whole blood setting and purified human CD4+ T cells revealed that PMA/CD3 stimulation induced a more pronounced Th1 specific, Lck and PKCθ dependent IFNγ production, whereas PMA/CD28 induced Th2 specific IL-5 and IL-13 production, independent of Lck activation. PMA/CD3-mediated skewing towards a Th1 phenotype was also reflected in mRNA expression of the master transcription factor Tbet, whereas PMA/CD28-mediated stimulation enhanced GATA3 mRNA expression in primary human CD4+ Tcells. Conclusions This study identifies stimulatory pathways and gene expression profiles for in vitro skewing of T helper cell

  4. Brassinosteroid signal transduction: An emerging picture

    Institute of Scientific and Technical Information of China (English)

    WANG Qiaomei; MA Ligeng

    2003-01-01

    Steroid hormones play essential roles in animal growth and development. Steroid signaling in animal system is focused on the direct gene regulation response mediated by its nuclear receptors. Recently, steroid hormones are also found in plants. Identification of BRI1 - a critical component of the plasma-membrane steroid receptor complex, and the related signal transduction pathway mediated by the membrane receptor have revealed an elementary picture of BR signaling from the cell surface perception to the activation of BR-responsive nuclear genes.

  5. Chlamydial plasmids and bacteriophages.

    Science.gov (United States)

    Pawlikowska-Warych, Małgorzata; Śliwa-Dominiak, Joanna; Deptuła, Wiesław

    2015-01-01

    Chlamydia are absolute pathogens of humans and animals; despite being rather well recognised, they are still open for discovery. One such discovery is the occurrence of extrachromosomal carriers of genetic information. In prokaryotes, such carriers include plasmids and bacteriophages, which are present only among some Chlamydia species. Plasmids were found exclusively in Chlamydia (C.) trachomatis, C. psittaci, C. pneumoniae, C. suis, C. felis, C. muridarum and C. caviae. In prokaryotic organisms, plasmids usually code for genes that facilitate survival of the bacteria in the environment (although they are not essential). In chlamydia, their role has not been definitely recognised, apart from the fact that they participate in the synthesis of glycogen and encode proteins responsible for their virulence. Furthermore, in C. suis it was evidenced that the plasmid is integrated in a genomic island and contains the tetracycline-resistance gene. Bacteriophages specific for chlamydia (chlamydiaphages) were detected only in six species: C. psittaci, C. abortus, C. felis, C. caviae C. pecorum and C. pneumoniae. These chlamydiaphages cause inhibition of the developmental cycle, and delay transformation of reticulate bodies (RBs) into elementary bodies (EBs), thus reducing the possibility of infecting other cells in time. Plasmids and bacteriophages can be used in the diagnostics of chlamydioses; although especially in the case of plasmids, they are already used for detection of chlamydial infections. In addition, bacteriophages could be used as therapeutic agents to replace antibiotics, potentially addressing the problem of increasing antibiotic-resistance among chlamydia.

  6. Inhibition of the CyclinD1 promoter in response to sonic hedgehog signaling pathway transduction is mediated by Gli1

    Science.gov (United States)

    Lin, Zhongxiao; Sheng, Hansong; You, Chaoguo; Cai, Ming; Zhang, Yiping; Yu, Li Sheng; Yu, Xiaoming; Lin, Jian; Zhang, Nu

    2017-01-01

    Medulloblastoma (MB) is the most common malignant tumor of the central nervous system in children. Accumulating evidence suggests a major role for the activation of the sonic hedgehog (SHH) signaling pathway in the development of MB cells; however, the mechanisms underlying the effect of this pathway on tumor survival and growth remain poorly understood. The Gli family zinc finger 1 (Gli1) transcription factor is considered as a mediator of the SHH signaling pathway in MB cells. Therefore, the present study investigated whether the SHH signaling pathway promotes the apoptosis of MB cells via downregulation of Gli1. GANT61, a novel Gli1 inhibitor, is known to have an in vitro activity against tumors. In the current study, Daoy cells were treated with different concentrations of GANT61 for 24 h, and the effect on cell proliferation was assayed by cell counting kit-8 assay. In addition, the cell cycle progression and apoptosis were assayed by flow cytometry analysis and hematoxylin-eosin (HE) staining. The effects of GANT61 treatment on SHH signaling pathway at the mRNA level were assayed by polymerase chain reaction (PCR). To further elucidate the inhibitory effects of GANT61 on the expression of Gli1 and CyclinD1, their protein levels were examined by western blot and immunofluorescence. The results indicated that GANT61 significantly inhibited the proliferation of Daoy cells in a dose-dependent manner, compared with the control group (PSHH pathway activity in MB, and may be a novel agent for use in combined chemotherapeutic regimens. PMID:28123507

  7. The insulin receptor substrate-1-related 4PS substrate but not the interleukin-2R gamma chain is involved in interleukin-13-mediated signal transduction.

    Science.gov (United States)

    Wang, L M; Michieli, P; Lie, W R; Liu, F; Lee, C C; Minty, A; Sun, X J; Levine, A; White, M F; Pierce, J H

    1995-12-01

    Interleukin-13 (IL-13) induced a potent mitogenic response in IL-3-dependent TF-1 cells and DNA synthesis to a lesser extent in MO7E and FDC-P1 cells. IL-13 stimulation of these lines, like IL-4 and insulin-like growth factor-1 (IGF-1), resulted in tyrosine phosphorylation of a 170-kD substrate. The tyrosine-phosphorylated 170-kD substrate strongly associated with the 85-kD subunit of phosphoinositol-3 (PI-3) kinase and with Grb-2. Anti-4PS serum readily detected the 170-kD substrate in lysates from both TF-1 and FDC-P1 cells stimulated with IL-13 or IL-4. These data provide evidence that IL-13 induces tyrosine phosphorylation of the 4PS substrate, providing an essential interface between the IL-13 receptor and signaling molecules containing SH2 domains. IL-13 and IL-4 stimulation of murine L cell fibroblasts, which endogenously express the IL-4 receptor (IL-4R alpha) and lack expression of the IL-2 receptor gamma subunit (IL-2R gamma), resulted in tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1)/4PS. Enhanced tyrosine phosphorylation of IRS-1/4PS was observed in response to IL-4, but not IL-13 treatment of L cells transfected with the IL-2R gamma chain. These results indicate that IL-13 does not use the IL-2R gamma subunit in its receptor complex and that expression of IL-2R gamma enhances, but is not absolutely required for mediating IL-4-induced tyrosine phosphorylation of IRS-1/4PS.

  8. Chemical-Induced Inhibition of Blue Light-Mediated Seedling Development Caused by Disruption of Upstream Signal Transduction Involving Cryptochromes in Arabidopsis thaliana.

    Science.gov (United States)

    Ong, Wen-Dee; Okubo-Kurihara, Emiko; Kurihara, Yukio; Shimada, Setsuko; Makita, Yuko; Kawashima, Mika; Honda, Kaori; Kondoh, Yasumitsu; Watanabe, Nobumoto; Osada, Hiroyuki; Cutler, Sean R; Sudesh, Kumar; Matsui, Minami

    2017-01-01

    Plants have a remarkable ability to perceive and respond to various wavelengths of light and initiate regulation of different cascades of light signaling and molecular components. While the perception of red light and the mechanisms of its signaling involving phytochromes are largely known, knowledge of the mechanisms of blue light signaling is still limited. Chemical genetics involves the use of diverse small active or synthetic molecules to evaluate biological processes. By combining chemicals and analyzing the effects they have on plant morphology, we identified a chemical, 3-bromo-7-nitroindazole (3B7N), that promotes hypocotyl elongation of wild-type Arabidopsis only under continuous blue light. Further evaluation with loss-of-function mutants confirmed that 3B7N inhibits photomorphogenesis through cryptochrome-mediated light signaling. Microarray analysis demonstrated that the effect of 3B7N treatment on gene expression in cry1cry2 is considerably smaller than that in the wild type, indicating that 3B7N specifically interrupts cryptochrome function in the control of seedling development in a light-dependent manner. We demonstrated that 3B7N directly binds to CRY1 protein using an in vitro binding assay. These results suggest that 3B7N is a novel chemical that directly inhibits plant cryptochrome function by physical binding. The application of 3B7N can be used on other plants to study further the blue light mechanism and the genetic control of cryptochromes in the growth and development of plant species. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  9. Signal transduction by the major histocompatibility complex class I molecule

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Skov, S; Bregenholt, S;

    1999-01-01

    Ligation of cell surface major histocompatibility class I (MHC-I) proteins by antibodies, or by their native counter receptor, the CD8 molecule, mediates transduction of signals into the cells. MHC-I-mediated signaling can lead to both increased and decreased activity of the MHC-I-expressing cell...

  10. Heat resistance mediated by a new plasmid encoded Clp ATPase, ClpK, as a possible novel mechanism for nosocomial persistence of Klebsiella pneumoniae

    DEFF Research Database (Denmark)

    Bojer, Martin Saxtorph; Struve, Carsten; Ingmer, Hanne;

    2010-01-01

    Klebsiella pneumoniae is an important opportunistic pathogen and a frequent cause of nosocomial infections. We have characterized a K. pneumoniae strain responsible for a series of critical infections in an intensive care unit over a two-year period. The strain was found to be remarkably thermoto......Klebsiella pneumoniae is an important opportunistic pathogen and a frequent cause of nosocomial infections. We have characterized a K. pneumoniae strain responsible for a series of critical infections in an intensive care unit over a two-year period. The strain was found to be remarkably...... resistant to lethal heat shock. Furthermore, one third of a collection of nosocomial K. pneumoniae isolates carry clpK and exhibit a heat resistant phenotype. The discovery of ClpK as a plasmid encoded factor and its profound impact on thermal stress survival sheds new light on the biological relevance...... of Clp ATPases in acquired environmental fitness and highlights the challenges of mobile genetic elements in fighting nosocomial infections....

  11. A Transient Three-plasmid Expression System for the Production of Hepatocytes Targeting Retroviral Vectors

    Institute of Scientific and Technical Information of China (English)

    Peng QI; Jinxiang HAN; Yanqin LU; Chuanxi WANG; Bo ZHU

    2007-01-01

    Targeting of retroviral vectors to specific cells was attempted through modifying the surface protein of the murine leukemia viruses (MLVs), but in many cases the protein function was affected, and it is difficult to achieve the targeted delivery. In this study, we have tried to engineer ecotropic Moloney murine leukemia viruses (MoMLV)-based retroviral vectors to transduce hepatocytes. A chimeric envelope (Env)expression plasmid was constructed containing the hepatitis B virus PreS2 peptide fused to aa +1 at the Nterminus of Env. Following simultaneous transfection of pgag-pol, pLEGFP and chimeric env plasmids into 293T cells, helper-free retrovirus stocks with the titer of approximately 104 infectious units/ml were achieved at 48 h post-transfection. These pseudotype vectors showed the normal host range of retrovirus, infecting host NIH 3T3 cells, although the efficiency was reduced compared with that of virions carrying wild-type ecotropic MoMLV envelope. In addition, the resultant pseudotype viruses could transduce human hepatoma cells mediated by polymerized human serum albumin with relatively high titers in comparison with those transductions without polymerized human serum albumin. This approach can be used to target hepatocytes selectively.

  12. Experimental study of electroporation-mediated plasmid gene expression in skin and incisional wound%电穿孔介导质粒基因在皮肤和线性伤口中的表达

    Institute of Scientific and Technical Information of China (English)

    高振; 宋楠; 武晓莉; 曾谊林; 刘伟

    2008-01-01

    Objective To explore the feasibility of electroporation mediated gone transfer in rat incisional wound.Methods 12 Sprague-Dawley rat's dorsal skins were electroporated(800 voltages in amplitude with 6 square wsve pulses,each lasting 20 milliseconds with 200 millisecond interval)after injection of plasmid DNA(1 μg/μl,in 100 μl PBS)containing enhanced green fluorescence protein(EGFP)gene.Electroporated skins were incisiomdly wounded 24 hours after electroporation.Specimens were harvested at day 2,4,6,14,then EGFP expression in dennis wag observed and quantitatively analyzed with integrated optical density(IOD)followed by H&E staimng.Results Eleetroporation can mediate EGFP expression in epidermis,dermis and pannicuhs muscle.The expression level in dermis Wag the highest at dav 2(IOD=3.50±1.45)and disappeared at day 14.EGFP expression Wag not found in dennis if no electroporation apphed after plasmid injection(IOD=0).Conclusion Electroporation can mediate plagmid gone expression in ineisional wound efficiently and widely.%目的 研究在线性伤口中进行电穿孔基因转染的可行性.方法 于12只Sprague-Dawley大鼠背部皮肤内注射增强型绿色荧光蛋白(EGFP)质粒(1 μg/μl)后,在注射局部进行电穿孔(800 V/cm,6个方波,每个持续20ms,间隔为200ms).电穿孔后24 h在电穿孔部位做线性切口,于第2、4、6、14天收集标本.观察EGFP表达并计算其真皮内积分光密度(IOD).然后进行HE染色.结果 电穿孔使EGFP在表皮、真皮以及肉膜内表达,真皮内表达量在第2天最高(IOD=3.50±1.45),至第14天消失,而单纯质粒注射真皮内无表达(IOD=0).结论 电穿孔能够介导质粒在线性伤口中高效而广泛地表达.

  13. 受体激酶介导的油菜素内酯信号转导途径%Receptor kinases mediated brassinosteroid signal transduction in plants

    Institute of Scientific and Technical Information of China (English)

    卫卓赟; 黎家

    2011-01-01

    油菜素内酯(brassinosteroids,BRs)是一类重要的类固醇激素,参与调控植物生长发育的许多过程.结合应用遗传学、生物化学以及蛋白质组学等研究手段现已基本阐明了BR信号转导的主要过程.BRI1作为受体在细胞表面感知BR,BRI1抑制子BKI1从质膜上解离下来,使BRI1与其共受体BAK1结合.BRI1和BAK1通过顺序磷酸化将BR信号完全激活.活化的BRI1将BSK磷酸化激活,BSK活化BSU1,BSU1将BIN2去磷酸化使其失活,解除BIN2对BES 1/BZR1的抑制功能.PP2A可以将BES1/BZR1去磷酸化激活,又可以将受体BRI1去磷酸化促使其降解.BR信号的传递最终使去磷酸化状态的BES1/BZR1在细胞内累积,激活BR信号通路下游的转录调控.%Brassinosteroids (BRs) are a class of steroidal hormones, playing essential roles in a wide range of processes during plant growth and development. The combined genetics, biochemistry and proteomics approaches have been employed to elucidate the major events in BR signaling pathway, mainly via a series of reversible phosphorylation and dephosphorylation processes. Perception of BR by the BR receptor BRI1 at cell surface leads to the dissociation of BRI1 kinase inhibitor BKI1 from the plasma membrane and association of BRI1 with its co-receptor BAK1. Sequential transphosphorylation of the paired BRJ1/BAK1 receptor kinases completely activates the BR signaling cascade. BRI1 phosphorylation of the BSK kinases leads to the activation of the BSU1 phosphatases, which can function to dephosphorylate and inactivate the BIN2 kinase. PP2A phosphatases play dual roles in mediating BR signaling pathway including dephosphorylating BES1/BZR1 transcription factors to promote their functions and dephosphorylating BRI1 to make it less stable. These events result in the accumulation of unphosphorylated BES1/BZR1 transcription factors in the nucleus and subsequent activation of the downstream transcriptional network.

  14. Chitosan coated polylactic acid nanoparticle-mediated combinatorial delivery of cisplatin and siRNA/Plasmid DNA chemosensitizes cisplatin-resistant human ovarian cancer cells.

    Science.gov (United States)

    Babu, Anish; Wang, Qi; Muralidharan, Ranganayaki; Shanker, Manish; Munshi, Anupama; Ramesh, Rajagopal

    2014-08-01

    Development of resistance toward anticancer drugs results in ineffective therapy leading to increased mortality. Therefore, overriding resistance and restoring sensitivity to anticancer drugs will improve treatment efficacy and reduce mortality. While numerous mechanisms for drug resistance in cancer have previously been demonstrated, recent studies implicate a role for proteasome and the autophagy regulatory protein P62/SQSTM1 (P62) in contributing to drug resistance. Specifically, reduction in the expression of the β5 subunit of the proteasome and/or enhanced P62 protein expression is known to contribute to cancer drug resistance such as cisplatin (CDDP) in ovarian cancer cells. Therefore, we hypothesized that restoration of β5 expression and/or suppression of P62 protein expression in CDDP-resistant ovarian cancer cells will lead to restoration of sensitivity to CDDP and enhanced cell killing. To test our hypothesis we developed a biodegradable multifunctional nanoparticle (MNP) system that codelivered P62siRNA, β5 plasmid DNA, and CDDP and tested its efficacy in CDDP resistant 2008/C13 ovarian cancer cells. MNP consisted of CDDP loaded polylactic acid nanoparticle as inner core and cationic chitosan (CS) consisting of ionically linked P62siRNA (siP62) and/or β5 expressing plasmid DNA (pβ5) as the outer layer. The MNPs were spherical in shape with a hydrodynamic diameter in the range of 280-350 nm, and demonstrated encapsulation efficiencies of 82% and 78.5% for CDDP and siRNA respectively. MNPs efficiently protected the siRNA and showed superior serum stability compared to naked siRNA as measured by gel retardation and spectrophotometry assays. The MNPs successfully delivered siP62 and pβ5 to cause P62 knockdown and restoration of β5 expression in 2008/C13 cells. Combined delivery of siP62, pβ5, and CDDP using the MNPs resulted in a marked reduction in the IC50 value of CDDP in 2008/C13 cells from 125 ± 1.3 μM to 98 ± 0.6 μM (P < 0.05; 21

  15. Plasmid-mediated AmpC: prevalence in community-acquired isolates in Amsterdam, the Netherlands, and risk factors for carriage.

    Directory of Open Access Journals (Sweden)

    E Ascelijn Reuland

    Full Text Available The objective of this study was to determine the prevalence of pAmpC beta-lactamases in community-acquired Gram negative bacteria in the Netherlands, and to identify possible risk factors for carriage of these strains.Fecal samples were obtained from community-dwelling volunteers. Participants also returned a questionnaire for analysis of risk factors. Screening for pAmpC was performed with selective enrichment broth and a selective screening agar. Confirmation of AmpC-production was performed with two double disc combination tests: cefotaxime and ceftazidime with either boronic acid or cloxacillin as inhibitor. Multiplex PCR was used as gold standard for detection of pAmpC. 16S rRNA PCR and AFLP were performed as required, plasmids were identified by PCR-based replicon typing. Questionnaire results were analyzed with SPSS, version 20.0.Fecal samples were obtained from 550 volunteers; mean age 51 years (range: 18-91, 61% were females. pAmpC was present in seven E. coli isolates (7/550, 1.3%, 0.6-2.7 95% CI: six CMY-2-like pAmpC and one DHA. ESBL-encoding genes were found in 52/550 (9.5%, 7.3-12.2 95% CI isolates; these were predominantly blaCTX-M genes. Two isolates had both ESBL and pAmpC. Admission to a hospital in the previous year was the only risk factor we identified.Our data indicate that the prevalence of pAmpC in the community seems still low. However, since pAmpC-producing isolates were not identified as ESBL producers by routine algorithms, there is consistent risk that further increase of their prevalence might go undetected.

  16. Specific binding of the replication protein of plasmid pPS10 to direct and inverted repeats is mediated by an HTH motif.

    Science.gov (United States)

    García de Viedma, D; Serrano-López, A; Díaz-Orejas, R

    1995-01-01

    The initiator protein of the plasmid pPS10, RepA, has a putative helix-turn-helix (HTH) motif at its C-terminal end. RepA dimers bind to an inverted repeat at the repA promoter (repAP) to autoregulate RepA synthesis. [D. García de Viedma, et al. (1996) EMBO J. in press]. RepA monomers bind to four direct repeats at the origin of replication (oriV) to initiate pPS10 replication This report shows that randomly generated mutations in RepA, associated with defficiencies in autoregulation, map either at the putative HTH motif or in its vicinity. These mutant proteins do not promote pPS10 replication and are severely affected in binding to both the repAP and oriV regions in vitro. Revertants of a mutant that map in the vicinity of the HTH motif have been obtained and correspond to a second amino acid substitution far upstream of the motif. However, reversion of mutants that map in the helices of the motif occurs less frequently, at least by an order of magnitude. All these data indicate that the helices of the HTH motif play an essential role in specific RepA-DNA interactions, although additional regions also seem to be involved in DNA binding activity. Some mutations have slightly different effects in replication and autoregulation, suggesting that the role of the HTH motif in the interaction of RepA dimers or monomers with their respective DNA targets (IR or DR) is not the same. Images PMID:8559664

  17. Presence of Glycopeptide-Encoding Plasmids in Enterococcal Isolates from Food and Humans in Denmark

    DEFF Research Database (Denmark)

    Migura, Lourdes Garcia; Valenzuela, Antonio Jesus Sanchez; Jensen, Lars Bogø

    2011-01-01

    developed techniques for classification of plasmids. Replicons associated with sex pheromone-inducible plasmids were detected in all GR E. faecalis, whereas GR Enterococcus faecium contained plasmids known to be widely distributed among enterococci. vanA resistance is common in E. faecium isolates from meat...... and animals in Europe and is rarely found in E. faecalis. This article describes the first characterization of MGE from vanA mediated E. faecalis, thus linking this resistance genotype to pheromone responding plasmids....

  18. Transconjugation and genotyping of the plasmid-mediated AmpC β-lactamase and extended-spectrum β-lactamase genes in Klebsiella pneumoniae

    Institute of Scientific and Technical Information of China (English)

    SHI Wei-feng; ZHOU Jun; QIN Jian-ping

    2009-01-01

    Backgroud AmpC β-lactamases and extended-spectrum β-lactamases (ESBLs) are becoming predominant causes of resistance to third and forth-generation cephalosporins in Klebsiella pneumoniae (K. Pneumoniae). It is very difficult to treat infectious diseases caused by multidrug-resistant K. Pneumoniae. The purpose of the present study was to investigate transconjugation and characteristics of β-lactamase genes in K. Pneumoniae producing AmpC β-lactamases and ESBLs.Methods AmpC β-lactamases were detected by three-dimension test and ESBLs by disc confirmatory test. Minimum inhibitory concentrations (MICs) were determined by agar dilution. Transfer of resistance to EC600 (Rif') was attempted by conjugation in broth and screened on agar containing cefotaxime (2 μg/ml) plus rifampin (1024 μg/ml). The genes encoding AmpC or ESBLs and their transconjugants were detected by PCR and verified by DNA sequencing. Results The resistant rates to ampicillin and piperacillin were 100% in 18 isolates of K. Pneumoniae. However, imipenem was still of great bactericidal activity on K. Pneumoniae, and its MIC50 was 0.5 μg/mL. Eleven β-lactamase genes, including TEM-1, TEM-11, SHV-13, SHV-28, CTX-M-9, CTX-M-22, CTX-M-55, OXA-1, LEN, OKP-6 and DHA-1, were found from 18 isolates. And at least one β-lactamase gene occurred in each isolate. To our surprise, there were six β-lactamase genes in the CZ04 strain. Among 18 isolates of K. Pneumoniae, the partial resistant genes in 8 isolates were conjugated successfully, which had 100% homological sequence with donors by sequence analysis. Compared with donors, 8 transconjugants had attained resistance to most β-lactams, including ampicillin, piperacillin, cefoxitin, cefotaxime and aztreonam, or even amikacin and gentamicin.Conclusions R plasmids can be easily transferred between the resistant and sensitive negative bacilli. It is very difficult to block and prevent the spread of antimicrobial resistance. So more attention should be paid

  19. 伤寒杆菌耐药质粒pRST98介导细菌毒力的研究%Studies on virulence mediated by drug resistant Salmonella typhi R plasmid

    Institute of Scientific and Technical Information of China (English)

    黄瑞; 吴淑燕; 闻玉梅

    2001-01-01

    Objective To explore the possibility of R plasmid (pRST98) encoding resistance to antimicrobial agents in S. typhi mediate virulence to its host bacteria. Methods pRST98 was transferred into a less virulent strain of S. typhimurium RIA for creating a transconjugant pRST98/RIA. The standard S. typhimurium virulence strain SR-11 which carries a 100kb virulence plasmid was used as a positive control, and RIA as a negative one. Infection with S. typhimurium was employed to assess the effect of pRST98 on the virulence of host bacteria by LD50 of peroral (p.o.) and intraperitoneal (i.p.) infection of BALB/c mice. After oral inoculation of the bacteria into BALB/c mice, mesenteric lymph nodes, spleen and liver were examined for quantity of bacteria and for the histopathological changes. The three strains were also studied for their in vitro adhesion and invasion of HEp-2, CHO and HeLa cells. Three chromosomally isogenic strains, i.e., plasmid-containing wild-type S. typhi strain, plasmid artificially cured strain and plasmid-reintroduced into the cured strain were compared for their resistance to the bactericidal activity present in human, rabbit and guinea pig sera. Results The p.o. and i.p. LD50 of pRST98/RIA was 700 and 75 folds less than that of RIA. The bacteria counting in mesenteric lymph nodes, spleen and liver were more (P<0.05) and more severe histopathological changes were observed in pRST98 harboring S. typhimurium. However, pRST98 did not affect the adhesion and invasion of S. typhimurium to the cells. Experiments showed that pRST98 mediated resistance to serum bactericidal effect in host bacteria (P<0.05). Conclusion This is the first report about a plasmid carrying genes coding for drug resistance and virulence in S. typhi. The data presented here suggested that novel virulence gene or related sequences, which are important in the pathogenesis of Salmonella infection could exist on pRST98.%目的研究伤寒杆菌耐药质粒pRST98能否介导

  20. 成骨细胞膜脂筏在TNFR1介导信号转导中的作用%The role of lipid raft in TNFR1-media-ted signal transduction in osteoblasts

    Institute of Scientific and Technical Information of China (English)

    王海芳; Fredrick M.Pavalko; 梅其炳

    2011-01-01

    目的:初步研究细胞膜脂筏在MC3T3成骨细胞TNFRl介导信号转导中的作用.方法:应用MCD(10 g/L,60 min)消耗细胞膜胆固醇,以TNF-α(10 μg/L)刺激MC313成骨细胞0、5、10、15、30 rain或以TNF-α+CHX(10ms/L)处理4 h诱导凋亡,以SDS-PAGE/Western blot法检测IKBot、ph-AKT、ph-ERK、ph-p38及caspase-3活性片段表达水平的变化,分析膜胆固醇在TNFRI介导信号转导中的作用.结果:MCD(10 g/L)处理60 min可将膜胆固醇水平减少至约35%.降低膜胆固醇水平不影响TNFRI介导的IKBa信号,但显著抑制TNFRl介导的AKT磷酸化激活;不影响TNFRl介导的caspase03活化和细胞凋亡;也不影响TNFRI介导的ERK和p38磷酸化激活.结论:消耗膜胆固醇可破坏脂筏结构,提示成骨细胞膜脂筏在TNFR1介导AKT激活的过程中发挥重要作用,而TNFR1介导的NF-kB和ERK、p38及凋亡信号通路的激活并不依赖脂筏.%AIM: To investigate the role of membrane cholesterol in TNFR1-mediated signal transduction in osteoblastic MC3T3 cells.METHODS: MCD binds cholesterol specifically and was commonly used to deplete cholesterol from cell plasma membrane.MC3T3 cells were serumstarved for 22 h, treated with MCD ( 10 g/L) for 60 min followed by TNF-α ( 10 μg/L) for 0, 5, 10, 15 or 30 min, or TNF-α plus CHX (10 mg/L) for 4 h to induce apoptosis,then TNFR1-mediated IκBα degradation, phosphorylation of AKT, ERK or p38, and processing of caspase-3 were analyzed by using SDS-PAGE/Western blotting method.RESULTS: MC3T3 cell membrane cholesterol level was reduced to 35% within 60 min by MCD ( 10 g/L).Reduction of MC3T3 cell surface cholesterol dramatically inhibited TNFR1-mediated AKT phosphorylation, while did not affect the degradation of IκBα, activation of ERK or p38, and processing of caspase-3 induced by TNF-α.CONCLUSION: Cholesterol depletion can destruct lipid rafts; therefore our results suggest that lipid raft is essential for TNFR1-mediated AKT phosphorylation, but is

  1. Highly active microbial phosphoantigen induces rapid yet sustained MEK/Erk- and PI-3K/Akt-mediated signal transduction in anti-tumor human gammadelta T-cells.

    Directory of Open Access Journals (Sweden)

    Daniel V Correia

    Full Text Available BACKGROUND: The unique responsiveness of Vgamma9Vdelta2 T-cells, the major gammadelta subset of human peripheral blood, to non-peptidic prenyl pyrophosphate antigens constitutes the basis of current gammadelta T-cell-based cancer immunotherapy strategies. However, the molecular mechanisms responsible for phosphoantigen-mediated activation of human gammadelta T-cells remain unclear. In particular, previous reports have described a very slow kinetics of activation of T-cell receptor (TCR-associated signal transduction pathways by isopentenyl pyrophosphate and bromohydrin pyrophosphate, seemingly incompatible with direct binding of these antigens to the Vgamma9Vdelta2 TCR. Here we have studied the most potent natural phosphoantigen yet identified, (E-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP, produced by Eubacteria and Protozoa, and examined its gammadelta T-cell activation and anti-tumor properties. METHODOLOGY/PRINCIPAL FINDINGS: We have performed a comparative study between HMB-PP and the anti-CD3epsilon monoclonal antibody OKT3, used as a reference inducer of bona fide TCR signaling, and followed multiple cellular and molecular gammadelta T-cell activation events. We show that HMB-PP activates MEK/Erk and PI-3K/Akt pathways as rapidly as OKT3, and induces an almost identical transcriptional profile in Vgamma9(+ T-cells. Moreover, MEK/Erk and PI-3K/Akt activities are indispensable for the cellular effects of HMB-PP, including gammadelta T-cell activation, proliferation and anti-tumor cytotoxicity, which are also abolished upon antibody blockade of the Vgamma9(+ TCR Surprisingly, HMB-PP treatment does not induce down-modulation of surface TCR levels, and thereby sustains gammadelta T-cell activation upon re-stimulation. This ultimately translates in potent human gammadelta T-cell anti-tumor function both in vitro and in vivo upon transplantation of human leukemia cells into lymphopenic mice, CONCLUSIONS/SIGNIFICANCE: The development of

  2. Signal transduction in the footsteps of goethe and schiller.

    Science.gov (United States)

    Friedrich, Karlheinz; Lindquist, Jonathan A; Entschladen, Frank; Serfling, Edgar; Thiel, Gerald; Kieser, Arnd; Giehl, Klaudia; Ehrhardt, Christina; Feller, Stephan M; Ullrich, Oliver; Schaper, Fred; Janssen, Ottmar; Hass, Ralf

    2009-02-04

    The historical town of Weimar in Thuringia, the "green heart of Germany" was the sphere of Goethe and Schiller, the two most famous representatives of German literature's classic era. Not yet entirely as influential as those two cultural icons, the Signal Transduction Society (STS) has nevertheless in the last decade established within the walls of Weimar an annual interdisciplinary Meeting on "Signal Transduction - Receptors, Mediators and Genes", which is well recognized as a most attractive opportunity to exchange results and ideas in the field.The 12th STS Meeting was held from October 28 to 31 and provided a state-of-the-art overview of various areas of signal transduction research in which progress is fast and discussion lively. This report is intended to share with the readers of CCS some highlights of the Meeting Workshops devoted to specific aspects of signal transduction.

  3. Signal Transduction in the Footsteps of Goethe and Schiller

    Directory of Open Access Journals (Sweden)

    Feller Stephan M

    2009-02-01

    Full Text Available Abstract The historical town of Weimar in Thuringia, the "green heart of Germany" was the sphere of Goethe and Schiller, the two most famous representatives of German literature's classic era. Not yet entirely as influential as those two cultural icons, the Signal Transduction Society (STS has nevertheless in the last decade established within the walls of Weimar an annual interdisciplinary Meeting on "Signal Transduction – Receptors, Mediators and Genes", which is well recognized as a most attractive opportunity to exchange results and ideas in the field. The 12th STS Meeting was held from October 28 to 31 and provided a state-of-the-art overview of various areas of signal transduction research in which progress is fast and discussion lively. This report is intended to share with the readers of CCS some highlights of the Meeting Workshops devoted to specific aspects of signal transduction.

  4. Mechanisms of plasmid segregation: have multicopy plasmids been overlooked?

    Science.gov (United States)

    Million-Weaver, Samuel; Camps, Manel

    2014-09-01

    Plasmids are self-replicating pieces of DNA typically bearing non-essential genes. Given that plasmids represent a metabolic burden to the host, mechanisms ensuring plasmid transmission to daughter cells are critical for their stable maintenance in the population. Here we review these mechanisms, focusing on two active partition strategies common to low-copy plasmids: par systems type I and type II. Both involve three components: an adaptor protein, a motor protein, and a centromere, which is a sequence area in the plasmid that is recognized by the adaptor protein. The centromere-bound adaptor nucleates polymerization of the motor, leading to filament formation, which can pull plasmids apart (par I) or push them towards opposite poles of the cell (par II). No such active partition mechanisms are known to occur in high copy number plasmids. In this case, vertical transmission is generally considered stochastic, due to the random distribution of plasmids in the cytoplasm. We discuss conceptual and experimental lines of evidence questioning the random distribution model and posit the existence of a mechanism for segregation in high copy number plasmids that moves plasmids to cell poles to facilitate transmission to daughter cells. This mechanism would involve chromosomally-encoded proteins and the plasmid origin of replication. Modulation of this proposed mechanism of segregation could provide new ways to enhance plasmid stability in the context of recombinant gene expression, which is limiting for large-scale protein production and for bioremediation.

  5. Plasmid-to-plasmid recombination in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Balganesh, M.; Setlow, J.K.

    1986-01-01

    No recombination between plasmids was observed after conjugal transfer of a plasmid into a cell carrying another plasmid. Two types of such recombination took place after transformation, one type being Rec/sup +/ dependent and suggesting a preferred site of recombination. The other much rarer type was at least partially Rec/sup +/ independent.

  6. 质粒介导细菌对喹诺酮类抗菌药物的耐药机制%Plasmid Mediated Mechanism of Bacterial Resistance to Quinolones

    Institute of Scientific and Technical Information of China (English)

    马晓波; 宋秀宇

    2009-01-01

    喹诺酮类抗菌药物是临床最为常用的抗菌药物之一,但其耐药的问题也日益严重。细菌对喹诺酮类抗菌药物的耐药机制有:(1)染色体介导的耐药,包括药物作用靶位的改变(特别是喹诺酮耐药决定区(QRDR)的基因突变]、外膜通透性的下降、主动外排作用。(2)质粒介导的耐药。质粒介导的喹诺酮类耐药(plasmid mediated quinolone resistance,PMQR)由qnr(quinolone resistance,后来更名为qnrA)、aac-(6’)-Ⅰb-cr及qepA(quinolone efflux proteinA)等参与。PMQR机制的发现使人们对细菌耐喹诺酮类药物的机制有了新的认识。

  7. In vitro activities and detection performances of cefmetazole and flomoxef for extended-spectrum β-lactamase and plasmid-mediated AmpC β-lactamase-producing Enterobacteriaceae.

    Science.gov (United States)

    Matsumura, Yasufumi; Yamamoto, Masaki; Nagao, Miki; Tanaka, Michio; Takakura, Shunji; Ichiyama, Satoshi

    2016-04-01

    To investigate the in vitro activities of cephamycins (cefmetazole and flomoxef) for extended-spectrum β-lactamase (ESBL)- and plasmid-mediated AmpC β-lactamase (pAmpC)-producing Enterobacteriaceae, a total of 574 third-generation cephalosporin-resistant clinical isolates were collected at a Japanese multicenter study. PCR and sequencing identified 394 isolates with only ESBL genes, 63 isolates with only pAmpC genes, and 6 isolates with both ESBL and pAmpC genes. blaCTX-M types predominated 95.5% of the ESBL genes, and blaCMY-2 predominated 91.3% of the pAmpC genes. The MIC50/90 values of cefmetazole and flomoxef were ≤ 1/4 and ≤ 1/≤ 1 μg/mL for isolates with only ESBL genes, respectively, and 16/>16 and 8/16 μg/mL for isolates with only pAmpC genes, respectively. Flomoxef ≥ 4 μg/mL had the best screening performance for the detection of isolates with pAmpC genes. Flomoxef had better in vitro activities against ESBL-producing Enterobacteriaceae and provided a clearer distinction between ESBL and pAmpC-producing Enterobacteriaceae compared to cefmetazole.

  8. Cefotaxime for the detection of extended-spectrum β-lactamase or plasmid-mediated AmpC β-lactamase and clinical characteristics of cefotaxime-non-susceptible Escherichia coli and Klebsiella pneumoniae bacteraemia.

    Science.gov (United States)

    Matsumura, Y; Yamamoto, M; Matsushima, A; Nagao, M; Ito, Y; Takakura, S; Ichiyama, S

    2012-08-01

    We investigated the performance of cefotaxime for the detection of extended-spectrum β-lactamase (ESBL) or plasmid-mediated AmpC β-lactamase (pAmpC) and the clinical characteristics of cefotaxime-non-susceptible Escherichia coli or Klebsiella pneumoniae (CTXNS-EK) bacteraemia. All of the consecutive bloodstream isolates between 2005 and 2010 in a Japanese university hospital were characterised using polymerase chain reaction (PCR). Risk factors and outcomes of CTXNS-EK were analysed by multivariate logistic regression analysis. We identified 58 CTXNS-EK (15.6%) from 249 E. coli and 122 K. pneumoniae. Cefotaxime with a minimum inhibitory concentration (MIC) of >1 μg/mL had a sensitivity of 98.3% and a specificity of 99.7% for the detection of ESBL or pAmpC. CTXNS-EK had increased from 4.5% in 2005 to 23% in 2009. Risk factors for CTXNS-EK were previous isolation of multidrug-resistant bacteria, use of oxyimino-cephalosporins or fluoroquinolones, and high Sequential Organ Failure Assessment (SOFA) score. Patients with CTXNS-EK bacteraemia less frequently received appropriate empirical therapy than patients with cefotaxime-susceptible EK bacteraemia (81% vs. 97%, pcefotaxime alone can identify ESBL or pAmpC producers. CTXNS-EK is an important and increasingly prevalent bacteraemia pathogen.

  9. Cell-penetrating Peptide YARA-mediated Transduction of Enhanced Green Fluorescent Protein into Human Vascular Smooth Muscle Cells%YARA介导增强型绿色荧光蛋白穿透人血管平滑肌细胞

    Institute of Scientific and Technical Information of China (English)

    陈思思; 王家宁; 黄永章; 郭凌郧; 孔霞

    2012-01-01

    Objective To investigate the penetrating ability of fusion protein YARA-EGFP with human vascular smooth muscle cells (HVSMC). Methods The prokaryotic expression plastnids YARA-EGFP was constructed and transformed into E. coli BL21 (DE3) to express fusion protein YARA-EGFP. The fusion protein YARA-EGFP was purified with Ni2 + -resin affinity chromatography and transduced into HVSMC. Results YARA-EGFP fusion protein could transduce into HVSMC and distribute in cytoplasm and nucleus after 6 h incubation. Conclusion The successful expression and purification of YARA-EGFP fusion protein can transduce into human vascular smooth muscle cells. This study provides a basis for the research on transduction of antiproliferative proteins such as p27 and p21 mediated by the cell-penetrating peptide, YARA,in protein therapy for the diabetic vascular diseases.%目的:研究细胞穿透肽YARA介导大分子蛋白穿透人血管平滑肌细施(human vascular smooth musle cells,HVSMC)细胞膜的能力.方法:用基因工程的方法制备并纯化YARA-EGFP融合蛋白,将其和培养的人平滑肌细胞共同孵育,在荧光显微镜下直接观察YARA介导目的蛋白EGFP转导入人平滑肌细胞的能力.结果:荧光显微镜下观察到,荧光蛋白穿透细胞膜进入并分布在入血管平滑肌细胞内.结论:YARA能有效携带目的蛋白进入人血管平滑肌细胞,这为将来用细胞穿透肽YARA介导有生物活性的大分子抗血管平滑肌细胞增殖,进行糖尿病血管病变的蛋白治疗奠定了基础.

  10. DNA sequence analysis of plasmids from multidrug resistant Salmonella enterica serotype Heidelberg isolates.

    Directory of Open Access Journals (Sweden)

    Jing Han

    Full Text Available Salmonella enterica serovar Heidelberg is among the most detected serovars in swine and poultry, ranks among the top five serotypes associated with human salmonellosis and is disproportionately associated with invasive infections and mortality in humans. Salmonella are known to carry plasmids associated with antimicrobial resistance and virulence. To identify plasmid-associated genes in multidrug resistant S. enterica serovar Heidelberg, antimicrobial resistance plasmids from five isolates were sequenced using the 454 LifeSciences pyrosequencing technology. Four of the isolates contained incompatibility group (Inc A/C multidrug resistance plasmids harboring at least eight antimicrobial resistance genes. Each of these strains also carried a second resistance plasmid including two IncFIB, an IncHI2 and a plasmid lacking an identified Inc group. The fifth isolate contained an IncI1 plasmid, encoding resistance to gentamicin, streptomycin and sulfonamides. Some of the IncA/C plasmids lacked the full concert of transfer genes and yet were able to be conjugally transferred, likely due to the transfer genes carried on the companion plasmids in the strains. Several non-IncA/C resistance plasmids also carried putative virulence genes. When the sequences were compared to previously sequenced plasmids, it was found that while all plasmids demonstrated some similarity to other plasmids, they were unique, often due to differences in mobile genetic elements in the plasmids. Our study suggests that Salmonella Heidelberg isolates harbor plasmids that co-select for antimicrobial resistance and virulence, along with genes that can mediate the transfer of plasmids within and among other bacterial isolates. Prevalence of such plasmids can complicate efforts to control the spread of S. enterica serovar Heidelberg in food animal and human populations.

  11. Countrywide dissemination of a DHA-1-type plasmid-mediated AmpC β-lactamase-producing Klebsiella pneumoniae ST11 international high-risk clone in Hungary, 2009-2013.

    Science.gov (United States)

    Kis, Zoltán; Tóth, Ákos; Jánvári, Laura; Damjanova, Ivelina

    2016-09-01

    The first plasmid-mediated AmpC β-lactamase-producing Klebsiella pneumoniae (pAmpC KP) isolate was detected in December 2009 in Hungary. Hungarian microbiological laboratories were asked to send all KP strains showing cefoxitin resistance and decreased susceptibility or resistance to any third-generation cephalosporins to the Reference Laboratories at the National Center for Epidemiology. Investigation was conducted in order to outline spatio-temporal distribution and genetic characterization of pAmpC-KP isolates in Hungary. Between December 2009 and December 2013, 312 consecutive KP clinical isolates were confirmed as producing pAmpCs. All isolates showed resistance to third-generation cephalosporins, aminoglycosides and fluoroquinolones, and 77 % were non-susceptible to at least one carbapenem. Analysis of β-lactamase genes showed blaDHA-1 in all and additionally blaCTX-M-15 in 90 % of isolates. PFGE typing revealed 12 pulsotypes; of these, KP053 (262/312) and KP070 (38/312) belonged to sequence type ST11 and comprised 96 % of the isolates. The blaDHA-1 and blaCTX-M-15 co-producing KP053/ST11 clone affected 234 patients and spread to 55 healthcare centres across Hungary during the study period. Three KP053 isolates were also resistant to colistin. In two of these, the mgrB gene was truncated by IS10R, while in the third isolate, insertional inactivation of mgrB by ISKPn14 was identified. Hungary is the first European country showing endemic spread of blaDHA-1 facilitated by the international high-risk clone ST11. The rapid countrywide spread of this multidrug-resistant clone seriously endangers Hungarian healthcare facilities and warrants strengthening of infection control practices and prudent use of carbapenems and colistin.

  12. Molecular characterization of extended-spectrum β-lactamase, plasmid-mediated AmpC cephalosporinase and carbapenemase genes among Enterobacteriaceae isolates in five medical centres of East and West Azerbaijan, Iran.

    Science.gov (United States)

    Sadeghi, Mohammad Reza; Ghotaslou, Reza; Akhi, Mohammad Taghi; Asgharzadeh, Mohammad; Hasani, Alka

    2016-11-01

    Very little is known about the occurrence and various types of extended-spectrum β-lactamase (ESBL), AmpC and carbapenemase in Iran. The aims of this study were to determine the prevalence of ESBLs, AmpCs and carbapenemase genes among Enterobacteriaceae in Azerbaijan and to characterize the genetic composition of the detected genes. A total of 307 Enterobacteriaceae isolates, recovered from five medical centres, were screened for ESBL, AmpC and carbapenemase activities by the disc diffusion method and phenotypic confirmatory tests. The 162 selected strains (third-generation cephalosporins, cefoxitin- or carbapenem-resistant strains with positive or negative phenotypic confirmatory tests) were selected for multiplex PCR screening for β-lactamase genes, and detected genes were confirmed by sequencing. Of 162 isolates, 156 harboured 1 to 6 β-lactamase genes of 41 types. The most prevalent genes were blaTEM-1 (29.9 %), followed by blaCTX-M-15 (25.7 %). Plasmid-mediated AmpC was detected in 66 strains (21.5 %) alone or in combination with other genes. Carbapenemase-encoding genes were detected in 18 strains (5.8 %) of 27 carbapenem-non-susceptible isolates including 11, 7, 3 and 1 cases of blaOXA-48, blaNDM-1, blaKPC-2 and blaKPC-3 genes, respectively. Interestingly, 148 (94.8 %) of 156 strains with any β-lactamase gene were found to have a multidrug-resistant pattern. The rate of resistance to β-lactams and multidrug-resistant Enterobacteriaceae is high in Azerbaijan. All positive strains for carbapenemase genes were resistant to all β-lactams. The present study reveals the high occurrence of CTX-M-type ESBLs followed by TEM and SHV variants among Enterobacteriaceae isolates. East Azerbaijan seems to be an alarming focus for OXA-48, NDM-1 and KPC dissemination.

  13. Characterization of Plasmid-Mediated Quinolone Resistance Determinants in High-Level Quinolone-Resistant Enterobacteriaceae Isolates from the Community: First Report of qnrD Gene in Algeria.

    Science.gov (United States)

    Yanat, Betitera; Machuca, Jesús; Díaz-De-Alba, Paula; Mezhoud, Halima; Touati, Abdelaziz; Pascual, Álvaro; Rodríguez-Martínez, José-Manuel

    2017-01-01

    The objective was to assess the prevalence of plasmid-mediated quinolone resistance (PMQR)-producing isolates in a collection of quinolone-resistant Enterobacteriaceae of community origin isolated in Bejaia, Algeria. A total of 141 nalidixic acid-resistant Enterobacteriaceae community isolates were collected in Bejaia (Northern Algeria) and screened for PMQR genes using polymerase chain reaction (PCR). For PMQR-positive strains, antimicrobial susceptibility testing was performed by broth microdilution and disk diffusion. Mutations in the quinolone resistance-determining regions of the target genes, gyrA and parC, were detected with a PCR-based method and sequencing. Southern blotting, conjugation and transformation assays and molecular typing by pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing were also performed. The prevalence of PMQR-producing Enterobacteriaceae isolates was 13.5% (19/141); 11 of these isolates produced Aac(6')-Ib-cr and 8 were qnr-positive (4 qnrB1-like, 2 qnrS1-like, and 2 qnrD1-like), including the association with aac(6')-Ib-cr gene in three cases. PMQR gene transfer by conjugation was successful in 6 of 19 isolates tested. PFGE revealed that most of the PMQR-positive Escherichia coli isolates were unrelated, except for two groups comprising two and four isolates, respectively, including the virulent multidrug-resistant clone E. coli ST131 that were clonally related. Our findings indicate that PMQR determinants are prevalent in Enterobacteriaceae isolates from the community studied. We describe the first report of the qnrD gene in Algeria.

  14. Prevalence of plasmid-mediated AmpC β-lactamase-producing Escherichia coli and spread of the ST131 clone among extended-spectrum β-lactamase-producing E. coli in Japan.

    Science.gov (United States)

    Matsumura, Yasufumi; Yamamoto, Masaki; Higuchi, Takeshi; Komori, Toshiaki; Tsuboi, Fusayuki; Hayashi, Akihiko; Sugimoto, Yoshihisa; Hotta, Gou; Matsushima, Aki; Nagao, Miki; Takakura, Shunji; Ichiyama, Satoshi

    2012-08-01

    In 2010, a total of 1327 clinical Escherichia coli isolates from five hospitals in the Kyoto and Shiga regions of Japan were analysed by PCR. The prevalences of plasmid-mediated AmpC β-lactamase (pAmpC)-producers, extended-spectrum β-lactamase (ESBL)-producers and co-producers of pAmpC and ESBL were 1.7%, 9.7% and 0.3%, respectively. Less than one-half of the pAmpC-producers were reported to be resistant to third-generation cephalosporins, cephamycins and β-lactam/β-lactam inhibitors using the old 2009 Clinical and Laboratory Standards Institute (CLSI) breakpoints. CMY-2 was the most prevalent pAmpC type (95%), and CTX-M-14 (38%), CTX-M-15 (26%) and CTX-M-27 (19%) were the most prevalent ESBL types. The worldwide O25b-ST131-B2 clone accounted for 11% of pAmpC-producers and 41% of ESBL-producers. The O25b-ST131-B2 clone was characterised by a CTX-M-27- or CTX-M-15-type ESBL and ciprofloxacin-non-susceptibility with quadruple mutations in the quinolone resistance-determining regions (S83L and D87N in GyrA and S80I and E84V in ParC). A significant proportion of pAmpC-producers and the O25b-ST131-B2 clone were found in Japan by a recent regional surveillance programme.

  15. TLR-4介导的鹅β-防御素1抗肠炎沙门菌感染的信号传导机制初探%Preliminary Exploration of the Signal Transduction Mechanisms of the Goose Beta Defensin 1 Resistance to Salmonella Enteritidis Infection Mediated by Toll-like Receptor 4

    Institute of Scientific and Technical Information of China (English)

    张名岳; 张可心; 辛胜男; 韩宗玺; 邵昱昊; 刘晓丽; 刘胜旺; 马得莹

    2012-01-01

    %) with ostrich AvBDl. The purified recombinant goose AvBDl exhibited extensive antimicrobial activity against twelve investigated bacteria strains, including Gram-positive and Gram-negative. At high salt ions conditions, antimicrobial activity of recombinant goose AvBDl protein against both Staphylococcus aureus and Proteus mirabilis decreased significantly and hemolysis activity of the recombinant protein was extremely low. Experimental results show that Salmonella enteritidis did induced goose AvBDl gene expression in bone marrow, at the same time goose AvBDl resistance to Salmonella enteritidis infection induces expression of TLR4 may be mediated signal transduction

  16. Persistence Mechanisms of Conjugative Plasmids

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Hansen, Lars H.; Sørensen, Søren Johannes

    2009-01-01

    Are plasmids selfish parasitic DNA molecules or an integrated part of the bacterial genome? This chapter reviews the current understanding of the persistence mechanisms of conjugative plasmids harbored by bacterial cells and populations. The diversity and intricacy of mechanisms affecting...... the successful propagation and long-term continued existence of these extra-chromosomal elements is extensive. Apart from the accessory genetic elements that may provide plasmid-harboring cells a selective advantage, special focus is placed on the mechanisms conjugative plasmids employ to ensure their stable...... maintenance in the host cell. These importantly include the ability to self-mobilize in a process termed conjugative transfer, which may occur across species barriers. Other plasmid stabilizing mechanisms include the multimer resolution system, active partitioning, and post-segregational-killing of plasmid...

  17. Partition-associated incompatibility caused by random assortment of pure plasmid clusters

    DEFF Research Database (Denmark)

    Ebersbach, Gitte; Sherratt, David J; Gerdes, Kenn;

    2005-01-01

    Summary Bacterial plasmids and chromosomes encode centromere-like partition loci that actively segregate DNA before cell division. The molecular mechanism behind DNA segregation in bacteria is largely unknown. Here we analyse the mechanism of partition-associated incompatibility for plasmid pB171......-lived pairing of plasmids. Instead, pure R1 and F foci were positioned along the length of the cell, and in a random order. Thus, our results raise the possibility that partition-mediated plasmid incompatibility is not caused by pairing of heterologous plasmids but instead by random positioning of pure plasmid...... clusters along the long axis of the cell. The strength of the incompatibility was correlated with the capability of the plasmids to compete for the mid-cell position....

  18. Molecular basis of mechanosensory transduction

    Science.gov (United States)

    Gillespie, Peter G.; Walker, Richard G.

    2001-09-01

    Mechanotransduction - a cell's conversion of a mechanical stimulus into an electrical signal - reveals vital features of an organism's environment. From hair cells and skin mechanoreceptors in vertebrates, to bristle receptors in flies and touch receptors in worms, mechanically sensitive cells are essential in the life of an organism. The scarcity of these cells and the uniqueness of their transduction mechanisms have conspired to slow molecular characterization of the ensembles that carry out mechanotransduction. But recent progress in both invertebrates and vertebrates is beginning to reveal the identities of proteins essential for transduction.

  19. Conjugative plasmids of Neisseria gonorrhoeae.

    Directory of Open Access Journals (Sweden)

    Emilia Pachulec

    Full Text Available Many clinical isolates of the human pathogen Neisseria gonorrhoeae contain conjugative plasmids. The host range of these plasmids is limited to Neisseria species, but presence of a tetracycline (tetM determinant inserted in several of these plasmids is an important cause of the rapid spread of tetracycline resistance. Previously plasmids with different backbones (Dutch and American type backbones and with and without different tetM determinants (Dutch and American type tetM determinants have been identified. Within the isolates tested, all plasmids with American or Dutch type tetM determinants contained a Dutch type plasmid backbone. This demonstrated that tetM determinants should not be used to differentiate between conjugal plasmid backbones. The nucleotide sequences of conjugative plasmids with Dutch type plasmid backbones either not containing the tetM determinant (pEP5233 or containing Dutch (pEP5289 or American (pEP5050 type tetM determinants were determined. Analysis of the backbone sequences showed that they belong to a novel IncP1 subfamily divergent from the IncP1alpha, beta, gamma, delta and epsilon subfamilies. The tetM determinants were inserted in a genetic load region found in all these plasmids. Insertion was accompanied by the insertion of a gene with an unknown function, and rearrangement of a toxin/antitoxin gene cluster. The genetic load region contains two toxin/antitoxins of the Zeta/Epsilon toxin/antitoxin family previously only found in Gram positive organisms and the virulence associated protein D of the VapD/VapX toxin/antitoxin family. Remarkably, presence of VapX of pJD1, a small cryptic neisserial plasmid, in the acceptor strain strongly increased the conjugation efficiency, suggesting that it functions as an antitoxin for the conjugative plasmid. The presence of the toxin and antitoxin on different plasmids might explain why the host range of this IncP1 plasmid is limited to Neisseria species. The isolated plasmids

  20. Plasmid Rolling-Circle Replication.

    Science.gov (United States)

    Ruiz-Masó, J A; MachóN, C; Bordanaba-Ruiseco, L; Espinosa, M; Coll, M; Del Solar, G

    2015-02-01

    Plasmids are DNA entities that undergo controlled replication independent of the chromosomal DNA, a crucial step that guarantees the prevalence of the plasmid in its host. DNA replication has to cope with the incapacity of the DNA polymerases to start de novo DNA synthesis, and different replication mechanisms offer diverse solutions to this problem. Rolling-circle replication (RCR) is a mechanism adopted by certain plasmids, among other genetic elements, that represents one of the simplest initiation strategies, that is, the nicking by a replication initiator protein on one parental strand to generate the primer for leading-strand initiation and a single priming site for lagging-strand synthesis. All RCR plasmid genomes consist of a number of basic elements: leading strand initiation and control, lagging strand origin, phenotypic determinants, and mobilization, generally in that order of frequency. RCR has been mainly characterized in Gram-positive bacterial plasmids, although it has also been described in Gram-negative bacterial or archaeal plasmids. Here we aim to provide an overview of the RCR plasmids' lifestyle, with emphasis on their characteristic traits, promiscuity, stability, utility as vectors, etc. While RCR is one of the best-characterized plasmid replication mechanisms, there are still many questions left unanswered, which will be pointed out along the way in this review.

  1. Meeting Report: Teaching Signal Transduction

    Science.gov (United States)

    Kramer, IJsbrand; Thomas, Geraint

    2006-01-01

    In July, 2005, the European Institute of Chemistry and Biology at the campus of the University of Bordeaux, France, hosted a focused week of seminars, workshops, and discussions around the theme of "teaching signal transduction." The purpose of the summer school was to offer both junior and senior university instructors a chance to reflect on the…

  2. Conjugation efficiency depends on intra and intercellular interactions between distinct plasmids: Plasmids promote the immigration of other plasmids but repress co-colonizing plasmids.

    Science.gov (United States)

    Gama, João Alves; Zilhão, Rita; Dionisio, Francisco

    2017-08-24

    Conjugative plasmids encode the genes responsible for the synthesis of conjugative pili and plasmid transfer. Expression of the conjugative machinery (including conjugative pili) may be costly to bacteria, not only due to the energetic/metabolic cost associated with their expression but also because they serve as receptors for certain viruses. Consequently, the presence of two plasmids in the same cell may be disadvantageous to each plasmid, because they may impose a higher fitness cost on the host. Therefore, plasmids may encode mechanisms to cope with co-resident plasmids. Moreover, it is possible that the transfer rate of a plasmid is affected by the presence of a distinct plasmid in the recipient cell. In this work, we measured transfer rates of twelve natural plasmids belonging to seven incompatibility groups in three situations, namely when: (i) donor cells contain a plasmid and recipient cells are plasmid-free; (ii) donor cells contain two unrelated plasmids and recipient cells are plasmid-free; and (iii) half of the cells contain a given plasmid and the other half contain another, unrelated, plasmid. In the third situation, recipient cells of a plasmid are the donor cells of the other plasmid. We show that there are more negative interactions (reduction of a plasmid's conjugative efficiency) between plasmids if they reside in the same cell than if they reside in different cells. However, if plasmids interacted intercellularly, the transfer rate of one of the plasmids was often higher (when the unrelated conjugative plasmid was present in the recipient cell) than if the recipient cell was plasmid-free - a positive effect. Experimental data retrieved from the study of mutant plasmids not expressing conjugative pili on the cell surface suggest that positive effects result from a higher efficiency of mating pair formation. Overall, our results suggest that negative interactions are significantly more frequent when plasmids occupy the same cell. Such

  3. Plasmid recombination in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.

    1982-01-01

    DNA recombination in exponential phase and competent Haemophilus influenzae was measured by an electron microscopic assay that relies on the conversion of plasmid RSF0885 monomers into multimeric forms. Dimer circles were present at a frequency of 2% in plasmid preparations from competent Rd (wild-type) cells; multimers were present at a frequency of 0.2% in preparations from exponential phase cells. Thus, plasmid recombination was stimulated in competent cells. Multimer formation occurred efficiently in cells of the transformation defective mutant rec2, implying that the rec2 gene product is not required for plasmid recombination. However, the absence of multimer plasmids in preparations from competent cells of the transformation defective mutant rec1 suggests that the rec1 gene product is required. Digestion of purified plasmids with restriction endonuclease PvuII, which makes a single cut in the monomer, revealed the presence of recombination intermediates composed of two linear plasmids joined to form two pairs of arms resembling the Greek letter chi. Length measurements of these arms taken from a population of recombination intermediates gave evidence that the plasmids were joined at sites of homology. The distributions of individual DNA strands, at the intersections of the four arms, could be resolved in some recombination intermediates and were of two types. The first type of junction appeared as a single-stranded arm appended to each corner. The second type of junction consisted of a single strand of DNA linking the two linear plasmids at a site of homology. The single-stranded linker was frequently situated at the edge of a short gap on one of the plasmids in the pair. The fine structures of the recombinational joints have been interpreted in terms of previously proposed models of recombination.

  4. Plasmid Classification in an Era of Whole-Genome Sequencing: Application in Studies of Antibiotic Resistance Epidemiology

    Science.gov (United States)

    Orlek, Alex; Stoesser, Nicole; Anjum, Muna F.; Doumith, Michel; Ellington, Matthew J.; Peto, Tim; Crook, Derrick; Woodford, Neil; Walker, A. Sarah; Phan, Hang; Sheppard, Anna E.

    2017-01-01

    Plasmids are extra-chromosomal genetic elements ubiquitous in bacteria, and commonly transmissible between host cells. Their genomes include variable repertoires of ‘accessory genes,’ such as antibiotic resistance genes, as well as ‘backbone’ loci which are largely conserved within plasmid families, and often involved in key plasmid-specific functions (e.g., replication, stable inheritance, mobility). Classifying plasmids into different types according to their phylogenetic relatedness provides insight into the epidemiology of plasmid-mediated antibiotic resistance. Current typing schemes exploit backbone loci associated with replication (replicon typing), or plasmid mobility (MOB typing). Conventional PCR-based methods for plasmid typing remain widely used. With the emergence of whole-genome sequencing (WGS), large datasets can be analyzed using in silico plasmid typing methods. However, short reads from popular high-throughput sequencers can be challenging to assemble, so complete plasmid sequences may not be accurately reconstructed. Therefore, localizing resistance genes to specific plasmids may be difficult, limiting epidemiological insight. Long-read sequencing will become increasingly popular as costs decline, especially when resolving accurate plasmid structures is the primary goal. This review discusses the application of plasmid classification in WGS-based studies of antibiotic resistance epidemiology; novel in silico plasmid analysis tools are highlighted. Due to the diverse and plastic nature of plasmid genomes, current typing schemes do not classify all plasmids, and identifying conserved, phylogenetically concordant genes for subtyping and phylogenetics is challenging. Analyzing plasmids as nodes in a network that represents gene-sharing relationships between plasmids provides a complementary way to assess plasmid diversity, and allows inferences about horizontal gene transfer to be made. PMID:28232822

  5. In Silico Detection and Typing of Plasmids using PlasmidFinder and Plasmid Multilocus Sequence Typing

    DEFF Research Database (Denmark)

    Carattoli, Alessandra; Zankari, Ea; García-Fernández, Aurora

    2014-01-01

    In the work presented here, we designed and developed two easy-to-use Web tools for in silico detection and characterization of whole-genome sequence (WGS) and whole-plasmid sequence data from members of the family Enterobacteriaceae. These tools will facilitate bacterial typing based on draft...... genomes of multidrug-resistant Enterobacteriaceae species by the rapid detection of known plasmid types. Replicon sequences from 559 fully sequenced plasmids associated with the family Enterobacteriaceae in the NCBI nucleotide database were collected to build a consensus database for integration...... sequences identified in the 559 fully sequenced plasmids. For plasmid multilocus sequence typing (pMLST) analysis, a database that is updated weekly was generated from www.pubmlst.org and integrated into a Web tool called pMLST. Both databases were evaluated using draft genomes from a collection...

  6. Recombinogenic engineering of conjugative plasmids with fluorescent marker cassettes

    DEFF Research Database (Denmark)

    Reisner, A.; Molin, Søren; Zechner, E.L.

    2002-01-01

    An efficient approach for the insertion of fluorescent marker genes with sequence specificity into conjugative plasmids in Escherichia coli is described. For this purpose, homologous recombination of linear double-stranded targeting DNA was mediated by the bacteriophage lambda recombination funct...

  7. Effects of maternal plasmid GHRH treatment on offspring growth

    Science.gov (United States)

    To differentiate prenatal effects of plasmid growth hormone-releasing hormone (GHRH) treatment from maternal effects mediated by lactation on long-term growth of offspring, a cross-fostering study was designed. Pregnant sows (n = 12) were untreated (n = 6), or received either a Wt-GHRH (n = 2), or H...

  8. Competing ParA structures space bacterial plasmids equally over the nucleoid.

    Directory of Open Access Journals (Sweden)

    Robert Ietswaart

    2014-12-01

    Full Text Available Low copy number plasmids in bacteria require segregation for stable inheritance through cell division. This is often achieved by a parABC locus, comprising an ATPase ParA, DNA-binding protein ParB and a parC region, encoding ParB-binding sites. These minimal components space plasmids equally over the nucleoid, yet the underlying mechanism is not understood. Here we investigate a model where ParA-ATP can dynamically associate to the nucleoid and is hydrolyzed by plasmid-associated ParB, thereby creating nucleoid-bound, self-organizing ParA concentration gradients. We show mathematically that differences between competing ParA concentrations on either side of a plasmid can specify regular plasmid positioning. Such positioning can be achieved regardless of the exact mechanism of plasmid movement, including plasmid diffusion with ParA-mediated immobilization or directed plasmid motion induced by ParB/parC-stimulated ParA structure disassembly. However, we find experimentally that parABC from Escherichia coli plasmid pB171 increases plasmid mobility, inconsistent with diffusion/immobilization. Instead our observations favor directed plasmid motion. Our model predicts less oscillatory ParA dynamics than previously believed, a prediction we verify experimentally. We also show that ParA localization and plasmid positioning depend on the underlying nucleoid morphology, indicating that the chromosomal architecture constrains ParA structure formation. Our directed motion model unifies previously contradictory models for plasmid segregation and provides a robust mechanistic basis for self-organized plasmid spacing that may be widely applicable.

  9. Complete genetic analysis of plasmids carrying mcr-1 and other resistance genes in an Escherichia coli isolate of animal origin.

    Science.gov (United States)

    Li, Ruichao; Xie, Miaomiao; Lv, Jingzhang; Wai-Chi Chan, Edward; Chen, Sheng

    2017-03-01

    To investigate the genetic features of three plasmids recovered from an MCR-1 and ESBL-producing Escherichia coli strain, HYEC7, and characterize the transmission mechanism of mcr-1 . The genetic profiles of three plasmids were determined by PCR, S1-PFGE, Southern hybridization and WGS analysis. The ability of the mcr-1 -bearing plasmid to undergo conjugation was also assessed. The mcr-1 -bearing transposon Tn 6330 was characterized by PCR and DNA sequencing. Complete sequences of three plasmids were obtained. A non-conjugative phage P7-like plasmid, pHYEC7- mcr1 , was found to harbour the mcr-1 -bearing transposon Tn 6330 , which could be excised from the plasmid by generating a circular intermediate harbouring mcr-1 and the IS Apl1 element. The insertion of the circular intermediate into another plasmid, pHYEC7-IncHI2, could form pHNSHP45-2, the original IncHI2-type mcr-1 -carrying plasmid that was reported. The third plasmid, pHYEC7-110, harboured two replicons, IncX1 and IncFIB, and comprised multiple antimicrobial resistance mobile elements, some of which were shared by pHYEC7-IncHI2. The Tn 6330 element located in the phage-like plasmid pHYEC7- mcr1 could be excised from the plasmid and formed a circular intermediate that could be integrated into plasmids containing the IS Apl1 element. This phenomenon indicated that Tn 6330 is a key element responsible for widespread dissemination of mcr-1 among various types of plasmids and bacterial chromosomes. The dissemination rate of such an element may be further enhanced upon translocation into phage-like vectors, which may also be transmitted via transduction events.

  10. Comparison of lentiviral and sleeping beauty mediated αβ T cell receptor gene transfer.

    Directory of Open Access Journals (Sweden)

    Anne-Christine Field

    Full Text Available Transfer of tumour antigen-specific receptors to T cells requires efficient delivery and integration of transgenes, and currently most clinical studies are using gamma retroviral or lentiviral systems. Whilst important proof-of-principle data has been generated for both chimeric antigen receptors and αβ T cell receptors, the current platforms are costly, time-consuming and relatively inflexible. Alternative, more cost-effective, Sleeping Beauty transposon-based plasmid systems could offer a pathway to accelerated clinical testing of a more diverse repertoire of recombinant high affinity T cell receptors. Nucleofection of hyperactive SB100X transposase-mediated stable transposition of an optimised murine-human chimeric T cell receptor specific for Wilm's tumour antigen from a Sleeping Beauty transposon plasmid. Whilst transfer efficiency was lower than that mediated by lentiviral transduction, cells could be readily enriched and expanded, and mediated effective target cells lysis in vitro and in vivo. Integration sites of transposed TCR genes in primary T cells were almost randomly distributed, contrasting the predilection of lentiviral vectors for transcriptionally active sites. The results support exploitation of the Sleeping Beauty plasmid based system as a flexible and adaptable platform for accelerated, early-phase assessment of T cell receptor gene therapies.

  11. Gibberellin Signal Transduction in Rice

    Institute of Scientific and Technical Information of China (English)

    Liu-Min Fan; Xiaoyan Feng; Yu Wang; Xing Wang Deng

    2007-01-01

    In the past decade, significant knowledge has accumulated regarding gibberellin (GA) signal transduction in rice as a result of studies using multiple approaches, particularly molecular genetics. The present review highlights the recent developments in the identification of GA signaling pathway components, the discovery of GA-induced destruction of GA signaling represser (DELLA protein), and the possible mechanism underlying the regulation of GA-responsive gene expression in rice.

  12. Postsymbiotic plasmid acquisition and evolution of the repA1-replicon in Buchnera aphidicola

    Science.gov (United States)

    Van Ham, Roeland C. H. J.; González-Candelas, Fernando; Silva, Francisco J.; Sabater, Beatriz; Moya, Andrés; Latorre, Amparo

    2000-01-01

    Buchnera aphidicola is an obligate, strictly vertically transmitted, bacterial symbiont of aphids. It supplies its host with essential amino acids, nutrients required by aphids but deficient in their diet of plant phloem sap. Several lineages of Buchnera show adaptation to their nutritional role in the form of plasmid-mediated amplification of key-genes involved in the biosynthesis of tryptophan (trpEG) and leucine (leuABCD). Phylogenetic analyses of these plasmid-encoded functions have thus far suggested the absence of horizontal plasmid exchange among lineages of Buchnera. Here, we describe three new Buchnera plasmids, obtained from species of the aphid host families Lachnidae and Pemphigidae. All three plasmids belong to the repA1 family of Buchnera plasmids, which is characterized by the presence of a repA1-replicon responsible for replication initiation. A comprehensive analysis of this family of plasmids unexpectedly revealed significantly incongruent phylogenies for different plasmid and chromosomally encoded loci. We infer from these incongruencies a case of horizontal plasmid transfer in Buchnera. This process may have been mediated by secondary endosymbionts, which occasionally undergo horizontal transmission in aphids. PMID:10984505

  13. In vivo comparison of transduction efficiency with recombinant adenovirus-mediated p53 in a human colon cancer mouse model by different delivery routes%rAd/p53不同给药途径治疗人类结肠癌荷瘤鼠模型p53导入效率的在体评价

    Institute of Scientific and Technical Information of China (English)

    Qi Xie; Biling Liang; ling Zhang; Qihua Yang; Xiongfei Gu; Jing Xu; Mingwang Chen

    2008-01-01

    Objective: To evaluate transduction efficiency with recombinant adenovirus-mediated p53 (rAd/p53) therapy in a human colon cancer mouse model by intra-tumoral injection and intra-arterial delivery. Methods: The tumor pieces of human colon cancer SW480 were implanted in the livers of 45 nude mice. These mice were administrated with rAd/p53 by intratu-moral injection and intra-arterial delivery. After 24 h, 48 h and 72 h rAd/p53 administration, 5 mice each group were killed with over anesthesia and their livers were removed. P53 expression and apoptosis of tumor and liver were assessed. Results: P53 expression and apoptosis of intratumoral administration group was higher than tail vein group and control group. Apoptosis and p53 expression of livers in three groups had no significant difference. Conclusion: p53 gene transduction efficiency and anticancer effect of tAd/p53 is much better by intra-tumoral injection than intra-arterial delivery.

  14. Mediatization

    DEFF Research Database (Denmark)

    Hjarvard, Stig

    2017-01-01

    Mediatization research shares media effects studies' ambition of answering the difficult questions with regard to whether and how media matter and influence contemporary culture and society. The two approaches nevertheless differ fundamentally in that mediatization research seeks answers...... to these general questions by distinguishing between two concepts: mediation and mediatization. The media effects tradition generally considers the effects of the media to be a result of individuals being exposed to media content, i.e. effects are seen as an outcome of mediated communication. Mediatization...... research is concerned with long-term structural changes involving media, culture, and society, i.e. the influences of the media are understood in relation to how media are implicated in social and cultural changes and how these processes come to create new conditions for human communication and interaction...

  15. Plasmid and chromosome partitioning: surprises from phylogeny

    DEFF Research Database (Denmark)

    Gerdes, Kenn; Møller-Jensen, Jakob; Bugge Jensen, Rasmus

    2000-01-01

    Plasmids encode partitioning genes (par) that are required for faithful plasmid segregation at cell division. Initially, par loci were identified on plasmids, but more recently they were also found on bacterial chromosomes. We present here a phylogenetic analysis of par loci from plasmids and chr...

  16. Multiple drug resistant carbapenemases producing Acinetobacter baumannii isolates harbours multiple R-plasmids

    Directory of Open Access Journals (Sweden)

    Rajagopalan Saranathan

    2014-01-01

    Full Text Available Background & objectives: The nosocomial human pathogen Acinetobacter baumannii has high propensity to develop resistance to antimicrobials and to become multidrug resistant (MDR, consequently complicating the treatment. This study was carried out to investigate the presence of resistant plasmids (R-plasmids among the clinical isolates of A. baumannii. In addition, the study was performed to check the presence of common β-lactamases encoding genes on these plasmids. Methods: A total of 55 clinical isolates of A. baumannii were included in the study and all were subjected to plasmid DNA isolation, followed by PCR to check the presence of resistance gene determinants such as blaOXA-23 , blaOXA-51, blaOXA-58 and blaIMP-1 on these plasmids that encode for oxacillinase (OXA and metallo-β-lactamase (MBL type of carbapenemases. Plasmid curing experiments were carried out on selected isolates using ethidium bromide and acridine orange as curing agents and the antibiotic resistance profiles were evaluated before and after curing. Results: All the isolates were identified as A. baumannii by 16SrDNA amplification and sequencing. Plasmid DNA isolated from these isolates showed the occurrence of multiple plasmids with size ranging from 500bp to ≥ 25 kb. The percentage of blaOXA-51 and blaOXA-23 on plasmids were found to be 78 and 42 per cent, respectively and 20 isolates (36% carried blaIMP-1 gene on plasmids. Significant difference was observed in the antibiograms of plasmid cured isolates when compared to their parental ones. The clinical isolates became susceptible to more than two antibiotic classes after curing of plasmids indicating plasmid borne resistance. Interpretation & conclusions: Our study determined the plasmid mediated resistance mechanisms and occurrence of different resistance genes on various plasmids isolated from MDR A. baumannii. The present findings showed the evidence for antibiotic resistance mediated through multiple plasmids in

  17. In vivo expansion of regulatory T cells with IL-2/IL-2 mAb complexes prevents anti-factor VIII immune responses in hemophilia A mice treated with factor VIII plasmid-mediated gene therapy.

    Science.gov (United States)

    Liu, Chao-Lien; Ye, Peiqing; Yen, Benjamin C; Miao, Carol H

    2011-08-01

    Generation of transgene-specific immune responses can constitute a major complication following gene therapy treatment. An in vivo approach to inducing selective expansion of Regulatory T (Treg) cells by injecting interleukin-2 (IL-2) mixed with a specific IL-2 monoclonal antibody (JES6-1) was adopted to modulate anti-factor VIII (anti-FVIII) immune responses. Three consecutive IL-2 complexes treatments combined with FVIII plasmid injection prevented anti-FVIII formation and achieved persistent, therapeutic-level of FVIII expression in hemophilia A (HemA) mice. The IL-2 complexes treatment expanded CD4(+)CD25(+)Foxp3(+) Treg cells five- to sevenfold on peak day, and they gradually returned to normal levels within 7-14 days without changing other lymphocyte populations. The transiently expanded Treg cells are highly activated and display suppressive function in vitro. Adoptive transfer of the expanded Treg cells protected recipient mice from generation of high-titer antibodies following FVIII plasmid challenge. Repeated plasmid transfer is applicable in tolerized mice without eliciting immune responses. Mice treated with IL-2 complexes mounted immune responses against both T-dependent and T-independent neoantigens, indicating that IL-2 complexes did not hamper the immune system for long. These results demonstrate the important role of Treg cells in suppressing anti-FVIII immune responses and the potential of developing Treg cell expansion therapies that induce long-term tolerance to FVIII.

  18. Use of sperm plasmid DNA lipofection combined with REMI (restriction enzyme-mediated insertion) for production of transgenic chickens expressing eGFP (enhanced green fluorescent protein) or human follicle-stimulating hormone.

    Science.gov (United States)

    Harel-Markowitz, Eliane; Gurevich, Michael; Shore, Laurence S; Katz, Adi; Stram, Yehuda; Shemesh, Mordechai

    2009-05-01

    Linearized p-eGFP (plasmid-enhanced green fluorescent protein) or p-hFSH (plasmid human FSH) sequences with the corresponding restriction enzyme were lipofected into sperm genomic DNA. Sperm transfected with p-eGFP were used for artificial insemination in hens, and in 17 out of 19 of the resultant chicks, the exogenous DNA was detected in their lymphocytes as determined by PCR and expressed in tissues as determined by (a) PCR, (b) specific emission of green fluorescence by the eGFP, and (c) Southern blot analysis. A complete homology was found between the Aequorea Victoria eGFP DNA and a 313-bp PCR product of extracted DNA from chick blood cells. Following insemination with sperm lipofected with p-hFSH, transgenic offspring were obtained for two generations as determined by detection of the transgene for human FSH (PCR) and expression of the gene (RT-PCR and quantitative real-time PCR) and the presence of the protein in blood (radioimmunoassay). Data demonstrate that lipofection of plasmid DNA with restriction enzyme is a highly efficient method for the production of transfected sperm to produce transgenic offspring by direct artificial insemination.

  19. The transcription factors CREB and c-Fos play key roles in NCAM-mediated neuritogenesis in PC12-E2 cells

    DEFF Research Database (Denmark)

    Jessen, U; Novitskaya, V; Pedersen, N;

    2001-01-01

    roles in this process by estimating NCAM-dependent neurite outgrowth from PC12-E2 cells grown in co-culture with NCAM-negative or NCAM-positive fibroblasts. PC12-E2 cells were transiently transfected with expression plasmids encoding wild-type or dominant negative forms of CREB and c-Fos or an activated...... thus present evidence that NCAM-mediated neurite outgrowth involves a series of signal transduction pathways, including the cAMP/PKA pathway, targeting c-Fos and CREB....

  20. Resolution of Multimeric Forms of Circular Plasmids and Chromosomes.

    Science.gov (United States)

    Crozat, Estelle; Fournes, Florian; Cornet, François; Hallet, Bernard; Rousseau, Philippe

    2014-10-01

    One of the disadvantages of circular plasmids and chromosomes is their high sensitivity to rearrangements caused by homologous recombination. Odd numbers of crossing-over occurring during or after replication of a circular replicon result in the formation of a dimeric molecule in which the two copies of the replicon are fused. If they are not converted back to monomers, the dimers of replicons may fail to correctly segregate at the time of cell division. Resolution of multimeric forms of circular plasmids and chromosomes is mediated by site-specific recombination, and the enzymes that catalyze this type of reaction fall into two families of proteins: the serine and tyrosine recombinase families. Here we give an overview of the variety of site-specific resolution systems found on circular plasmids and chromosomes.

  1. Multiple Pathways of Plasmid DNA Transfer in Helicobacter pylori

    Science.gov (United States)

    Rohrer, Stefanie; Holsten, Lea; Weiss, Evelyn; Benghezal, Mohammed; Fischer, Wolfgang; Haas, Rainer

    2012-01-01

    Many Helicobacter pylori (Hp) strains carry cryptic plasmids of different size and gene content, the function of which is not well understood. A subgroup of these plasmids (e.g. pHel4, pHel12), contain a mobilisation region, but no cognate type IV secretion system (T4SS) for conjugative transfer. Instead, certain H. pylori strains (e.g. strain P12 carrying plasmid pHel12) can harbour up to four T4SSs in their genome (cag-T4SS, comB, tfs3, tfs4). Here, we show that such indigenous plasmids can be efficiently transferred between H. pylori strains, even in the presence of extracellular DNaseI eliminating natural transformation. Knockout of a plasmid-encoded mobA relaxase gene significantly reduced plasmid DNA transfer in the presence of DNaseI, suggesting a DNA conjugation or mobilisation process. To identify the T4SS involved in this conjugative DNA transfer, each individual T4SS was consecutively deleted from the bacterial chromosome. Using a marker-free counterselectable gene deletion procedure (rpsL counterselection method), a P12 mutant strain was finally obtained with no single T4SS (P12ΔT4SS). Mating experiments using these mutants identified the comB T4SS in the recipient strain as the major mediator of plasmid DNA transfer between H. pylori strains, both in a DNaseI-sensitive (natural transformation) as well as a DNaseI-resistant manner (conjugative transfer). However, transfer of a pHel12::cat plasmid from a P12ΔT4SS donor strain into a P12ΔT4SS recipient strain provided evidence for the existence of a third, T4SS-independent mechanism of DNA transfer. This novel type of plasmid DNA transfer, designated as alternate DNaseI-Resistant (ADR) mechanism, is observed at a rather low frequency under in vitro conditions. Taken together, our study describes for the first time the existence of three distinct pathways of plasmid DNA transfer between H. pylori underscoring the importance of horizontal gene transfer for this species. PMID:23029142

  2. Type 3 Fimbriae Encoded on Plasmids Are Expressed from a Unique Promoter without Affecting Host Motility, Facilitating an Exceptional Phenotype That Enhances Conjugal Plasmid Transfer

    Science.gov (United States)

    Madsen, Jonas Stenløkke; Riber, Leise; Kot, Witold; Basfeld, Alrun; Burmølle, Mette; Hansen, Lars Hestbjerg; Sørensen, Søren Johannes

    2016-01-01

    Horizontal gene transfer (HGT), the transmission of genetic material to a recipient that is not the progeny of the donor, is fundamental in bacterial evolution. HGT is often mediated by mobile genetic elements such as conjugative plasmids, which may be in conflict with the chromosomal elements of the genome because they are independent replicons that may petition their own evolutionary strategy. Here we study differences between type 3 fimbriae encoded on wild type plasmids and in chromosomes. Using known and newly characterized plasmids we show that the expression of type 3 fimbriae encoded on plasmids is systematically different, as MrkH, a c-di-GMP dependent transcriptional activator is not needed for strong expression of the fimbriae. MrkH is required for expression of type 3 fimbriae of the Klebsiella pneumoniae chromosome, wherefrom the fimbriae operon (mrkABCDF) of plasmids is believed to have originated. We find that mrkABCDFs of plasmids are highly expressed via a unique promoter that differs from the original Klebsiella promoter resulting in fundamental behavioral consequences. Plasmid associated mrkABCDFs did not influence the swimming behavior of the host, that hereby acquired an exceptional phenotype being able to both actively swim (planktonic behavior) and express biofilm associated fimbriae (sessile behavior). We show that this exceptional phenotype enhances the conjugal transfer of the plasmid. PMID:27627107

  3. Toll like receptor 4 and its mediated signal transduction with acute anterior uveitis%Toll样受体-4及其介导的信号转导与急性前葡萄膜炎

    Institute of Scientific and Technical Information of China (English)

    王婧; 卢弘

    2011-01-01

    急性前葡萄膜炎是最常见的葡萄膜炎,其发病机制尚不明确.近年来研究认为,G阴性细菌感染启动、Toll样受体-4介导的信号转导在急性前葡萄膜炎的发病机制中发挥重要作用.本文对目前脂多糖-Toll样受体-4信号转导通路及通过对该通路干预来调控炎症的相关研究进展进行综述,以期为前葡萄膜炎的治疗提供新的思路.%Acute anterior uveitis, which pathogenesis is still not explicit, is the most common uveitis. Recent years,some researchers believe that Gram-negative bacterium infection initiating,toll like receptor 4 signal transduction may play an important role in the pathogenesis of acute anterior uveitis. In this article, we review the present li-popolysaccharide-Toll like receptor 4 pathways and intervention for the way ,in the hope of supplying some new ideas for the treatment of acute anterior uveitis.

  4. Identification of photoperception and light signal transduction pathways in citrus

    Directory of Open Access Journals (Sweden)

    Vera Quecini

    2007-01-01

    Full Text Available Studies employing model species have elucidated several aspects of photoperception and light signal transduction that control plant development. However, the information available for economically important crops is scarce. Citrus genome databases of expressed sequence tags (EST were investigated in order to identify genes coding for functionally characterized proteins responsible for light-regulated developmental control in model plants. Approximately 176,200 EST sequences from 53 libraries were queried and all bona fide and putative photoreceptor gene families were found in citrus species. We have identified 53 orthologs for several families of transcriptional regulators and cytoplasmic proteins mediating photoreceptor-induced responses although some important Arabidopsis phytochrome- and cryptochrome-signaling components are absent from citrus sequence databases. The main gene families responsible for phototropin-mediated signal transduction were present in citrus transcriptome, including general regulatory factors (14-3-3 proteins, scaffolding elements and auxin-responsive transcription factors and transporters. A working model of light perception, signal transduction and response-eliciting in citrus is proposed based on the identified key components. These results demonstrate the power of comparative genomics between model systems and economically important crop species to elucidate several aspects of plant physiology and metabolism.

  5. First characterisation of plasmid-mediated quinolone resistance-qnrS1 co-expressed bla CTX-M-15 and bla DHA-1 genes in clinical strain of Morganella morganii recovered from a Tunisian Intensive Care Unit

    Directory of Open Access Journals (Sweden)

    S Mahrouki

    2012-01-01

    Full Text Available Purpose: Aim of this study was to show the emergence of the qnr genes among fluoroquinolone-resistant, AMPC and ESBL (extended-spectrum-beta-lactamase co-producing Morganella morganii isolate. Materials and Methods: A multi resistant Morganella morganii SM12012 isolate was recovered from pus from a patient hospitalized in the intensive care unit at the Military hospital, Tunisia. Antibiotic susceptibility was tested with the agar disk diffusion method according to Clinical and Laboratory Standards Institute guidelines. ESBLs were detected using a standard double-disk synergy test. The characterization of beta-lactamases and associated resistance genes were performed by isoelectric focusing, polymerase chain reaction and nucleotide sequencing. Results: The antimicrobial susceptibility testing showed the high resistance to penicillins, cephalosporins (MICs: 64-512 μg/ml and fluoroquinolones (MICs: 32-512 μg/ml. But M. morganii SM12012 isolate remained susceptible to carbapenems (MICs: 4-<0.25 μg/ml. The double-disk synergy test confirmed the phenotype of extended-spectrum β-lactamases (ESBLs. Three identical β-lactamases with pI values of 6.5, 7.8 and superior to 8.6 were detected after isoelectric focusing analysis. These β-lactamases genes can be successfully transferred by the conjugative plasmid. Molecular analysis demonstrated the co-production of bla DHA-1, bla CTX-M-15 and qnrS1 genes on the same plasmid. The detection of an associated chromosomal quinolone resistance revealed the presence of a parC mutation at codon 80 (Ser80-lle80. Conclusion: This is the first report in Tunisia of nosocomial infection due to the production of CTX-M-15 and DHA-1 β-lactamases in M. morganii isolate with the association of quinolone plasmid resistance. The incidence of these strains invites continuous monitoring of such multidrug-resistant strains and the further study of their epidemiologic evolution.

  6. Conjugation of plasmids of Neisseria gonorrhoeae to other Neisseria species: potential reservoirs for the beta-lactamase plasmid.

    Science.gov (United States)

    Genco, C A; Knapp, J S; Clark, V L

    1984-09-01

    The discovery that penicillinase production in Neisseria gonorrhoeae was plasmid mediated and the spread of the beta-lactamase encoding plasmids in gonococcal isolates since 1976, raise the possibility that a nonpathogenic indigenous bacterium could serve as a reservoir for these plasmids. We initiated studies to define the ability of commensal Neisseria species and Branhamella catarrhalis strains, as well as strains of the pathogen Neisseria meningitidis, to serve as recipients in conjugation with Neisseria gonorrhoeae. We found that with N. gonorrhoeae as the donor, 3 of 5 Neisseria cinerea, 2 of 5 Neisseria flava, 0 of 1 Neisseria flavescens, 1 of 3 Neisseria subflava, 0 of 6 B. catarrhalis, 0 of 7 Neisseria lactamica, 1 of 5 Neisseria mucosa, 1 of 7 Neisseria perflava/sicca, and 0 of 13 N. meningitidis strains gave detectable conjugation frequencies (greater than 10(-8). N. cinerea was the only species found to maintain the gonococcal conjugal plasmid (pLE2451). A N. cinerea transconjugant containing pLE2451 was observed to transfer both the beta-lactamase plasmid and pLE2451 to N. gonorrhoeae at high frequency.

  7. Key features of mcr-1-bearing plasmids from Escherichia coli isolated from humans and food.

    Science.gov (United States)

    Zurfluh, Katrin; Nüesch-Inderbinen, Magdalena; Klumpp, Jochen; Poirel, Laurent; Nordmann, Patrice; Stephan, Roger

    2017-01-01

    Mcr-1-harboring Enterobacteriaceae are reported worldwide since their first discovery in 2015. However, a limited number of studies are available that compared full-length plasmid sequences of human and animal origins. In this study, mcr-1-bearing plasmids from seven Escherichia coli isolates recovered from patients (n = 3), poultry meat (n = 2) and turkey meat (n = 2) in Switzerland were further analyzed and compared. Isolates were characterized by multilocus sequence typing (MLST). The mcr-1-bearing plasmids were transferred by transformation into reference strain E. coli DH5α and MCR-1-producing transformants were selected on LB-agar supplemented with 2 mg/L colistin. Purified plasmids were then sequenced and compared. MLST revealed six distinct STs, illustrating the high clonal diversity among mcr-1-positive E. coli isolates of different origins. Two different mcr-1-positive plasmids were identified from a single E. coli ST48 human isolate. All other isolates possessed a single mcr-1 harboring plasmid. Transferable IncI2 (size ca. 60-61 kb) and IncX4 (size ca. 33-35 kb) type plasmids each bearing mcr-1 were found associated with human and food isolates. None of the mcr-1-positive IncI2 and IncX4 plasmids possessed any additional resistance determinants. Surprisingly, all but one of the sequenced mcr-1-positive plasmids lacked the ISApl1 element, which is a key element mediating acquisition of mcr-1 into various plasmid backbones. There is strong evidence that the food chain may be an important transmission route for mcr-1-bearing plasmids. Our data suggest that some "epidemic" plasmids rather than specific E. coli clones might be responsible for the spread of the mcr-1 gene along the food chain.

  8. 大肠埃希菌产质粒介导AmpC酶基因在中国分布的Meta分析%Distribution of plasmid mediated AmpC genes of β-lactamase-producing Escherichia coli in China: A meta analysis

    Institute of Scientific and Technical Information of China (English)

    茅国峰; 徐儿

    2016-01-01

    AmpC酶基因的阳性率差异具有统计学意义(P<0.05).%Objective To analyze the distribution of plasmid-mediated AmpC genes of beta-lactamase producing Escherichia coli in China.Methods Retrieving four Chinese database,EMbase,PubMed and foreign periodicals database,retrieval time for the database until May 2015,by two independent researchers strictly according to the inclusion and exclusion criteria selected studies and extracted relevant data of screening of the ESBLs genes in E.coli,the research literature by Stata 12.1 single group rate of meta analysis,analysis includes the detection rate of all kinds of plasmid mediated AmpC enzyme gene of E.coli and the distribution of different regions.Results A total of 35 Chinese and 4 foreign literatures were included,and the data were analyzed by random effects model,merge result display:(1) the E.coli plasmid mediated by the positive rate of AmpC enzyme gene was 5.0% (95%CI:4.0%-7.0%).The positive rate of plasmid mediated AmpC enzyme gene in E.coli was 5.0% in 2005-2008 (95%CI:3.0%-7.0%).The positive rate of plasmid mediated AmpC enzyme gene in E.coli was 6.0% in 2009-2013 (95%CI:4.0%-8.0%),the differences in two time periods of AmpC enzyme gene positive rate had statistical significance (P<0.05);(2) The detection rate of DHA primer amplification gene was 43.0% (95%CI:28.0%-59.0%).The detection rate of CIT primer amplification gene was 52.0% (95%CI:37.0%-66.0%).The detection rate of EBC primer amplification gene was 23.0% (95%CI:10.0%-37.0%);(3) The positive rate of plasmid mediated AmpC enzyme gene of E.coli was 8.0% (95%CI:5.0%-11.0%) in the north of China.The positive rate of plasmid mediated AmpC enzyme gene in E.coli was 5.0% (95%CI:1.0%-9.0%) in the southeast of China.The positive rate of plasmid mediated AmpC enzyme gene in E.coli was 6.0% (95%CI:2.0%-10.0%) in the central and western regions.The positive rate of plasmid mediated Amp

  9. Cellular semiotics and signal transduction

    DEFF Research Database (Denmark)

    Bruni, Luis Emilio

    2007-01-01

    to the processes of sign interpretation and transmission between organisms of the same or different species). In Biosemiotics it is customary to recognise the cell as the most elementary integration unit for semiosis. Therefore intra and intercellular communication constitute the departure point for the study......Semiosis, the processes of production, communication and interpretation of signs - coding and de-coding - takes place within and between organisms. The term "endosemiosis" refers to the processes of interpretation and sign transmission inside an organism (as opposed to "exosemiosis", which refers...... considering semiotic logic in order to construct our understanding of living phenomena. Given the central integrating role of signal transduction in physiological and ecological studies, this chapter outlines its semiotic implications. The multi-modality and modularity of signal molecules and relative...

  10. 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

  11. Co-resident plasmids travel together.

    Science.gov (United States)

    Gama, João Alves; Zilhão, Rita; Dionisio, Francisco

    2017-08-24

    Conjugative plasmids encode genes that enable them to transfer, by conjugation, from a given host cell to another cell. Conjugative transfer, despite being an important feature of conjugative plasmids, is not constitutive for most plasmids, the reason being that genes involved in horizontal transfer are mostly repressed. Only upon their transient de-repression are plasmids able to transfer horizontally. If host cells harbour multiple plasmids, their simultaneous transfer depends on simultaneous transient de-repression of all plasmids. If de-repression of different plasmids was random and independent events, simultaneous de-repression should be a rare event because the probability of simultaneous de-repression would be the product of the probabilities of de-repression of each plasmid. Some previous observations support this hypothesis, while others show that co-transfer of plasmids is more frequent than this reasoning indicates. Here, we show that co-transfer of multiple plasmids mainly results from non-independent events: the probability that all plasmids within a cell become de-repressed is much higher than if de-repression of plasmids genes were independent. We found a simple model for the probability of co-transfer: the plasmid having the lowest conjugation rates is the one who limits co-transfer. In this sense, cells receiving the plasmid with the lower transfer rate also receive the other plasmid. If de-repression happens simultaneously on co-resident plasmids, common cues may stimulate de-repression of distinct plasmids. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. The Chlamydophila felis plasmid is highly conserved.

    Science.gov (United States)

    Harley, Ross; Day, Sarinder; Di Rocco, Camillo; Helps, Chris

    2010-11-20

    The presence of a plasmid in the Chlamydiaceae is both species and strain specific. Knowledge of the prevalence of the plasmid in different Chlamydia species is important for future studies aiming to investigate the role of the plasmid in chlamydial biology and disease. Although strains of Chlamydophila felis with or without the plasmid have been identified, only a small number of laboratory-adapted strains have been analysed and the prevalence of the plasmid in field isolates has not been determined. This study aimed to determine the prevalence of the plasmid in C. felis-positive conjunctival and oropharyngeal clinical samples submitted for routine diagnosis of C. felis by real-time (Q)PCR. DNA extracts from four laboratory-adapted strains were also analysed. QPCR assays targeting regions of C. felis plasmid genes pCF01, pCF02 and pCF03 were developed for the detection of plasmid DNA. QPCR analysis of DNA extracts from C. felis-positive clinical samples found evidence of plasmid DNA in 591 of 595 samples representing 561 of 564 (99.5%) clinical cases. Plasmid DNA was also detected by QPCR in laboratory-adapted strains 1497V, K2487 and K2490, but not strain 905. We conclude that the plasmid is highly conserved in C. felis, and plasmid-deficient strains represent a rare but important population for future studies of chlamydial plasmid function.

  13. Mechanisms of UV-induced signal transduction

    Energy Technology Data Exchange (ETDEWEB)

    Kulms, D.; Schwarz, T. [Univ. Muenster, Muenster (Germany). Ludwing Boltzmann Inst. for Cell Biology and Immunobiology of the Skin

    2002-04-01

    Ultraviolet radiation (UV) causes a variety of biological effects that can be either beneficial or harmful for human health. To exert these effects on a cellular basis, UV uses a variety of signaling pathways. DNA is the major chromophore for UVB. Thus, nuclear DNA damage has been detected to be a major mediator of numerous UVB effects, and experimental reduction of DNA damage is associated with a loss of these effects. On the other hand, UV has been found to utilize molecular components within the cytoplasm or at the cell membrane for signaling. UV can directly activate cell surface receptors, kinases, and transcription factors. The nuclear and extranuclear signaling pathways are generated independently and have been recently recognized to be not mutually exclusive but to contribute to various UV effects in an independent and additive way. Further knowledge of how these signaling pathways relate to each other will certainly increase our understanding of how UV acts as a pathogen. The following review will briefly discuss current aspects of the mechanisms involved in UV-induced signal transduction. (author)

  14. Mobilization of Bacillus thuringiensis plasmid pTX14-3.

    Science.gov (United States)

    Andrup, L; Bendixen, H H; Jensen, G B

    1995-05-01

    The Bacillus thuringiensis subsp. israelensis (Bti) plasmid pTX14-3 has been reported to contain a gene, mob14-3, with considerable homology to genes encoding mobilization proteins from other gram-positive bacteria. We have used the aggregation-mediated conjugation system recently discovered in Bti to compare the mobilization kinetics of different derivatives of plasmid pTX14-3. Plasmid pTX14-3 has been found to replicate by the rolling-circle mechanism and to contain a locus suppressing the formation of high-molecular-weight DNA. We found that deleting a DNA fragment containing this locus increased the transfer frequency about twofold. The mobilization frequency of the plasmid containing the intact mob14-3 gene did not indicate a mobilization-enhancing activity of the encoded polypeptide. However, the presence of the mob14-3 gene seemed to increase the stability of the plasmid in exponential growth.

  15. Molecular relationship among fosfomycin-resistant plasmids and clinical impact of fosfomycin resistance.

    Science.gov (United States)

    Mendoza, M C; Teran, F J; Mendez, F J; Hardisson, C

    1988-10-01

    We have been carrying out a surveillance programme on plasmid-mediated fosfomycin resistance in our community over the last decade and have isolated and characterized several varieties of conjugative plasmids from different enterobacteriae. In this work we show that seven varieties of plasmids are related with the Inc M group, and carry the same For determinant which encodes a modifying enzyme. The comparative study on their R-phenotype, restriction analysis and DNA-DNA hybridization showed different degrees of molecular relationship among them. The spread of For-plasmids as well as the fosfomycin resistance by other mechanisms seems to be low in spite of the great For-plasmid diversity found.

  16. Architectures and representations for string transduction

    NARCIS (Netherlands)

    Chrupala, Grzegorz

    2015-01-01

    String transduction problems are ubiquitous in natural language processing: they include transliteration, grapheme-to-phoneme conversion, text normalization and translation. String transduction can be reduced to the simpler problems of sequence labeling by expressing the target string as a sequence

  17. Human hematopoietic cell culture, transduction, and analyses

    DEFF Research Database (Denmark)

    Bonde, Jesper; Wirthlin, Louisa; Kohn, Donald B;

    2008-01-01

    This unit provides methods for introducing genes into human hematopoietic progenitor cells. The Basic Protocol describes isolation of CD34(+) cells, transduction of these cells with a retroviral vector on fibronectin-coated plates, assaying the efficiency of transduction, and establishing long...

  18. PLASMIDS FROM ANAEROCELLUM THERMOPHILUM AND USES THEREOF

    DEFF Research Database (Denmark)

    2003-01-01

    The present invention concerns the isolation of plasmids from extremely thermophilic anaerobic microorganisms and their use in genetic transformation of thermophilic and mesophilic microorganisms. More particular the invention concerns the use of thermostable plasmid vectors as tools for creating...

  19. Sentra, a database of signal transduction proteins.

    Energy Technology Data Exchange (ETDEWEB)

    Maltsev, N.; Marland, E.; Yu, G. X.; Bhatnagar, S.; Lusk, R.; Mathematics and Computer Science

    2002-01-01

    Sentra (http://www-wit.mcs.anl.gov/sentra) is a database of signal transduction proteins with the emphasis on microbial signal transduction. The database was updated to include classes of signal transduction systems modulated by either phosphorylation or methylation reactions such as PAS proteins and serine/threonine kinases, as well as the classical two-component histidine kinases and methyl-accepting chemotaxis proteins. Currently, Sentra contains signal transduction proteins from 43 completely sequenced prokaryotic genomes as well as sequences from SWISS-PROT and TrEMBL. Signal transduction proteins are annotated with information describing conserved domains, paralogous and orthologous sequences, and conserved chromosomal gene clusters. The newly developed user interface supports flexible search capabilities and extensive visualization of the data.

  20. SENTRA, a database of signal transduction proteins.

    Energy Technology Data Exchange (ETDEWEB)

    D' Souza, M.; Romine, M. F.; Maltsev, N.; Mathematics and Computer Science; PNNL

    2000-01-01

    SENTRA, available via URL http://wit.mcs.anl.gov/WIT2/Sentra/, is a database of proteins associated with microbial signal transduction. The database currently includes the classical two-component signal transduction pathway proteins and methyl-accepting chemotaxis proteins, but will be expanded to also include other classes of signal transduction systems that are modulated by phosphorylation or methylation reactions. Although the majority of database entries are from prokaryotic systems, eukaroytic proteins with bacterial-like signal transduction domains are also included. Currently SENTRA contains signal transduction proteins in 34 complete and almost completely sequenced prokaryotic genomes, as well as sequences from 243 organisms available in public databases (SWISS-PROT and EMBL). The analysis was carried out within the framework of the WIT2 system, which is designed and implemented to support genetic sequence analysis and comparative analysis of sequenced genomes.

  1. Plasmid required for virulence of Agrobacterium tumefaciens

    Energy Technology Data Exchange (ETDEWEB)

    Watson, B.; Currier, T.C.; Gordon, M.P.; Chilton, M.D.; Nester, E.W.

    1975-07-01

    The irreversible loss of crown gall-inducing ability of Agrobacterium tumefaciens strain C-58 during growth at 37/sup 0/C is shown to be due to loss of a large plasmid (1.2 x 10/sup 8/ daltons). The gene responsible for this high rate of plasmid loss at elevated temperatures seems to be located on the plasmid. In addition, another spontaneous avirulent variant, A. tumefaciens strain IIBNV6, is shown to lack the virulence plasmid which its virulent sibling strain, IIBV7, possesses. Deoxyribonucleic acid reassociation measurements prove that the plasmid is eliminated, not integrated into the chromosome, in both of the avirulent derivatives. Transfer of virulence from donor strain C-58 to avirulent recipient strain A136 results from the transfer of a plasmid, which appears identical to the donor plasmid by deoxyribonucleic acid reassociation measurements. The transfer of virulence in another cross, K27 x A136, was also shown to result from the transfer of a large plasmid. These findings establish unequivocally that the large plasmid determines virulence. Two additional genetic determinants have been located on the virulence plasmid of A. tumefaciens strain C-58: the ability to utilize nopaline and sensitivity to a bacteriocin produced by strain 84. The latter trait can be exploited for selection of avirulent plasmid-free derivatives of strain C-58. The trait of nopaline utilization appears to be on the virulence plasmid also in strains IIBV7 and K27.

  2. Plasmids expressing interleukin-10 short hairpin RNA mediate IL-10 knockdown and enhance tumor necrosis factor alpha and interferon gamma expressions in response to porcine reproductive and respiratory syndrome virus.

    Science.gov (United States)

    Charerntantanakul, Wasin; Kasinrerk, Watchara

    2012-04-15

    Porcine reproductive and respiratory syndrome virus (PRRSV) has been suggested to exploit interleukin-10 (IL-10) to suppress immune defense of infected pigs. The present study constructed plasmids encoding selected short hairpin RNA specific to porcine IL-10 mRNA (pIL-10sh) to knockdown IL-10 transcription and investigated the suppressive effect of PRRSV-induced IL-10 on various immune marker expressions. Naïve blood monocytes from eight PRRSV-seronegative pigs were transfected with pIL-10sh and pNeg (plasmid vector) prior to PRRSV inoculation and subsequent lipopolysaccharide (LPS) stimulation. The mRNA expressions of IL-10, IL-1β, IL-12p40, tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), transforming growth factor beta (TGFβ), CD80, and CD86 were evaluated by real-time PCR. The IL-10, TNFα, and IFNγ protein productions were determined by ELISA. Compared with non-transfected monocyte control, transfection with selected pIL-10sh (pIL-10sh1), but not other pIL-10sh nor pNeg, significantly reduced IL-10 expression and significantly enhanced TNFα and IFNγ expressions. Slight increases in IL-1β, IL-12p40, CD80, and CD86 expressions were also observed. Neither pIL-10sh1 nor pNeg transfection affected TGFβ expression. Our results indicate that PRRSV does exploit IL-10 to suppress the expressions of pro-inflammatory cytokines, mainly TNFα and IFNγ, and co-stimulatory molecules, CD80 and CD86.

  3. Instability of multiple drug resistance plasmids in Salmonella typhimurium isolated from poultry.

    Science.gov (United States)

    Brown, D J; Threlfall, E J; Rowe, B

    1991-04-01

    Plasmids in five strains of Salmonella typhimurium resistant to ampicillin, chloramphenicol, gentamicin, neomycin/kanamycin, streptomycin, sulphonamides, tetracyclines and trimethoprim (ACGKSSuTTm), CGKSSuTTm, ACSSuT or CSSuT which had been isolated from poultry in the first 3 months of 1989 have been characterized and compared with plasmids in two strains of R-types ACGKSSuTTm and ASSuTTm isolated from two patients later in the year. With the exception of the human isolate of R-type ASSuTTm, all strains carried two non-conjugative plasmids, one coding for SSu and belonging to incompatibility group Q, and a second coding for multiple resistance and belonging to the FIme incompatibility group. The human isolate of R-type ASSuTTm did not carry the IncQ SSu plasmid but like the poultry isolates, carried a non-conjugative FIme plasmid. Restriction endonuclease digestion with the enzymes EcoR I, Pst I and Hind III demonstrated that the FIme plasmids from strains of different R-types showed a high degree of homology but exhibited numerous fragment size polymorphisms. The restriction digest fingerprint of plasmids in the human isolate of R-type ACGKSSuTTm was indistinguishable from a poultry isolate of the same R-type. Analysis of segregants of one of the poultry isolates of R-type ACGKSSuTTm demonstrated that resistance determinants could be rapidly lost from the FIme plasmid to give rise to a number of R-types and fingerprint patterns. Loss of tetracycline resistance from this plasmid appeared to be correlated with the integration of other plasmid-mediated resistances into the bacterial chromosome. Evidence is presented for the rapid loss of antimicrobial resistance determinants from a multiple resistance plasmid of the FIme incompatibility group in response to withdrawal of antibiotic selective pressure.

  4. Ordering the mob: Insights into replicon and MOB typing schemes from analysis of a curated dataset of publicly available plasmids.

    Science.gov (United States)

    Orlek, Alex; Phan, Hang; Sheppard, Anna E; Doumith, Michel; Ellington, Matthew; Peto, Tim; Crook, Derrick; Walker, A Sarah; Woodford, Neil; Anjum, Muna F; Stoesser, Nicole

    2017-03-09

    Plasmid typing can provide insights into the epidemiology and transmission of plasmid-mediated antibiotic resistance. The principal plasmid typing schemes are replicon typing and MOB typing, which utilize variation in replication loci and relaxase proteins respectively. Previous studies investigating the proportion of plasmids assigned a type by these schemes ('typeability') have yielded conflicting results; moreover, thousands of plasmid sequences have been added to NCBI in recent years, without consistent annotation to indicate which sequences represent complete plasmids. Here, a curated dataset of complete Enterobacteriaceae plasmids from NCBI was compiled, and used to assess the typeability and concordance of in silico replicon and MOB typing schemes. Concordance was assessed at hierarchical replicon type resolutions, from replicon family-level to plasmid multilocus sequence type (pMLST)-level, where available. We found that 85% and 65% of the curated plasmids could be replicon and MOB typed, respectively. Overall, plasmid size and the number of resistance genes were significant independent predictors of replicon and MOB typing success. We found some degree of non-concordance between replicon families and MOB types, which was only partly resolved when partitioning plasmids into finer-resolution groups (replicon and pMLST types). In some cases, non-concordance was attributed to ambiguous boundaries between MOBP and MOBQ types; in other cases, backbone mosaicism was considered a more plausible explanation. β-lactamase resistance genes tended not to show fidelity to a particular plasmid type, though some previously reported associations were supported. Overall, replicon and MOB typing schemes are likely to continue playing an important role in plasmid analysis, but their performance is constrained by the diverse and dynamic nature of plasmid genomes.

  5. Polymer-enhanced adenoviral transduction of CAR-negative bladder cancer cells.

    Science.gov (United States)

    Kasman, Laura M; Barua, Sutapa; Lu, Ping; Rege, Kaushal; Voelkel-Johnson, Christina

    2009-01-01

    The application of adenoviral gene therapy for cancer is limited by immune clearance of the virus as well as poor transduction efficiency, since the protein used for viral entry (CAR) serves physiological functions in adhesion and is frequently decreased among cancer cells. Cationic polymers have been used to enhance adenoviral gene delivery, but novel polymers with low toxicity are needed to realize this approach. We recently identified polymers that were characterized by high transfection efficiency of plasmid DNA and a low toxicity profile. In this study we evaluated the novel cationic polymer EGDE-3,3' for its potential to increase adenoviral transduction of the CAR-negative bladder cancer cell line TCCSUP. The amount of adenovirus required to transduce 50-60% of the cells was reduced 100-fold when Ad.GFP was preincubated with the EGDE-3,3' polymer. Polyethyleneimine (pEI), a positively charged polymer currently used as a standard for enhancing adenoviral transduction, also increased infectivity, but transgene expression was consistently higher with EGDE-3,3'. In addition, EGDE-3,3'-supplemented transduction of an adenovirus expressing an apoptosis inducing transgene, Ad.GFP-TRAIL, significantly enhanced the amount of cell death. Thus, our results indicate that novel biocompatible polymers may be useful in improving the delivery of adenoviral gene therapy.

  6. Origin and Evolution of Rickettsial Plasmids.

    Science.gov (United States)

    El Karkouri, Khalid; Pontarotti, Pierre; Raoult, Didier; Fournier, Pierre-Edouard

    2016-01-01

    Rickettsia species are strictly intracellular bacteria that have undergone a reductive genomic evolution. Despite their allopatric lifestyle, almost half of the 26 currently validated Rickettsia species have plasmids. In order to study the origin, evolutionary history and putative roles of rickettsial plasmids, we investigated the evolutionary processes that have shaped 20 plasmids belonging to 11 species, using comparative genomics and phylogenetic analysis between rickettsial, microbial and non-microbial genomes. Plasmids were differentially present among Rickettsia species. The 11 species had 1 to 4 plasmid (s) with a size ranging from 12 kb to 83 kb. We reconstructed pRICO, the last common ancestor of the current rickettsial plasmids. pRICO was vertically inherited mainly from Rickettsia/Orientia chromosomes and diverged vertically into a single or multiple plasmid(s) in each species. These plasmids also underwent a reductive evolution by progressive gene loss, similar to that observed in rickettsial chromosomes, possibly leading to cryptic plasmids or complete plasmid loss. Moreover, rickettsial plasmids exhibited ORFans, recent gene duplications and evidence of horizontal gene transfer events with rickettsial and non-rickettsial genomes mainly from the α/γ-proteobacteria lineages. Genes related to maintenance and plasticity of plasmids, and to adaptation and resistance to stress mostly evolved under vertical and/or horizontal processes. Those involved in nucleotide/carbohydrate transport and metabolism were under the influence of vertical evolution only, whereas genes involved in cell wall/membrane/envelope biogenesis, cycle control, amino acid/lipid/coenzyme and secondary metabolites biosynthesis, transport and metabolism underwent mainly horizontal transfer events. Rickettsial plasmids had a complex evolution, starting with a vertical inheritance followed by a reductive evolution associated with increased complexity via horizontal gene transfer as well as

  7. Signaling transduction pathways involved in basophil adhesion and histamine release

    DEFF Research Database (Denmark)

    Sha, Quan; Poulsen, Lars K.; Gerwien, Jens

    2006-01-01

    Little is known about basophil with respect to the different signaling transduction pathways involved in spontaneous, cytokine or anti-IgE induced adhesion and how this compares to IgE-dependent and IgE-independent mediator secretion. The purpose of the present study was to investigate the roles...... of beta1 and beta2 integrins in basophil adhesion as well as hosphatidylinositol 3-kinase (PI3K), src-kinases and extracellular signal regulated kinase (ERK) 1/2 in basophil adhesion and histamine release (HR)....

  8. Chlamydophila felis: plasmid detection in Italian isolates.

    Science.gov (United States)

    Di Francesco, Antonietta; Donati, Manuela; Salvatore, Daniela; Cevenini, Roberto; Di Paolo, Maria; Baldelli, Raffaella

    2010-04-01

    Plasmids have been detected in the majority of strains in the genus Chlamydia and in many Chlamydophila species. Previous studies showed that FP Pring and FP Cello Chlamydophila felis strains have an extrachromosomial plasmid, whereas the FP Baker strain does not. Azuma et al. recently sequenced the entire genomic DNA sequence of the Japanese Cp. felis strain Fe/C-56 and described a 7,552 base pair circular plasmid. In the present study a highly conserved plasmid gene was detected in 11 Italian Cp. felis isolates, showing 100% nucleotide identity with the plasmid gene of Fe/C-56 Cp. felis strain.

  9. Transposon Insertion Reveals pRM, a Plasmid of Rickettsia monacensis▿

    OpenAIRE

    Baldridge, Gerald D.; Burkhardt, Nicole Y.; Felsheim, Roderick F.; Timothy J Kurtti; Munderloh, Ulrike G.

    2007-01-01

    Until the recent discovery of pRF in Rickettsia felis, the obligate intracellular bacteria of the genus Rickettsia (Rickettsiales: Rickettsiaceae) were thought not to possess plasmids. We describe pRM, a plasmid from Rickettsia monacensis, which was detected by pulsed-field gel electrophoresis and Southern blot analyses of DNA from two independent R. monacensis populations transformed by transposon-mediated insertion of coupled green fluorescent protein and chloramphenicol acetyltransferase m...

  10. COMP-angiopoietin 1 increases proliferation, differentiation, and migration of stem-like cells through Tie-2-mediated activation of p38 MAPK and PI3K/Akt signal transduction pathways

    Energy Technology Data Exchange (ETDEWEB)

    Kook, Sung-Ho [Cluster for Craniofacial Development and Regeneration Research, Institute of Oral Biosciences and School of Dentistry, Chonbuk National University, Jeonju (Korea, Republic of); Lim, Shin-Saeng [School of Dentistry and Dental Research Institute, Seoul National University, Seoul (Korea, Republic of); Cho, Eui-Sic; Lee, Young-Hoon; Han, Seong-Kyu; Lee, Kyung-Yeol [Cluster for Craniofacial Development and Regeneration Research, Institute of Oral Biosciences and School of Dentistry, Chonbuk National University, Jeonju (Korea, Republic of); Kwon, Jungkee [College of Veterinary Medicine, Chonbuk National University, Jeonju (Korea, Republic of); Hwang, Jae-Won; Bae, Cheol-Hyeon [Cluster for Craniofacial Development and Regeneration Research, Institute of Oral Biosciences and School of Dentistry, Chonbuk National University, Jeonju (Korea, Republic of); Seo, Young-Kwon [Research Institute of Biotechnology, Dongguk University, Seoul (Korea, Republic of); Lee, Jeong-Chae, E-mail: leejc88@jbnu.ac.kr [Cluster for Craniofacial Development and Regeneration Research, Institute of Oral Biosciences and School of Dentistry, Chonbuk National University, Jeonju (Korea, Republic of)

    2014-12-12

    Highlights: • COMP-Ang1 induces Tie-2 activation in BMMSCs, but not in primary osteoblasts. • Tie-2 knockdown inhibits COMP-Ang1-stimulated proliferation and osteoblastogenesis. • Tie-2 knockdown prevents COMP-Ang1-induced activation of PI3K/Akt and p38 MAPK. • COMP-Ang1 induces migration of cells via activation of PI3K/Akt and CXCR4 pathways. • COMP-Ang1 stimulates in vivo migration of PDLSCs into a calvarial defect site of rats. - Abstract: Recombinant COMP-Ang1, a chimera of angiopoietin-1 (Ang1) and a short coiled-coil domain of cartilage oligomeric matrix protein (COMP), is under consideration as a therapeutic agent capable of inducing the homing of cells with increased angiogenesis. However, the potentials of COMP-Ang1 to stimulate migration of mesenchymal stem cells (MSCs) and the associated mechanisms are not completely understood. We examined the potential of COMP-Ang1 on bone marrow (BM)-MSCs, human periodontal ligament stem cells (PDLSCs), and calvarial osteoblasts. COMP-Ang1 augmented Tie-2 induction at protein and mRNA levels and increased proliferation and expression of runt-related transcription factor 2 (Runx2), osterix, and CXCR4 in BMMSCs, but not in osteoblasts. The COMP-Ang1-mediated increases were inhibited by Tie-2 knockdown and by treating inhibitors of phosphoinositide 3-kinase (PI3K), LY294002, or p38 mitogen-activated protein kinase (MAPK), SB203580. Phosphorylation of p38 MAPK and Akt was prevented by siRNA-mediated silencing of Tie-2. COMP-Ang1 also induced in vitro migration of BMMSCs and PDLSCs. The induced migration was suppressed by Tie-2 knockdown and by CXCR4-specific peptide antagonist or LY294002, but not by SB203580. Furthermore, COMP-Ang1 stimulated the migration of PDLSCs into calvarial defect site of rats. Collectively, our results demonstrate that COMP-Ang1-stimulated proliferation, differentiation, and migration of progenitor cells may involve the Tie-2-mediated activation of p38 MAPK and PI3K/Akt pathways.

  11. The role of FIS in the Rcd checkpoint and stable maintenance of plasmid ColE1.

    Science.gov (United States)

    Blaby, I K; Summers, D K

    2009-08-01

    Escherichia coli plasmid ColE1 lacks active partitioning, and copies are distributed randomly to daughter cells at division. The plasmid is maintained stably in the bacterial population as long as its copy number remains high. The accumulation of plasmid dimers and higher multimers depresses copy number, and is an important cause of multicopy plasmid instability. ColE1 dimers are restored to the monomeric state by site-specific recombination, which requires the host-encoded proteins XerCD, ArgR and PepA acting at the plasmid cer site. In addition, a 70 nt RNA expressed from the cer site of plasmid dimers delays the division of dimer-containing cells. Here, we report that the global regulator FIS binds to cer in a sequence-specific manner, close to the Rcd promoter (P(cer)). FIS is not required for plasmid dimer resolution, but is essential for repression of P(cer) in plasmid monomers. Repression also requires the XerCD recombinase, but not ArgR or PepA. We propose a model for monomer-dimer control of P(cer) in which the promoter is repressed in plasmid monomers by the concerted action of FIS and XerCD. Rcd transcription is triggered in plasmid dimers by the lifting of XerCD-mediated repression in the synaptic complex.

  12. 质粒shRNA体内干扰Klotho基因对窦房结通道基因的影响%Study on the Effect of Klotho Gene Interferred by Plasmid-mediated Short Hairpin RNA (shRNA) on Sinoatrial Node Pacing Channel Gene

    Institute of Scientific and Technical Information of China (English)

    蔡盈盈; 汪汉; 侯言彬; 房晨鹂; 田鹏; 王贵华; 李璐; 邓珏琳

    2013-01-01

    通过质粒shRNA体内干扰,研究Klotho基因与窦房结起搏通道相关基因HCN4及HCN2之间的关系,为病窦综合征的研究提供新思路.取C57BL/6J小鼠20只,分为4组,每组5只,分别为:质粒shRNA 24 h组、质粒shRNA 12 h组、生理盐水24 h组、生理盐水12h组.质粒shRNA组经尾静脉注射质粒shRNA 50 μL(1 μg质粒/μL),生理盐水组经尾静脉注射生理盐水50 μL.分别于注射12h及24 h后取窦房结周围组织,行RT-PCR检测各组小鼠的Klotho、HCN2、HCN4基因的mRNA水平.RT-PCR结果显示:与生理盐水12h组比较,shRNA 12 h组的klotho、HCN4和HCN2的mRNA表达量明显降低,均有统计学差异(P<0.05).以上结果提示,小鼠Klotho基因和窦房结起搏基因可能存在一定关系.%The study was aimed to assess the effect of Klotho gene and sinoatrial node pacing channel gene (HCN4and HCN2) for studying sick sinus syndrome,with Klotho gene under the interference of Plasmid-mediated short hairpin RNA.Twenty-five C57BL/6J mice were divided into four groups,i.e,plasmid shRNA 24h group,plasmid shRNA 12h group,sodium chloride 24h group and sodium chloride 12h group.Plasmid shRNA 50μL (1μg/μL) and sodium chloride 50μl were respectively injected according to mice vena caudalis into those in plasmid shRNA group and sodium chloride group.After 12h or 24h respectively,all mice were executed and their sinoatrial node tissues were cut.The mRNA of Klotho,HCN4 and HCN2 gene were detected by RT-PCR.The results of RT-PCR showed that Klotho,HCN4 and HCN2 mRNA levels were lower compared with those in sodium chloride 12h group after 12h interference interval.The results indicated that there might be the a certain relationship between Klotho gene and sinoatrial node pacing channel gene.

  13. The sensory transduction pathways in bacterial chemotaxis

    Science.gov (United States)

    Taylor, Barry L.

    1989-01-01

    Bacterial chemotaxis is a useful model for investigating in molecular detail the behavioral response of cells to changes in their environment. Peritrichously flagellated bacteria such as coli and typhimurium swim by rotating helical flagella in a counterclockwise direction. If flagellar rotation is briefly reversed, the bacteria tumble and change the direction of swimming. The bacteria continuously sample the environment and use a temporal sensing mechanism to compare the present and immediate past environments. Bacteria respond to a broad range of stimuli including changes in temperature, oxygen concentration, pH and osmotic strength. Bacteria are attracted to potential sources of nutrition such as sugars and amino acids and are repelled by other chemicals. In the methylation-dependent pathways for sensory transduction and adaptation in E. coli and S. typhimurium, chemoeffectors bind to transducing proteins that span the plasma membrane. The transducing proteins are postulated to control the rate of autophosphorylation of the CheA protein, which in turn phosphorylates the CheY protein. The phospho-CheY protein binds to the switch on the flagellar motor and is the signal for clockwise rotation of the motor. Adaptation to an attractant is achieved by increasing methylation of the transducing protein until the attractant stimulus is cancelled. Responses to oxygen and certain sugars involve methylation-independent pathways in which adaption occurs without methylation of a transducing protein. Taxis toward oxygen is mediated by the electron transport system and changes in the proton motive force. Recent studies have shown that the methylation-independent pathway converges with the methylation-dependent pathway at or before the CheA protein.

  14. Monocytes-derived macrophages mediated stable expression of human brain-derived neurotrophic factor, a novel therapeutic strategy for neuroAIDS.

    Directory of Open Access Journals (Sweden)

    Jing Tong

    Full Text Available HIV-1 associated dementia remains a significant public health burden. Clinical and experimental research has shown that reduced levels of brain-derived neurotrophic factor (BDNF may be a risk factor for neurological complications associated with HIV-1 infection. We are actively testing genetically modified macrophages for their possible use as the cell-based gene delivery vehicle for the central nervous system (CNS. It can be an advantage to use the natural homing/migratory properties of monocyte-derived macrophages to deliver potentially neuroprotective BDNF into the CNS, as a non-invasive manner. Lentiviral-mediated gene transfer of human (hBDNF plasmid was constructed and characterized. Defective lentiviral stocks were generated by transient transfection of 293T cells with lentiviral transfer plasmid together with packaging and envelope plasmids. High titer lentiviral vector stocks were harvested and used to transduce human neuronal cell lines, primary cultures of human peripheral mononocyte-derived macrophages (hMDM and murine myeloid monocyte-derived macrophages (mMDM. These transduced cells were tested for hBDNF expression, stability, and neuroprotective activity. The GenomeLab GeXP Genetic Analysis System was used to evaluate transduced cells for any adverse effects by assessing gene profiles of 24 reference genes. High titer vectors were prepared for efficient transduction of neuronal cell lines, hMDM, and mMDM. Stable secretion of high levels of hBDNF was detected in supernatants of transduced cells using western blot and ELISA. The conditioned media containing hBDNF were shown to be protective to neuronal and monocytic cell lines from TNF-α and HIV-1 Tat mediated cytotoxicity. Lentiviral vector-mediated gene transduction of hMDM and mMDM resulted in high-level, stable expression of the neuroprotective factorBDNF in vitro. These findings form the basis for future research on the potential use of BDNF as a novel therapy for neuroAIDS.

  15. Monocytes-derived macrophages mediated stable expression of human brain-derived neurotrophic factor, a novel therapeutic strategy for neuroAIDS.

    Science.gov (United States)

    Tong, Jing; Buch, Shilpa; Yao, Honghong; Wu, Chengxiang; Tong, Hsin-I; Wang, Youwei; Lu, Yuanan

    2014-01-01

    HIV-1 associated dementia remains a significant public health burden. Clinical and experimental research has shown that reduced levels of brain-derived neurotrophic factor (BDNF) may be a risk factor for neurological complications associated with HIV-1 infection. We are actively testing genetically modified macrophages for their possible use as the cell-based gene delivery vehicle for the central nervous system (CNS). It can be an advantage to use the natural homing/migratory properties of monocyte-derived macrophages to deliver potentially neuroprotective BDNF into the CNS, as a non-invasive manner. Lentiviral-mediated gene transfer of human (h)BDNF plasmid was constructed and characterized. Defective lentiviral stocks were generated by transient transfection of 293T cells with lentiviral transfer plasmid together with packaging and envelope plasmids. High titer lentiviral vector stocks were harvested and used to transduce human neuronal cell lines, primary cultures of human peripheral mononocyte-derived macrophages (hMDM) and murine myeloid monocyte-derived macrophages (mMDM). These transduced cells were tested for hBDNF expression, stability, and neuroprotective activity. The GenomeLab GeXP Genetic Analysis System was used to evaluate transduced cells for any adverse effects by assessing gene profiles of 24 reference genes. High titer vectors were prepared for efficient transduction of neuronal cell lines, hMDM, and mMDM. Stable secretion of high levels of hBDNF was detected in supernatants of transduced cells using western blot and ELISA. The conditioned media containing hBDNF were shown to be protective to neuronal and monocytic cell lines from TNF-α and HIV-1 Tat mediated cytotoxicity. Lentiviral vector-mediated gene transduction of hMDM and mMDM resulted in high-level, stable expression of the neuroprotective factorBDNF in vitro. These findings form the basis for future research on the potential use of BDNF as a novel therapy for neuroAIDS.

  16. Calcium insensitivity of FA-6, a cell line derived from a pancreatic cancer associated with humoral hypercalcemia, is mediated by the significantly reduced expression of the Calcium Sensitive Receptor transduction component p38 MAPK

    Directory of Open Access Journals (Sweden)

    Fairfax Benjamin

    2006-11-01

    Full Text Available Abstract The Calcium-Sensing Receptor is a key component of Calcium/Parathyroid hormone homeostatic system that helps maintain appropriate plasma Ca2+ concentrations. It also has a number of non-homeostatic functions, including cell cycle regulation through the p38 MAPK pathway, and recent studies have indicated that it is required for Ca2+ mediated growth arrest in pancreatic carcinoma cells. Some pancreatic cancers produce pathogenic amounts of parathyroid like hormones, however, which significantly increase Ca2+ plasma concentrations and might be expected to block further cell growth. In this study we have investigated the expression and function of the p38 MAPK signaling pathway in Ca2+ sensitive (T3M-4 and insensitive (FA6 pancreatic cancer cell lines. FA-6 cells, which are derived from a pancreatic adenocarcinoma that secretes a parathyroid hormone related peptide, exhibit only very low levels of p38 MAPK expression, relative to T3M-4 cells. Transfecting FA-6 cells with a p38 MAPK expression construct greatly increases their sensitivity to Ca2+. Furthermore, the reduction of p38 MAPK in T3M-4 cells significantly reduces the extent to which high levels of Ca2+ inhibit proliferation. These results suggest that the low levels of p38 MAPK expression in FA-6 cells may serve to reduce their sensitivity to high concentrations of external Ca2+ that would otherwise block proliferation.

  17. Transduction of chemical into electrical energy.

    Science.gov (United States)

    Nachmansohn, D

    1976-01-01

    The paper recalls some fundamental notions, developed by Otto Meyerhof, which were used in the analysis of the transduction of chemical into mechanical energy during muscular contraction. These notions formed the basis of the approach to the analysis of the transduction of chemical into electrical energy, i.e., the very principle underlying nerve and muscle excitability and bioelectricity. Instrumental for this purpose was the use, since 1937, of electric organs of fish, a tissue highly specialized for bioelectrogenesis.

  18. New and Redesigned pRS Plasmid Shuttle Vectors for Genetic Manipulation of Saccharomycescerevisiae.

    Science.gov (United States)

    Chee, Mark K; Haase, Steven B

    2012-05-01

    We have constructed a set of 42 plasmid shuttle vectors based on the widely used pRS series for use in the budding yeast Saccharomyces cerevisiae and the bacterium Escherichia coli. This set of pRSII plasmids includes new shuttle vectors that can be used with histidine and adenine auxotrophic laboratory yeast strains carrying mutations in the genes HIS2 and ADE1, respectively. Our pRSII plasmids also include updated versions of commonly used pRS plasmids from which common restriction sites that occur within their yeast-selectable biosynthetic marker genes have been removed to increase the availability of unique restriction sites within their polylinker regions. Hence, our pRSII plasmids are a complete set of integrating, centromere and 2μ episomal plasmids with the biosynthetic marker genes ADE2, HIS3, TRP1, LEU2, URA3, HIS2, and ADE1 and a standardized selection of at least 16 unique restriction sites in their polylinkers. Additionally, we have expanded the range of drug selection options that can be used for PCR-mediated homologous replacement using pRS plasmid templates by replacing the G418-resistance kanMX4 cassette of pRS400 with MX4 cassettes encoding resistance to phleomycin, hygromycin B, nourseothricin, and bialaphos. Finally, in the process of generating the new plasmids, we have determined several errors in existing publicly available sequences for several commonly used yeast plasmids. Using our updated sequences, we constructed pRS plasmid backbones with a unique restriction site for inserting new markers to facilitate future expansion of the pRS series.

  19. An epidemiological survey on Escherichia coli producing plasmid-mediated AmpC enzyme and regulation of expression of AmpC enzyme%产质粒介导AmpC酶大肠埃希菌的流行病学调查及其酶的表达调控

    Institute of Scientific and Technical Information of China (English)

    邓江锦; 张文林; 张志祥; 李小庆

    2013-01-01

    Objective To explore the expression and regulation of Escherichia coli producing plasmid-mediated AmpC enzymes.Methods 30 strains of E.Coli producing plasmid-mediated AmpC enzyme were isolated.An epidemiological survey were conducted on Escherichia coli producing plasmidmediated AmpC enzyme,and transfer bonding test was carried out.6 primer bacterial plasmid DNA extracted for the multiplex PCR amplification were set.Results According to the epidemiological survey,a total of 20 strains producing β-lactamase were detected,5 of which produced AmpC β-lactamase enzymes and extended-spectrum β-lactamase enzyme,12 only produced extended-spectrum β-lactamase enzymes,and 3 produced lactamase AmpC β alone.5 strains were transferred successfully.PCR amplification test and the Genbank database sequence alignment analysis showed that 6 strains were amplified to produce plasmidmediated ampC gene,4 of which were CIT and 2 of which were type DHA.Conclusions Clinically,strains of Escherichia coli producing plasmid AmpC enzyme widely spread.Control of the regulatory mechanism of high expression is helpful in guiding clinical antibiotic uses.%目的 研究产质粒介导AmpC酶大肠埃希菌的流行病学状况及其酶的表达调控.方法 选择临床中分离到的疑似产质粒介导AmpC酶的大肠埃希菌30株,对产质粒介导AmpC酶大肠埃希菌的流行病学特征进行调查分析,并行转移接合试验.用6组引物对提取的细菌质粒DNA进行多重PCR扩增,从而对质粒介导的AmpC酶的表达调控进行分析.结果 流行病学调查,共检出20株产β-内酰胺酶菌株,其中有5株菌株同时产AmpC β-内酰胺酶和超广谱β-内酰胺酶,12株只产超广谱β-内酰胺酶,3株只产AmpC β-内酰胺酶,5株转移接合成功.PCR扩增试验后,与Genbank数据库进行序列比对分析,发现有6株扩增产生质粒介导的AmpC基因,其中CIT型4株,DHA型2株.结论 产质粒AmpC酶大肠埃希菌在临床上广泛传播,把握

  20. Comparative Sequence Analysis of Multidrug-Resistant IncA/C Plasmids from Salmonella enterica.

    Science.gov (United States)

    Hoffmann, Maria; Pettengill, James B; Gonzalez-Escalona, Narjol; Miller, John; Ayers, Sherry L; Zhao, Shaohua; Allard, Marc W; McDermott, Patrick F; Brown, Eric W; Monday, Steven R

    2017-01-01

    Determinants of multidrug resistance (MDR) are often encoded on mobile elements, such as plasmids, transposons, and integrons, which have the potential to transfer among foodborne pathogens, as well as to other virulent pathogens, increasing the threats these traits pose to human and veterinary health. Our understanding of MDR among Salmonella has been limited by the lack of closed plasmid genomes for comparisons across resistance phenotypes, due to difficulties in effectively separating the DNA of these high-molecular weight, low-copy-number plasmids from chromosomal DNA. To resolve this problem, we demonstrate an efficient protocol for isolating, sequencing and closing IncA/C plasmids from Salmonella sp. using single molecule real-time sequencing on a Pacific Biosciences (Pacbio) RS II Sequencer. We obtained six Salmonella enterica isolates from poultry, representing six different serovars, each exhibiting the MDR-Ampc resistance profile. Salmonella plasmids were obtained using a modified mini preparation and transformed with Escherichia coli DH10Br. A Qiagen Large-Construct kit™ was used to recover highly concentrated and purified plasmid DNA that was sequenced using PacBio technology. These six closed IncA/C plasmids ranged in size from 104 to 191 kb and shared a stable, conserved backbone containing 98 core genes, with only six differences among those core genes. The plasmids encoded a number of antimicrobial resistance genes, including those for quaternary ammonium compounds and mercury. We then compared our six IncA/C plasmid sequences: first with 14 IncA/C plasmids derived from S. enterica available at the National Center for Biotechnology Information (NCBI), and then with an additional 38 IncA/C plasmids derived from different taxa. These comparisons allowed us to build an evolutionary picture of how antimicrobial resistance may be mediated by this common plasmid backbone. Our project provides detailed genetic information about resistance genes in

  1. Genomic and Functional Characterization of qnr-Encoding Plasmids from Municipal Wastewater Biosolid Klebsiella pneumoniae Isolates.

    Science.gov (United States)

    Kaplan, Ella; Sela, Noa; Doron-Faigenboim, Adi; Navon-Venezia, Shiri; Jurkevitch, Edouard; Cytryn, Eddie

    2015-01-01

    Municipal wastewater treatment facilities are considered to be "hotspots" for antibiotic resistance, since they conjoin high densities of environmental and fecal bacteria with selective pressure in the form of sub-therapeutic concentrations of antibiotics. Discharged effluents and biosolids from these facilities can disseminate antibiotic resistant genes to terrestrial and aquatic environments, potentially contributing to the increasing global trend in antibiotic resistance. This phenomenon is especially pertinent when resistance genes are associated with mobile genetic elements such as conjugative plasmids, which can be transferred between bacterial phyla. Fluoroquinolones are among the most abundant antibiotic compounds detected in wastewater treatment facilities, especially in biosolids, where due to their hydrophobic properties they accumulate to concentrations that may exceed 40 mg/L. Although fluoroquinolone resistance is traditionally associated with mutations in the gyrA/topoisomerase IV genes, there is increasing evidence of plasmid-mediated quinolone resistance, which is primarily encoded on qnr genes. In this study, we sequenced seven qnr-harboring plasmids from a diverse collection of Klebsiella strains, isolated from dewatered biosolids from a large wastewater treatment facility in Israel. One of the plasmids, termed pKPSH-11XL was a large (185.4 kbp), multi-drug resistance, IncF-type plasmid that harbored qnrB and 10 additional antibiotic resistance genes that conferred resistance to five different antibiotic families. It was highly similar to the pKPN3-like plasmid family that has been detected in multidrug resistant clinical Klebsiella isolates. In contrast, the six additional plasmids were much smaller (7-9 Kbp) and harbored a qnrS -type gene. These plasmids were highly similar to each other and closely resembled pGNB2, a plasmid isolated from a German wastewater treatment facility. Comparative genome analyses of pKPSH-11XL and other pKPN3-like

  2. Modeling of biological doses and mechanical effects on bone transduction

    CERN Document Server

    Rieger, Romain; Jennane, Rachid; 10.1016/j.jtbi.2011.01.003

    2012-01-01

    Shear stress, hormones like parathyroid and mineral elements like calcium mediate the amplitude of stimulus signal which affects the rate of bone remodeling. The current study investigates the theoretical effects of different metabolic doses in stimulus signal level on bone. The model was built considering the osteocyte as the sensing center mediated by coupled mechanical shear stress and some biological factors. The proposed enhanced model was developed based on previously published works dealing with different aspects of bone transduction. It describes the effects of physiological doses variations of Calcium, Parathyroid Hormone, Nitric Oxide and Prostaglandin E2 on the stimulus level sensed by osteocytes in response to applied shear stress generated by interstitial fluid flow. We retained the metabolic factors (Parathyroid Hormone, Nitric Oxide, and Prostaglandin E2) as parameters of bone cell mechanosensitivity because stimulation/inhibition of induced pathways stimulates osteogenic response in vivo. We t...

  3. Evaluation of a DNA microarray for rapid detection of the most prevalent extended-spectrum β-lactamases, plasmid-mediated cephalosporinases and carbapenemases in Enterobacteriaceae, Pseudomonas and Acinetobacter.

    Science.gov (United States)

    Bogaerts, Pierre; Cuzon, Gaelle; Evrard, Stéphanie; Hoebeke, Martin; Naas, Thierry; Glupczynski, Youri

    2016-08-01

    The dissemination of Gram-negative bacteria (GNB) producing extended-spectrum β-lactamases (ESBLs), plasmid-encoded cephalosporinases (pAmpCs) and carbapenemases is a matter of great clinical concern. In this study, we evaluated a new low-density DNA array 'Check-MDR CT103 XL' (Check-Points, Wageningen, The Netherlands) that identifies the most clinically relevant β-lactamase genes of ESBLs (blaTEM, blaSHV, blaCTX-M, blaBEL, blaPER, blaGES and blaVEB), pAmpCs (blaCMY-2-like, blaDHA, blaFOX, blaACC-1, blaACT/MIR and blaCMY-1-like/MOX) and carbapenemases (blaKPC, blaOXA-48, blaVIM, blaIMP, blaNDM, blaGIM, blaSPM and blaOXA-23, -24 and -58) in cultured bacteria. In total, 223 GNB isolates with well-characterised resistance mechanisms to β-lactams were analysed. A specificity and sensitivity of 100% were recorded for most bla genes, with a slightly lower signal observed for blaIMP. The Check-MDR CT103 XL array proved highly accurate for the identification of epidemiologically relevant ESBL, pAmpC and carbapenemase genes harboured in Enterobacteriaceae, Pseudomonas and Acinetobacter spp. The Check-MDR CT103 XL assay is a significant improvement compared with Check-MDR CT103 and it highlights the ability of this array to evolve rapidly to adjust to the current needs for the detection of resistance mechanisms to β-lactam agents.

  4. Identification of plasmid-mediated carbapenem-hydrolyzing β-lactamase KPC-2 in Enterobacteriaceae%肠杆菌科细菌中质粒介导的KPC-2型碳青霉烯酶的检测

    Institute of Scientific and Technical Information of China (English)

    张嵘; 蔡加昌; 周宏伟; 陈功祥

    2008-01-01

    属的细菌之间传播.%Objective To investigate the molecular epidemiology and mechanism of earbapenem resistance of Serratia marcescens,Klebsiella pneumoniae and Escherichia coli isolates from intensive care units(ICUs).Methods Twenty-one S.marcescens,ten K.pneumoniae and one E.coli isolates with carbapenem resistance or reduced carbapenem susceptibility were recovered from two ICUs in our hospital from April 2006 to Febmary 2007.Pulsed-field gel electrophoresis(PFGE)and enterobacterial repetitive intergenic consensus-PCR(ERIC-PCR)were performed to analyze the molecular epidemiology of isolates.Antibiotic susceptibilities were determined bv agar dilution method.Conjugation experiments were carried out in mixed broth cultures.Plasmid DNA was obtained bv using an alkalinelysis technique and was digested by various endonucleases.Elimination of plasmid from S.marcesceus isolates were performed by repeated SDS treatment.The crude β-lactamase extracts of original isolates and E.coli transconjugants were subjected to isoelectric focusing(IEF);Specific PCRs and DNA sequencing were preformed to confirm the genotype of β-lactamases.Results ERIC-PCR indicated that all S.marcescens isolates belonged to a clonal strain.PFGE indicated that ten K. pneumoniae isolates were indistinguishable or closely related to each other.The MICs of imipenem and meropenero for all isolates were 2 to 8 μg/ml except K.pneumoniae K10(128 and 256 μg/ml).Conjugation studies with E.coli(EC600)resulted in the transfer of reduced carbapenem susceptibility from original isolates(MICs:from≤0.125 μg/ml to 1-2μg/ml).IEF,PCR and DNA sequence analysis confirmed that S.marcescens isolates produced KPC-2(pI of 6.7)and a β-lactamase(pI 6.5).k pneumoniae isolates produced TEM-1(pI 5.4),KPC-2,CTX-M-14(pI 7.9),and a β-lactamase(pI 7.3).E.coli El produced KPC-2,CTX-M-15(pI 9.0),and a β-laetamase(pI 7.3).Only a KPC-2 was detected in E.coli transeonjugants.Plasmid restricfion analysis using EcoR Ⅰ,Hind Ⅲ,and Bcu Ⅰ showed identical

  5. A Rebeccamycin Analog Provides Plasmid-Encoded Niche Defense.

    Science.gov (United States)

    Van Arnam, Ethan B; Ruzzini, Antonio C; Sit, Clarissa S; Currie, Cameron R; Clardy, Jon

    2015-11-18

    Bacterial symbionts of fungus-growing ants occupy a highly specialized ecological niche and face the constant existential threat of displacement by another strain of ant-adapted bacteria. As part of a systematic study of the small molecules underlying this fraternal competition, we discovered an analog of the antitumor agent rebeccamycin, a member of the increasingly important indolocarbazole family. While several gene clusters consistent with this molecule's newly reported modification had previously been identified in metagenomic studies, the metabolite itself has been cryptic. The biosynthetic gene cluster for 9-methoxyrebeccamycin is encoded on a plasmid in a manner reminiscent of plasmid-derived peptide antimicrobials that commonly mediate antagonism among closely related Gram-negative bacteria.

  6. Current trends in separation of plasmid DNA vaccines: a review.

    Science.gov (United States)

    Ghanem, Ashraf; Healey, Robert; Adly, Frady G

    2013-01-14

    Plasmid DNA (pDNA)-based vaccines offer more rapid avenues for development and production if compared to those of conventional virus-based vaccines. They do not rely on time- or labour-intensive cell culture processes and allow greater flexibility in shipping and storage. Stimulating antibodies and cell-mediated components of the immune system are considered as some of the major advantages associated with the use of pDNA vaccines. This review summarizes the current trends in the purification of pDNA vaccines for practical and analytical applications. Special attention is paid to chromatographic techniques aimed at reducing the steps of final purification, post primary isolation and intermediate recovery, in order to reduce the number of steps necessary to reach a purified end product from the crude plasmid.

  7. Persistence of Antibiotic Resistance Plasmids in Biofilms

    Science.gov (United States)

    2014-10-01

    plasmids* in*populations*of* Gram > negative *bacteria*grown*in*biofilms*and*well>mixed*liquid*cultures.** * Task2:*Characterize*the*evolution*of*plasmid...R.! Edwards.! 2005.! Overview! of! nosocomial! infections! caused! by! gramP negative ! bacilli .!Clin.!Infect.!Dis.!41:848P854.! LoftiePEaton,!W.,!A... negative ! interaction!between!one!of! its!chromosomal!segments!and!the!plasmid! by!simply!deleting!the!appropriate!chromosomal!segment.!! 7. None

  8. Regular cellular distribution of plasmids by oscillating and filament-forming ParA ATPase of plasmid pB171

    DEFF Research Database (Denmark)

    Ebersbach, Gitte; Ringgaard, Simon; Møller-Jensen, Jakob;

    2006-01-01

    with each other in a bacterial two-hybrid assay but do not interact with FtsZ, eight other essential cell division proteins or MreB actin. Based on these observations, we propose a simple model for how oscillating ParA filaments can mediate regular cellular distribution of plasmids. The model functions......Centromere-like loci from bacteria segregate plasmids to progeny cells before cell division. The ParA ATPase (a MinD homologue) of the par2 locus from plasmid pB171 forms oscillating helical structures over the nucleoid. Here we show that par2 distributes plasmid foci regularly along the length...... of the cell even in cells with many plasmids. In vitro, ParA binds ATP and ADP and has a cooperative ATPase activity. Moreover, ParA forms ATP-dependent filaments and cables, suggesting that ParA can provide the mechanical force for the observed regular distribution of plasmids. ParA and ParB interact...

  9. Regular Cellular Distribution of Plasmids by Oscillating and Filament-forming ParA ATPase of Plasmid pB171

    DEFF Research Database (Denmark)

    Ebersbach, Gitte; Ringgaard, Simon; Møller-Jensen, Jakob;

    2006-01-01

    with each other in a bacterial two-hybrid assay but do not interact with FtsZ, eight other essential cell division proteins or MreB actin. Based on these observations, we propose a simple model for how oscillating ParA filaments can mediate regular cellular distribution of plasmids. The model functions......Centromere-like loci from bacteria segregate plasmids to progeny cells before cell division. The ParA ATPase (a MinD homologue) of the par2 locus from plasmid pB171 forms oscillating helical structures over the nucleoid. Here we show that par2 distributes plasmid foci regularly along the length...... of the cell even in cells with many plasmids. In vitro, ParA binds ATP and ADP and has a cooperative ATPase activity. Moreover, ParA forms ATP-dependent filaments and cables, suggesting that ParA can provide the mechanical force for the observed regular distribution of plasmids. ParA and ParB interact...

  10. Plasmid profiles of Moraxella bovis isolates.

    Science.gov (United States)

    McDonald, T J; Pugh, G W

    1986-04-01

    Two-hundred isolates of Moraxella bovis were selected at random and examined for the presence of plasmid DNA by a rapid alkaline-detergent lysis method. All isolates contained from 1 to 6 plasmids, with varying agarose-gel electrophoretic migration patterns. Most (80%) isolates carried 2 to 4 plasmids, which ranged in molecular weight from 2.6 to 80 megadaltons. Seemingly, plasmid profiles can be used as a simple, reliable epizootiologic tool to establish a strain identification scheme for M bovis.

  11. Plasmid transfer systems in the rhizobia.

    Science.gov (United States)

    Ding, Hao; Hynes, Michael F

    2009-08-01

    Rhizobia are agriculturally important bacteria that can form nitrogen-fixing nodules on the roots of leguminous plants. Agricultural application of rhizobial inoculants can play an important role in increasing leguminous crop yields. In temperate rhizobia, genes involved in nodulation and nitrogen fixation are usually located on one or more large plasmids (pSyms) or on symbiotic islands. In addition, other large plasmids of rhizobia carry genes that are beneficial for survival and competition of rhizobia in the rhizosphere. Conjugative transfer of these large plasmids thus plays an important role in the evolution of rhizobia. Therefore, understanding the mechanism of conjugative transfer of large rhizobial plasmids provides foundations for maintaining, monitoring, and predicting the behaviour of these plasmids during field release events. In this minireview, we summarize two types of known rhizobial conjugative plasmids, including quorum sensing regulated plasmids and RctA-repressed plasmids. We provide evidence for the existence of a third type of conjugative plasmid, including pRleVF39c in Rhizobium leguminosarum bv. viciae strain VF39SM, and we provide a comparison of the different types of conjugation genes found in members of the rhizobia that have had their genomes sequenced so far.

  12. Nitrogen fixation by Klebsiella pneumoniae is inhibited by certain multicopy hybrid nif plasmids.

    Science.gov (United States)

    Riedel, G E; Brown, S E; Ausubel, F M

    1983-01-01

    In our studies of nif gene regulation, we have observed that certain hybrid nif plasmids drastically inhibit the expression of the chromosomal nif genes of Klebsiella pneumonia. Wild-type (Nif+) K. pneumoniae strains that acquire certain hybrid nif plasmids also acquire the Nif- phenotype; these strains lose 90 to 99% of all detectable nitrogen fixation activity and grow poorly (or not at all) on solid media with N2 as the sole nitrogen source. We describe experiments which defined this inhibition of the Nif+ phenotype by hybrid nif plasmids and identify and characterize four nif DNA regions associated with this inhibition. We show that plasmids carrying these nif regions could recombine with, but not complement, nif chromosomal mutations. Our results suggest that inhibition of the Nif+ phenotype will provide a useful bioassay for some of the factors that mediate nif gene expression.

  13. Effect of chromosome homology an plasmid transformation and plasmid conjugal transfer in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Balganesh, M.; Setlow, J.K.

    1984-05-14

    The pairing between plasmid and the homologous part of the chromosome associated with plasmid establishment may differ from the pairing which results from integration of a homologous region of the plasmid into the chromosome. Thus the rate of novobiocin transformation decreases with duplication of the chromosomal portion in pMB2, but the rate of establishment of the plasmid increases with this duplication. A model to explain these data is given. 17 references, 5 figures, 4 tables.

  14. A dnaN plasmid shuffle strain for rapid in vivo analysis of mutant Escherichia coli β clamps provides insight into the role of clamp in umuDC-mediated cold sensitivity.

    Directory of Open Access Journals (Sweden)

    Vignesh M P Babu

    Full Text Available The E. coli umuDC gene products participate in two temporally distinct roles: UmuD2C acts in a DNA damage checkpoint control, while UmuD'2C, also known as DNA polymerase V (Pol V, catalyzes replication past DNA lesions via a process termed translesion DNA synthesis. These different roles of the umuDC gene products are managed in part by the dnaN-encoded β sliding clamp protein. Co-overexpression of the β clamp and Pol V severely blocked E. coli growth at 30°C. We previously used a genetic assay that was independent of the ability of β clamp to support E. coli viability to isolate 8 mutant clamp proteins (βQ61K, βS107L, βD150N, βG157S, βV170M, βE202K, βM204K and βP363S that failed to block growth at 30°C when co-overexpressed with Pol V. It was unknown whether these mutant clamps were capable of supporting E. coli viability and normal umuDC functions in vivo. The goals of this study were to answer these questions. To this end, we developed a novel dnaN plasmid shuffle assay. Using this assay, βD150N and βP363S were unable to support E. coli viability. The remaining 6 mutant clamps, each of which supported viability, were indistinguishable from β+ with respect to umuDC functions in vivo. In light of these findings, we analyzed phenotypes of strains overexpressing either β clamp or Pol V alone. The strain overexpressing β+, but not those expressing mutant β clamps, displayed slowed growth irrespective of the incubation temperature. Moreover, growth of the Pol V-expressing strain was modestly slowed at 30°, but not 42°C. Taken together, these results suggest the mutant clamps were identified due to their inability to slow growth rather than an inability to interact with Pol V. They further suggest that cold sensitivity is due, at least in part, to the combination of their individual effects on growth at 30°C.

  15. A dnaN plasmid shuffle strain for rapid in vivo analysis of mutant Escherichia coli β clamps provides insight into the role of clamp in umuDC-mediated cold sensitivity.

    Science.gov (United States)

    Babu, Vignesh M P; Sutton, Mark D

    2014-01-01

    The E. coli umuDC gene products participate in two temporally distinct roles: UmuD2C acts in a DNA damage checkpoint control, while UmuD'2C, also known as DNA polymerase V (Pol V), catalyzes replication past DNA lesions via a process termed translesion DNA synthesis. These different roles of the umuDC gene products are managed in part by the dnaN-encoded β sliding clamp protein. Co-overexpression of the β clamp and Pol V severely blocked E. coli growth at 30°C. We previously used a genetic assay that was independent of the ability of β clamp to support E. coli viability to isolate 8 mutant clamp proteins (βQ61K, βS107L, βD150N, βG157S, βV170M, βE202K, βM204K and βP363S) that failed to block growth at 30°C when co-overexpressed with Pol V. It was unknown whether these mutant clamps were capable of supporting E. coli viability and normal umuDC functions in vivo. The goals of this study were to answer these questions. To this end, we developed a novel dnaN plasmid shuffle assay. Using this assay, βD150N and βP363S were unable to support E. coli viability. The remaining 6 mutant clamps, each of which supported viability, were indistinguishable from β+ with respect to umuDC functions in vivo. In light of these findings, we analyzed phenotypes of strains overexpressing either β clamp or Pol V alone. The strain overexpressing β+, but not those expressing mutant β clamps, displayed slowed growth irrespective of the incubation temperature. Moreover, growth of the Pol V-expressing strain was modestly slowed at 30°, but not 42°C. Taken together, these results suggest the mutant clamps were identified due to their inability to slow growth rather than an inability to interact with Pol V. They further suggest that cold sensitivity is due, at least in part, to the combination of their individual effects on growth at 30°C.

  16. The cornucopia of intestinal chemosensory transduction

    Directory of Open Access Journals (Sweden)

    Paul P Bertrand

    2009-12-01

    Full Text Available The chemosensory transduction mechanisms that the gastrointestinal (GI tract uses to detect chemical and nutrient stimuli are poorly understood. The GI tract is presented with a wide variety of stimuli including potentially harmful chemicals or toxins as well as 'normal' stimuli including nutrients, bacteria and mechanical forces. Sensory transduction is at its simplest the conversion of these stimuli into a neural code in afferent nerves. Much of the information encoded is used by the enteric nervous system (ENS to generate local reflexes while complementary information is sent to the central nervous system (CNS via afferents or by release of hormones to affect behaviour. This review focuses on the chemosensory transduction mechanisms present in the GI tract. It examines the expression and localisation of the machinery for chemosensory transduction. It summarises the types of cells which might be involved in detecting stimuli and releasing neuroactive transmitters. Finally, it highlights the idea that chemosensory transduction mechanisms in the GI tract utilise many overlapping and complementary mechanisms for detecting and transducing stimuli into reflex action.

  17. Distribution characteristics and drug resistant analysis of plasmid-mediated quinolone drug resistant gene qnr of Klebsiella pneumoniae isolates separated from sputum samples in our hospital%痰标本中质粒介导喹诺酮耐药基因qnr在肺炎克雷伯菌中的分布特征及耐药分析

    Institute of Scientific and Technical Information of China (English)

    菅凌燕; 何晓静; 于莹

    2012-01-01

    目的:了解从痰标本中分离出的肺炎克雷伯菌对16种抗茵药物的耐药性,以及研究由质粒介导的喹诺酮类耐药基因qnr在肺炎克雷伯菌中的存在情况.方法:用PCR及直接测序的方法对135株肺炎克雷伯菌进行qnr基因检测,并用K-B纸片法检测其对16种抗茵药物的体外抗菌活性.另外,用琼脂平皿二倍稀释法检测阳性菌株对左氧氟沙星的MIC值.结果:135株肺炎克雷伯菌中,9株(6.6%)检出qnr基因.阳性菌株均对亚胺培南敏感且对多种抗生素耐药,其中2株qnr阳性菌株对左氧氟沙星敏感.结论:肺炎克雷伯菌中存在质粒介导喹诺酮类耐药基因qnr基因,qnr阳性菌株呈现多重耐药.临床工作中,应加强对耐药基因的监测,降低细菌耐药的发生.%OBJECTIVE To explore the drug resistant characteristics to 16 kinds of antibiotics and the distribution of plasmid-mediated quinolone drug resistant gene qnr of Klebsiella pneumoniae isolates separated from sputum samples. METHODS By using PCR and direct sequencing method, the gene qnr of Klebsiella pneumoniae was detected. Then, the antibacterial activities of 16 kinds of antibiotics on Klebsiella pneumoniae isolates in vitro were studied. Finally, was detected the MIC value of levofloxacin on gene qnr positive Klebsiella pneumoniae isolates with agar plate two-fold dilution method. RESULTS Among all 135 Klebsiella pneumoniae isolates, 9 Klebsiella pneumoniae isolates was determined with gene qnr. These isolates were all sensitive to imipenem and resistant to the other kinds of antibiotics. There were also 2 Klebsiella pneumoniae isolates sensitive to levofloxacin. CONCLUSION There are plasmid-mediated quinolone drug resistant gene qnr in our hospital. Qnr positive i-solates were multi-drug resistant. In clinic, we should pay attention to monitor on drug resistant genes and decrease the frequencies of drug resistant.

  18. Detection and analysis of plasmid-mediated carbapenem-hydrolyzing enzyme KPC-2 in carbapenem-resistant Escherichia coli%大肠埃希菌质粒型碳青霉烯酶KPC-2检测和分析

    Institute of Scientific and Technical Information of China (English)

    蒯守刚; 邵海枫; 王卫萍; 史利宁; 张小卫; 范明

    2009-01-01

    Objective To study molecular epidemiology and carbapenem-resistance mechanism of four Escherichia coli strains isolated from general surgery wards. Methods Antibiotic susceptibility was carried out by K-B gar diffusion and agar dilution methods. Carbapenemases were screened by three dimensional test and EDTA-Na_2-disk synergy test. Pulsed-field gel electropboresis (PFGE) was performed to analyze molecular epidemiology of isolates. Plasmid was extracted by using an alkalinelysis technique. Conjunction experiment, transformation assay, specific PCR and DNA sequencing were performed to confirm carbapenemase genotype and its transmission mechanism Results Four Escherichia coli isolates were resistant to most antimicrobials including carbapenem. PFGE showed that the four isolates belong to four different clonal strains. Specific PCR and DNA sequence analysis identified that carbapenem resistance in four clinical isolates was mediated by KPC-2 encoded on an approximately 56 000 bp plasmid, and this plasmid did not harbor aminoglycosides and fluorquinolones resistant genes. Conclusion Four Escherichia coli isolates with carbapenem resistance are obtained from our hospital, and KPC-2 plasmid is main cause of carbapenem resistance in these isolates.%目的 研究普外科病区出现的4株碳青霉烯类药物耐药大肠埃希菌的分子流行病学特征及耐药机制.方法 用K-B纸片法和琼脂稀释法进行药物敏感试验,三维酶抑制试验和EDTA-Na_2协同试验分析酶的性质,通过脉冲场琼脂糖凝胶电泳(PFGE)分析耐药株的分子流行病学特征,特异性PCR及序列分析、接合试验、碱裂解法提取质粒和质粒转化试验研究碳青霉烯耐药的分子机制.结果 4株大肠埃希菌对包括碳青霉烯在内的多种抗菌药物广泛耐药,PFGE显示4株分离株属于不同的克隆型,对碳青霉烯类药物的耐药主要由相对分子质量约56 000的质粒携带的KPC-2基因介导,转化试验显示对氨基

  19. Genomic and functional characterization of qnr-encoding plasmids from municipal wastewater biosolid Klebsiella pneumoniae isolates

    Directory of Open Access Journals (Sweden)

    Ella eKaplan

    2015-12-01

    Full Text Available Municipal wastewater treatment facilities are considered to be hotspots for antibiotic resistance since they conjoin high densities of environmental and fecal bacteria with selective pressure in the form of sub-therapeutic concentrations of antibiotics. Discharged effluents and biosolids from these facilities can disseminate antibiotic resistant genes to terrestrial and aquatic environments, potentially contributing to the increasing global trend in antibiotic resistance. This phenomenon is especially pertinent when resistance genes are associated with mobile genetic elements such as conjugative plasmids, which can be transferred between bacterial phyla. Fluoroquinolones are among the most abundant antibiotic compounds detected in wastewater treatment facilities, especially in biosolids, where due to their hydrophobic properties they accumulate to concentrations that may exceed 40 mg/L. Although fluoroquinolone resistance is traditionally associated with mutations in the gyrA/topoisomerase IV genes, there is increasing evidence of plasmid-mediated quinolone resistance, which is primarily encoded on qnr genes. In this study, we sequenced seven qnr-harboring plasmids from a diverse collection of Klebsiella strains, isolated from dewatered biosolids from a large wastewater treatment facility in Israel. One of the plasmids, termed pKPSH-11XL was a large (185.4 kbp, multi-drug resistance, IncF-type plasmid that harbored qnrB and 10 additional antibiotic resistance genes that conferred resistance to 5 different antibiotic families. It was highly similar to the pKPN3-like plasmid family that has been detected in multidrug resistant clinical Klebsiella isolates. In contrast, the six additional plasmids were much smaller (7-9Kbp and harbored a qnrS -type gene. These plasmids were highly similar to each other and closely resembled pGNB2, a plasmid isolated from a German wastewater treatment facility. Comparative genome analyses of pKPSH-11XL and other p

  20. The expression of a plasmid-specified exported protein causes structural plasmid instability in Bacillus subtilis

    NARCIS (Netherlands)

    Cordes, C.; Meima, R; Twiest, B; Kazemier, B; Venema, G; vanDijl, JM; Bron, S

    The rolling-circle plasmid pGP1 was used to study the effects of the expression of a plasmid-specified exported protein on structural plasmid stability in Bacillus subtilis. pGP1 contains a fusion between the Bacillus licheniformis penP gene, encoding a C-terminally truncated penicillinase, and the

  1. 摩根摩根菌中质粒介导KPC-2型碳青霉烯酶的检测%Detection of plasmid-mediated carbapenem-hydrolyzing β-lactamase KPC-2 in Morganella morganii

    Institute of Scientific and Technical Information of China (English)

    杨玮; 蔡加昌; 胡燕燕; 周宏伟; 张嵘; 陈功祥

    2011-01-01

    Objective To investigate the molecular epidemiology and mechanisms of carbapenem resistance of Morganella morganii.Methods Seven carbapenem-non-susceptible M.morganii were isolated from Hangzhou Traditional Chinese Medicine Hospital from October 2010 to February 2011.Pulsed-field gel electrophoresis (PFGE) was performed to analysis the molecular epidemiology of isolates.Antibiotic susceptibilities were determined by agar dilution method.Conjugation experiments were carried out in mixed broth cultures.Plasmid DNA was obtained by an alkalinelysis technique and examined by electrophoresis.Specific PCRs and DNA sequencing were preformed to confirm the genotype of β-lactamases.Results PFGE indicated that 6 M.morganii isolates from emergency care unit were indistinguishable or closely related and 1 isolate from intensive care unit was distinguishable.Seven M.morganii showed similar antibiotic susceptibility patterns.M.morganii isolates were resistant to imipenem,were susceptible to meropenem,and were susceptible or intermediate resistant to ertapenem,with MICs of 8 μg/ml,1 μg/ml,and 0.25-0.50 μg/ml,respectively.M.morganii isolates were resistant to penicillins,aztreonam,and ciprofloxacin,were resistant or susceptible to cephalosporins,and were susceptible to amikacin.E.coli (EC600) acquired an approximately 60 kb plasmid from M.morganii by conjugation studies and resistant or intermediate resistant to carbapenems and other β-lactams.PCRs and DNA sequence analysis confirmed that all M.morganii isolates and their E. coli transconjugants produced the KPC-2 carbapenemase and carried the qnrS1 gene.Conclusion It is the first detection of KPC-2 in M.morganii isolates.Production of KPC-2 mainly contributed to the carbapenem resistance in M.morganii.%目的 研究碳青霉烯耐药摩根摩根菌的分子流行病学及其耐药机制.方法 2010年10月-2011年2月从杭州市中医院分离到7株碳青霉烯不敏感的摩根摩根菌.脉冲场凝胶电泳(PFGE)分析

  2. Origin-of-transfer sequences facilitate mobilisation of non-conjugative antimicrobial-resistance plasmids in Staphylococcus aureus

    Science.gov (United States)

    O'Brien, Frances G.; Yui Eto, Karina; Murphy, Riley J. T.; Fairhurst, Heather M.; Coombs, Geoffrey W.; Grubb, Warren B.; Ramsay, Joshua P.

    2015-01-01

    Staphylococcus aureus is a common cause of hospital, community and livestock-associated infections and is increasingly resistant to multiple antimicrobials. A significant proportion of antimicrobial-resistance genes are plasmid-borne, but only a minority of S. aureus plasmids encode proteins required for conjugative transfer or Mob relaxase proteins required for mobilisation. The pWBG749 family of S. aureus conjugative plasmids can facilitate the horizontal transfer of diverse antimicrobial-resistance plasmids that lack Mob genes. Here we reveal that these mobilisable plasmids carry copies of the pWBG749 origin-of-transfer (oriT) sequence and that these oriT sequences facilitate mobilisation by pWBG749. Sequences resembling the pWBG749 oriT were identified on half of all sequenced S. aureus plasmids, including the most prevalent large antimicrobial-resistance/virulence-gene plasmids, pIB485, pMW2 and pUSA300HOUMR. oriT sequences formed five subfamilies with distinct inverted-repeat-2 (IR2) sequences. pWBG749-family plasmids encoding each IR2 were identified and pWBG749 mobilisation was found to be specific for plasmids carrying matching IR2 sequences. Specificity of mobilisation was conferred by a putative ribbon-helix-helix-protein gene smpO. Several plasmids carried 2–3 oriT variants and pWBG749-mediated recombination occurred between distinct oriT sites during mobilisation. These observations suggest this relaxase-in trans mechanism of mobilisation by pWBG749-family plasmids is a common mechanism of plasmid dissemination in S. aureus. PMID:26243776

  3. cAMP依赖性蛋白激酶信号传导通路在大鼠CRH性发热机制中的作用%The cAMP-mediated protein kinase signal transduction pathway is involved in the pyrogenic effect of CRH in rats

    Institute of Scientific and Technical Information of China (English)

    王华东; 王彦平; 屈洋; 戚仁斌; 陆大祥; 李楚杰; 颜亮

    2001-01-01

    目的观察cAMP依赖性蛋白激酶信号传导通路在大鼠CRH性发热机制中的作用。 方法第三脑室注射促肾上腺皮质激素释放激素(CRH),腺苷酸环化酶抑制剂DDA或cAMP依赖性蛋白 激酶抑制剂Rp-cAMPS,测定大鼠结肠温度。用放射免疫分析法测定下丘脑cAMP含量,并用离体实验观 察CRH对下丘脑cAMP含量的影响。 结果第三脑室微量注射CRH(2.5μg,5.0μg,10μg)引起大鼠结肠温度和下丘脑cAMP水平明显升高,下 丘脑cAMP水平与大鼠2小时发热反应指数之间存在明显的正相关(r=0.994,P<0.01)。离体实验同样 观察到CRH可引起下丘脑cAMP水平明显升高。事先向第三脑室微量注射腺苷酸环化酶抑制剂DDA 30μg或cAMP依赖性蛋白激酶抑制剂Rp-cAMPs 15μg均显著抑制中枢注射CRH引起的发热反应。 结论这些结果证明,cAMP参与了大鼠CRH性发热的中枢机制,cAMP依赖性蛋白激酶可能在CRH性发 热机制中发挥重要作用,cAMP依赖性蛋白激酶信号传导通路可能介导中枢注射CRH对大鼠的致热作用。%To determine whether the cyclic adenosine monophosphate (cAMP) mediated protein kinase signal transduction pathway is involved in the pyrogenic action of corticotropin releasing hormone (CRH) in rats. Methods Corticotropin releasing hormone, 2', 3 '-dideoxyadenosine (DDA) and adenosine-3', 5' (cyclic) monophosphorothionate, Rp-lsomer (Rp-cAMPS), were administered intracerebroventricularly (i.c.v.). The colonic temperature was measured using a thermistor, and the content of cAMP in the hypothalamus was determined by radioimmunoassay. Hypethalemic incubation was used to assess the effects of CRH on the content of cAMP in the hypothalamus in vitro. Results Microinjection (i.c.v.) of CRH (2.5 μg, 5.0 μg and 10 μg) caused increases in colonic temperature and the hypothalemus cAMP level in conscious rats. CRH increased hypothalemus cAMP level in vitro. The pyrogenic effects of

  4. Bioinformatics analyses for signal transduction networks

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Research in signaling networks contributes to a deeper understanding of organism living activities. With the development of experimental methods in the signal transduction field, more and more mechanisms of signaling pathways have been discovered. This paper introduces such popular bioin-formatics analysis methods for signaling networks as the common mechanism of signaling pathways and database resource on the Internet, summerizes the methods of analyzing the structural properties of networks, including structural Motif finding and automated pathways generation, and discusses the modeling and simulation of signaling networks in detail, as well as the research situation and tendency in this area. Now the investigation of signal transduction is developing from small-scale experiments to large-scale network analysis, and dynamic simulation of networks is closer to the real system. With the investigation going deeper than ever, the bioinformatics analysis of signal transduction would have immense space for development and application.

  5. Downstream reporter gene imaging for signal transduction pathway of dopamine type 2 receptor

    Energy Technology Data Exchange (ETDEWEB)

    Le, Uyenchi N.; Min, Jung Joon; Moon, Sung Min; Bom, Hee Seung [School of Midicine, Chonnam National University, Gwangju (Korea, Republic of)

    2004-07-01

    The Dopamine 2 receptor (D2R) signal pathway regulates gene expression by phosphorylation of proteins including cAMP reponse element-binding protein (CREB), a transcription factor. In this study, we developed a reporter strategy using the GAL4 fusion CREB to assess the phosphorylation of CREB, one of the targets of the D2R signal transduction pathway. We used three plasmids: GAL4 fusion transactivator (pCMV-CREB), firefly luciferase reporter with GAL4 binding sites (pG5-FLUC), and D2R plasmid (pCMV-D2R). Group 1 293T cells were transiently transfected with pCMV-CREB and pG5-FLUC, and group 2 cells were transfected with all three plasmids. Transfected cells were stimulated with different concentrations of dopamine (0-200 M). For animal studies, group 1 and 2 cells (1x10{sup 6}) were subcutaneously injected on the left and right thigh of six nude mice, respectively. Dopamine stimiulation was performed with intraperitoneal injection of L-DOPA incombination with carbidopa, a peripheral DOPA decarboxylase inhibitor. Bioluminescence optical imaging studies were performed before and after L-DOPA injection. In cell culture studies, group 1 cells showed strong luciferase activity which implies direct activation of the signaling pathway due to growth factors contained in culture medium. Group 2 cells showed strong luciferase activity and a further increase after administration of dopamine. In animal studies, group 1 and 2 cells showed bioluminescence signal before L-DOPA injection, but signal from group 2 cells significantly increased 12 h after L-DOPA injection. The signal from group 1 cells disappeared thereafter, but group 2 cells continued to show signal until 36 h of L-DOPA injection. This study demonstrates imaging of the D2R signal transduction pathway and should be useful for noninvasive imaging of downstream effects of G-coupled protein pathways.

  6. Mechanism of DNA Segregation in Prokaryotes: Replicon Pairing by parC of Plasmid R1

    Science.gov (United States)

    Jensen, Rasmus Bugge; Lurz, Rudi; Gerdes, Kenn

    1998-07-01

    Prokaryotic chromosomes and plasmids encode partitioning systems that are required for DNA segregation at cell division. The systems are thought to be functionally analogous to eukaryotic centromeres and to play a general role in DNA segregation. The parA system of plasmid R1 encodes two proteins ParM and ParR, and a cis-acting centromere-like site denoted parC. The ParR protein binds to parC in vivo and in vitro. The ParM protein is an ATPase that interacts with ParR specifically bound to parC. Using electron microscopy, we show here that parC mediates efficient pairing of plasmid molecules. The pairing requires binding of ParR to parC and is stimulated by the ParM ATPase. The ParM mediated stimulation of plasmid pairing is dependent on ATP hydrolysis by ParM. Using a ligation kinetics assay, we find that ParR stimulates ligation of parC-containing DNA fragments. The rate-of-ligation was increased by wild type ParM protein but not by mutant ParM protein deficient in the ATPase activity. Thus, two independent assays show that parC mediates pairing of plasmid molecules in vitro. These results are consistent with the proposal that replicon pairing is part of the mechanism of DNA segregation in prokaryotes.

  7. Purinergic mechanosensory transduction and visceral pain

    Directory of Open Access Journals (Sweden)

    Burnstock Geoffrey

    2009-11-01

    Full Text Available Abstract In this review, evidence is presented to support the hypothesis that mechanosensory transduction occurs in tubes and sacs and can initiate visceral pain. Experimental evidence for this mechanism in urinary bladder, ureter, gut, lung, uterus, tooth-pulp and tongue is reviewed. Potential therapeutic strategies are considered for the treatment of visceral pain in such conditions as renal colic, interstitial cystitis and inflammatory bowel disease by agents that interfere with mechanosensory transduction in the organs considered, including P2X3 and P2X2/3 receptor antagonists that are orally bioavailable and stable in vivo and agents that inhibit or enhance ATP release and breakdown.

  8. Fluoroquinolone induction of phage-mediated gene transfer in multidrug-resistant Salmonella.

    Science.gov (United States)

    Bearson, Bradley L; Brunelle, Brian W

    2015-08-01

    Fluoroquinolones are broad-spectrum antibiotics that inhibit bacterial DNA gyrase and topoisomerase activity, which can cause DNA damage and result in bacterial cell death. In response to DNA damage, bacteria induce an SOS response to stimulate DNA repair. However, the SOS response may also induce prophage with production of infectious virions. Salmonella strains typically contain multiple prophages, and certain strains including phage types DT120 and DT104 contain prophage that upon induction are capable of generalised transduction. In this study, strains of multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium DT120 and DT104 were exposed to fluoroquinolones important for use in human and veterinary disease therapy to determine whether prophage(s) are induced that could facilitate phage-mediated gene transfer. Cultures of MDR S. Typhimurium DT120 and DT104 containing a kanamycin resistance plasmid were lysed after exposure to fluoroquinolones (ciprofloxacin, enrofloxacin and danofloxacin). Bacterial cell lysates were able to transfer the plasmid to a recipient kanamycin-susceptible Salmonella strain by generalised transduction. In addition, exposure of DT120 to ciprofloxacin induced the recA gene of the bacterial SOS response and genes encoded in a P22-like generalised transducing prophage. This research indicates that fluoroquinolone exposure of MDR Salmonella can facilitate horizontal gene transfer, suggesting that fluoroquinolone usage in human and veterinary medicine may have unintended consequences, including the induction of phage-mediated gene transfer from MDR Salmonella. Stimulation of gene transfer following bacterial exposure to fluoroquinolones should be considered an adverse effect, and clinical decisions regarding antibiotic selection for infectious disease therapy should include this potential risk. Published by Elsevier B.V.

  9. Suppression of tumorigenicity and metastatic potential of melanoma cells by transduction of interferon gene

    Directory of Open Access Journals (Sweden)

    Lykhova A. A.

    2014-01-01

    Full Text Available The aim of this study was to investigate an inhibitory effect of baculovirus-mediated transduction of the murine interferon-beta gene on mouse melanoma in vitro and in vivo. Methods. Studies were performed on B16 mouse melanoma (MM-4 cell line. Transduction, immunocytochemical and tumor cell biology approaches have been used in this study. Results. Transduction of MM-4 cells by the recombinant baculovirus with IFN-beta gene is accompanied by morphological changes of tumor cells, suppression of cell proliferation, significant inhibition of platting efficiency of cells and their colonies formation in semisolid agar. Moreover, transduction of melanoma MM-4 cells by the baculovirus IFN-transgene leads to inhibition of tumorigenicity and metastatic ability of the cells in vivo. The intravenous administration of recombinant baculovirus vector with IFN gene inhibits growth of metastases induced in the lungs of mice by intravenously injected tumor cells. Conclusions. Transduction of mouse melanoma cells by the recombinant baculovirus with murine IFN-beta gene inhibits their proliferative potential, tumorigenicity and metastatic activity.

  10. Neuro-protective effects of CNTF on hippocampal neurons via an unknown signal transduction pathway

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    In our previous study, we proposed that there may be an unknown pathway in the upper stream of the known signal transduction pathway of Ciliary neurotrophic factor (CNTF) that mediates the neuro-protective function of CNTF. In the present experiment, we observed that the neuro-protective function of the non-classic signal transduction pathway in a L-NMDA (a glutamic acid ion type receptor atagonist) induced hippocampal neuron injury model, using primary culture rat hippocampal neurons, continuous photography and gp130 immunohistochemical assay. The results showed that L-NMDA induced injurious reaction of hippocampal neurons, and CNTF was able to inhibit the toxic action of L-NMDA on hippocampal neurons. Additionally, when JAK/STATs in the known classic signal transduction pathway of CNTF were blocked by PTPi-2, the protective effect of CNTF against L-NMDA injury still existed. L-NMDA caused a rapid increase in the concentration of hippocampal intracellular free [Ca2+]i. CNTF was able to attenuate L-NMDA-induced elevation of [Ca2+]i, and blocking JAK/STATs in the known classic signal trans- duction pathway of CNTF did not affect L-NMDA- induced elevation of [Ca2+]i, indicating that, apart from the known classic signal transduction pathway, there may be some other transduction pathways for CNTF to exert the protective effect on hippocampal neurons, and this pathway is related to [Ca2+].

  11. Analysis of nitrated proteins in Saccharomyces cerevisiae involved in mating signal transduction.

    Science.gov (United States)

    Kang, Jeong Won; Lee, Na Young; Cho, Kyung-Cho; Lee, Min Young; Choi, Do-Young; Park, Sang-Hyun; Kim, Kwang Pyo

    2015-01-01

    Protein tyrosine nitration (PTN) is a PTM that regulates signal transduction and inflammatory responses, and is related to neurodegenerative and cardiovascular diseases. The cellular function of PTN remains unclear because the low stoichiometry of PTN limits the identification and quantification of nitrated peptides. Effective enrichment is an important aspect of PTN analysis. In this study, we analyzed the in vivo nitroproteome elicited by mating signal transduction in Saccharomyces cerevisiae using a novel chemical enrichment method followed by LC-MS/MS. Nitroproteome profiling successfully identified changes in the nitration states of 14 proteins during mating signal transduction in S. cerevisiae, making this the first reported in vivo nitroproteome in yeast. We investigated the biological functions of these nitroproteins and their relationships to mating signal transduction in S. cerevisiae using a protein-protein interaction network. Our results suggest that PTN and denitration may be involved in nonreactive nitrogen species-mediated signal transduction and can provide clues for understanding the functional roles of PTN in vivo.

  12. 重组腺病毒气管途径反复转染大鼠肺组织人类eNOS基因的转导效果%Efficiency of transduction of recombinant adenovirus-mediated human endothelial nitric oxide synthase gene into lung tissue by repeated intratracheal transfection in rats

    Institute of Scientific and Technical Information of China (English)

    周锦; 曹惠鹃; 张铁铮; 金强; 王俊科

    2012-01-01

    Objective To investigate the efficiency of transduction of recombinant adenovirus-mediated human endothelial nitric oxide synthase (eNOS) into lung tissue by repeated intratracheal transfection in rats.Methods Sixty 3-4 month old male Wistar rats weighing 220-280 g were randomly divided into 2 groups:control group (group C,n =10) and eNOS gene transduction group (group T,n =50).The animals were anesthetized with intraperitoneal 10% chloral hydrate 35 mg/kg,tracheally intubated and mechanically ventilated (VT 2.5 ml,RR 60 bpm,FiO2 1.0).Recombinant adenovirus carrying human eNOS gene was given as gift by Professor Gerard from Texas University,Southwest Medical Center.In group T 50 μl of the recombinant adenovirus in concentration of 5 × 109 PFU/ml was instilled into trachea every 5 minutes for 12 times,while in group C equal volume of vector conservation solution was instilled instead.Pulmonary arterial blood samples were obtained at 2,5,7,14 and 21 d after intratracheal transfection (n =10 at each time point) for determination of serum NO concentration.The animals were immediately sacrificed after blood sample collection for determination of expression of eNOS protein in the lung tissue and RNA.The eNOS expression in the trachea,bronchus,lung,liver,spleen and kidney was detected by immuno-histochemistry.Results The serum NO concentrations were significantly higher at all time points in group T than in group C.The eNOS expression was detected in the epithelial cells of trachea and bronchi,and endothelial cells of alveoli and pulmonary blood vessels in group T but not in group C.eNOS expression was not detected in liver,spleen and kidney at 7 d after intratracheal transfection in group T.Conclusion Human eNOS gene mediated by recombinant adenovirus was transducted into rat lung tissue with normal enzyme activity by repeated intratracheal administration without being detected in distant organs.%目的 重组腺病毒气管途径反复转染大鼠肺组织人类内

  13. 枸橼酸杆菌中质粒介导喹诺酮耐药基因的检测%Study of plasmid-mediated quinolone resistance determinants in Citrobacter freundii

    Institute of Scientific and Technical Information of China (English)

    邵宜波; 李旭; 胡立芬; 谢琴秀

    2013-01-01

    determine whether the qnr-carrying plasmids were self-transferable.The susceptibility of the positive isolates and transconjugants were tested by agar dilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines.The minimum inhibitory concentrations (MIC) of ciprofloxacin and levofloxacin were determined by E-test strips.Results Among the 31 Citrobacter strains,the qnr genes were detected in 8 isolates (25.8%),among which,6 carried qnrB.Aac-(6′)-Ib-cr and qepA were not identified in these isolates.The qnr genes were transferred from four clinical isolates to their transconjugants.Sequence analysis identified one novel qnrB variant (qnrB24).The resistant rate of qnr-positive clinical isolates to quinolone was 87.5 %.Most of them were also resistant to various other antibiotics,including cefotaxime (75.0 %),amikacin (7.5 %),ceftazidime (62.5 %),cefapime (37.5 %),and gentamycin (87.5 %).All qnr positive strains were susceptible to imipenem.MIC of all transconjugants showed reduced susceptibility to fluoroquinolones,with MIC increased by 10-23 folds.Conclusions Our study shows that qnr gene has occurred in Citrobacter freundii isolates from Anhui Province,China.QnrB is most prevalent in these isolates.Most qnr positive isolates are resistant to commonly used antimicrobial agents.

  14. Energy transduction in lactic acid bacteria

    NARCIS (Netherlands)

    Poolman, Bert

    In the discovery of some general principles of energy transduction, lactic acid bacteria have played an important role. In this review, the energy transducing processes of lactic acid bacteria are discussed with the emphasis on the major developments of the past 5 years. This work not only includes

  15. Inhibitors targeting two-component signal transduction.

    Science.gov (United States)

    Watanabe, Takafumi; Okada, Ario; Gotoh, Yasuhiro; Utsumi, Ryutaro

    2008-01-01

    A two-component signal transduction system (TCS) is an attractive target for antibacterial agents. In this chapter, we review the TCS inhibitors developed during the past decade and introduce novel drug discovery systems to isolate the inhibitors of the YycG/YycF system, an essential TCS for bacterial growth, in an effort to develop a new class of antibacterial agents.

  16. Clinical study on safety and immunogenicity of therapeutic dual-plasmid HBV DNA vaccine mediated by in vivo electroporation%电脉冲介导的治疗性双质粒HBV DNA疫苗的临床安全性及免疫原性研究

    Institute of Scientific and Technical Information of China (English)

    杨海燕; 陈光明; 崔一民; 赵侠; 梅川; 饶桂荣; 莫国玉; 杨若才; 杨富强

    2013-01-01

    目的 观察电脉冲(EP)介导的治疗性双质粒HBV DNA疫苗的安全性及免疫原性.方法 将30名健康志愿者随机分为低(1mg)、中(2mg)、高(4mg)三个剂量组(n=10).于0、4、12、24周肌注联合EP导入治疗性双质粒HBVDNA疫苗.每个剂量组随机再分成两组(n=5),分别使用两种不同输出电压(36V和60V)导入DNA疫苗.观察受试者HBVDNA疫苗给药前后生命体征,物理学诊断指标(心电图、胸透、B超),实验室检查指标(血、尿常规、血液生化、凝血酶原时间、甲状腺功能、肿瘤标记物),免疫学检测指标[干扰素y(IFN-γ)、抗核抗体(ANA)、抗双链DNA抗体]、HBV血清标记物(HBsAg、HBcAb、HBeAg、HBeAb、HBV DNA)及抗HBs等的变化.结果 所有志愿者接受疫苗后,耐受性良好、生命体征平稳,个别受试者出现一过性体检指标升高或轻度异常,怀疑与用药有关,但均能自行缓解或恢复;抗HBs在大剂量36V组中有升高趋势,其中1例受试者给药后达17.22mU/ml.结论 EP介导的治疗性双质粒HBVDNA疫苗在低、中、高三个剂量组中均显示较好的耐受性和安全性,且在大剂量组中具有一定的体液免疫原性.%Objective To evaluate the safety and immunogenicity of the therapeutic dual-plasmid HBV DNA vaccine mediated by electroporation (EP) in vivo against the hepatitis B virus in healthy adult volunteers. Methods The enrolled 30 healthy volunteers were randomly divided into three dosage groups (10 volunteers in each group), namely: high-dose (4mg), middle-dose (2mg) and low-dose (1mg) groups. Volunteers received four intramuscular injections of HBV DNA vaccine mediated by in vivo EP at the 0, 4th, 12th and 24th week. Each dose group was further divided into 2 sub-groups (5 persons/per group) with different EP frequencies, i.e. 36 and 60 volt. The changes in response was determined by physical diagnosis (ECG, chest X-ray, type-B ultrasound), lab findings (blood and urine routine, blood

  17. 电穿孔介导的pIRES-hVEGF165-EGFP转染对牵引成骨过程中早期血管生成的影响%Effect of electroporation mediated transfecting recombinant plasmid pIRES-hVEGF165-EGFP on angiogene-sis of distraction area during early mandibular distraction osteogenesis

    Institute of Scientific and Technical Information of China (English)

    吴国平; 黎德平; 何小川; 李盛华; 杨智慧; 廖毅; 郭力

    2010-01-01

    目的 探讨电穿孔介导的基因治疗对下领骨牵引成骨过程中早期血管生成的影响.方法 32只新西兰大白兔随机分为4组:质粒+电穿孔组(A组),质粒组(B组),生理盐水+电穿孔组(C组),空白对照组(D组).各组动物分别于注射后1、3、7、14 d处死,取牵引区组织进行组织学检查、电镜观察、CD34免疫组织化学染色及微血管密度检测.结果 A、B组血管内皮细胞呈增殖活跃状态;C、D组多数血管内皮细胞部分呈现退变及凋亡早期改变.免疫组化染色发现,转染后第1天血管壁内皮细胞浆CD34表达较弱;第3、7、14天,牵引区肉芽组织血管内皮细胞均出现CD34阳性表达.A组CD34阳性表达较B组强,A、B组的CD34表达持续阳性且呈上升趋势;C、D组表达最弱,CD34阳性表达维持在第1天水平上平稳波动.结论 电穿孔介导的pIRES-hVEGF165-EGFP重组质粒体内转染能够促进牵引区早期微血管的生成,使局部血管增生、渗入,增加骨断端的血流量.对调节和促进骨的生长和修复过程具有重要作用.%Objective To explore the effect of electroporation mediated gene therapy on angiogene-sis of the distraction area during early mandibular distraction osteogenesis (DO). Methods Thirty-two New-Zeland rabbits were randomly divided into 4 groups: group A: recombinant plasmid pIRES-VEGF165-EGFP and electroporation; group B: recombinant plasmid pIRES-VEGF165-EGFP; group C: normal saline (NS) and electroporation and group D: control group. The rabbits were sacrificed at 1d, 3d, 7d and 14d after injection, respectively. The distraction area tissue was removed for histological examination and electron microscopy, and immunohistochemical stain for CD34 was performed to detect the microvessel density. Results Generation of vascular endothelial cells (VEC) in the group A and group B were active, and majority of VEC in groups C and D took on early change of cataplasia and apoptosis. The

  18. Role of TLR3 in the immunogenicity of replicon plasmid-based vaccines.

    Science.gov (United States)

    Diebold, S S; Schulz, O; Alexopoulou, L; Leitner, W W; Flavell, R A; Reis e Sousa, C

    2009-03-01

    Replicon plasmids encoding an alphavirus RNA replicase constitute an alternative to conventional DNA plasmids with promise for DNA vaccination in humans. Replicase activity amplifies the levels of transgene mRNA through a copying process involving double-stranded (ds) RNA intermediates, which contribute to vaccine immunogenicity by activating innate antiviral responses. Toll-like receptor 3 (TLR3) is a dsRNA innate immune receptor expressed by antigen-presenting dendritic cells (DCs). Here, we test the hypothesis that TLR3 is necessary for the immunogenicity of replicon plasmid-based DNA vaccines. We show that mouse CD8 alpha(+) DC phagocytose dying replicon plasmid-transfected cells in vitro and are activated in a TLR3-dependent manner by dsRNA present within those cells. However, we find that cytotoxic T-cell responses to a replicon plasmid intramuscular vaccine are not diminished in the absence of TLR3 in vivo. Our results underscore the potential role of TLR3 in mediating immune activation by dsRNA-bearing replicon plasmid-transfected cells and indicate that other innate sensing pathways can compensate for TLR3 absence in vivo.

  19. Genomics of high molecular weight plasmids isolated from an on-farm biopurification system.

    Science.gov (United States)

    Martini, María C; Wibberg, Daniel; Lozano, Mauricio; Torres Tejerizo, Gonzalo; Albicoro, Francisco J; Jaenicke, Sebastian; van Elsas, Jan Dirk; Petroni, Alejandro; Garcillán-Barcia, M Pilar; de la Cruz, Fernando; Schlüter, Andreas; Pühler, Alfred; Pistorio, Mariano; Lagares, Antonio; Del Papa, María F

    2016-06-20

    The use of biopurification systems (BPS) constitutes an efficient strategy to eliminate pesticides from polluted wastewaters from farm activities. BPS environments contain a high microbial density and diversity facilitating the exchange of information among bacteria, mediated by mobile genetic elements (MGEs), which play a key role in bacterial adaptation and evolution in such environments. Here we sequenced and characterized high-molecular-weight plasmids from a bacterial collection of an on-farm BPS. The high-throughput-sequencing of the plasmid pool yielded a total of several Mb sequence information. Assembly of the sequence data resulted in six complete replicons. Using in silico analyses we identified plasmid replication genes whose encoding proteins represent 13 different Pfam families, as well as proteins involved in plasmid conjugation, indicating a large diversity of plasmid replicons and suggesting the occurrence of horizontal gene transfer (HGT) events within the habitat analyzed. In addition, genes conferring resistance to 10 classes of antimicrobial compounds and those encoding enzymes potentially involved in pesticide and aromatic hydrocarbon degradation were found. Global analysis of the plasmid pool suggest that the analyzed BPS represents a key environment for further studies addressing the dissemination of MGEs carrying catabolic genes and pathway assembly regarding degradation capabilities.

  20. Signal Transduction and Intracellular Trafficking by the Interleukin 36 Receptor*

    Science.gov (United States)

    Saha, Siddhartha S.; Singh, Divyendu; Raymond, Ernest L.; Ganesan, Rajkumar; Caviness, Gary; Grimaldi, Christine; Woska, Joseph R.; Mennerich, Detlev; Brown, Su-Ellen; Mbow, M. Lamine; Kao, C. Cheng

    2015-01-01

    Improper signaling of the IL-36 receptor (IL-36R), a member of the IL-1 receptor family, has been associated with various inflammation-associated diseases. However, the requirements for IL-36R signal transduction remain poorly characterized. This work seeks to define the requirements for IL-36R signaling and intracellular trafficking. In the absence of cognate agonists, IL-36R was endocytosed and recycled to the plasma membrane. In the presence of IL-36, IL-36R increased accumulation in LAMP1+ lysosomes. Endocytosis predominantly used a clathrin-mediated pathway, and the accumulation of the IL-36R in lysosomes did not result in increased receptor turnover. The ubiquitin-binding Tollip protein contributed to IL-36R signaling and increased the accumulation of both subunits of the IL-36R. PMID:26269592

  1. Molecular biology of thermosensory transduction in C. elegans.

    Science.gov (United States)

    Aoki, Ichiro; Mori, Ikue

    2015-10-01

    As the environmental temperature prominently influences diverse biological aspects of the animals, thermosensation and the subsequent information processing in the nervous system has attracted much attention in biology. Thermotaxis in the nematode Caenorhabditis elegans is an ideal behavioral paradigm by which to address the molecular mechanism underlying thermosensory transduction. Molecular genetic analysis in combination with other physiological and behavioral studies revealed that sensation of ambient temperature is mediated mainly by cyclic guanosine monophosphate (cGMP) signaling in thermosensory neurons. The information of the previously perceived temperature is also stored within the thermosensory neurons, and the consequence of the comparison between the past and the present temperature is conveyed to the downstream interneurons to further regulate the motor-circuits that encode the locomotion.

  2. The signal transduction mechanisms on the intestinal mucosa of rat following irradiation

    Energy Technology Data Exchange (ETDEWEB)

    You, J. H.; Kim, S. S.; Lee, K. J.; Lee, J. S. [Ewha Womans Univ., Seoul (Korea, Republic of). Coll. of Medicine

    1997-06-01

    Phospholipase C(PLC) isozymes play significant roles in signal transduction mechanism. The exact mechanisms of these signal transduction following irradiation, however, were not clearly documented. Thus, this study was planned to determine the biological significance of PLC, ras oncoprotein, EGFR, and PKC in damage and regeneration of rat intestinal mucosa following irradiation. Sixty Sprague-Dawley rats were irradiated to entire body with a single dose of 8Gy. The rats were divided into 5 groups according to the sacrifice days after irradiation. The expression of PLC, ras oncoprotein, EGRF PKC in each group were examined by the immunoblotting and immunohistochemistry. The histopathologic findings were observed using H and E stain, and the mitoses for the evidence of regeneration were counted using the light microscopy and PCNA kit. The phosphoinositide(PI) hydrolyzing activity assay was also done for the indirect evaluation of PLC-{gamma}1 activity. In the immunohistochemistry, the expression of PLC-{beta} was negative for all groups. The expression of PLC-{gamma}1 was highest in the group III followed by group II in the proliferative zone of mucosa. The expression of PKC-{delta}1 was strongly positive in group I followed by group II in the damaged surface epithelium. The above findings were also confirmed in the immunoblotting study. In the immunoblotting study, the expressions of PLC-{beta}, PLC-{gamma}1, and PLC-{delta}1 were the same as the results of immunohistochemistry. The expression of ras oncoprotein was weakly positive in groups II, III and IV and the expression of PKC was weakly positive in the group II and III. PLC-{gamma}1 mediated signal transduction including ras oncoprotein, EGFR, and PKC play a significant role in mucosal regeneration after irradiation. PLC-{delta}1 mediated signal transduction might have an important role in mucosal damage after irradiation. Further studies will be necessary to confirm the signal transduction mediating the PLC

  3. The Plasmid-Encoded Regulator Activates Factors Conferring Lysozyme Resistance on Enteropathogenic Escherichia coli Strains▿

    Science.gov (United States)

    Salinger, Nina; Kokona, Bashkim; Fairman, Robert; Okeke, Iruka N.

    2009-01-01

    We demonstrate that enhanced lysozyme resistance of enteropathogenic Escherichia coli requires the plasmid-encoded regulator, Per, and is mediated by factors outside the locus for enterocyte effacement. EspC, a Per-activated serine protease autotransporter protein, conferred enhanced resistance on nonpathogenic E. coli, and a second Per-regulated, espC-independent lysozyme resistance mechanism was identified. PMID:18997020

  4. The plasmid-encoded regulator activates factors conferring lysozyme resistance on enteropathogenic Escherichia coli strains.

    Science.gov (United States)

    Salinger, Nina; Kokona, Bashkim; Fairman, Robert; Okeke, Iruka N

    2009-01-01

    We demonstrate that enhanced lysozyme resistance of enteropathogenic Escherichia coli requires the plasmid-encoded regulator, Per, and is mediated by factors outside the locus for enterocyte effacement. EspC, a Per-activated serine protease autotransporter protein, conferred enhanced resistance on nonpathogenic E. coli, and a second Per-regulated, espC-independent lysozyme resistance mechanism was identified.

  5. Sequencing of IncX-plasmids suggests ubiquity of mobile forms of a biofilm-promoting gene cassette recruited from Klebsiella pneumoniae.

    Directory of Open Access Journals (Sweden)

    Mette Burmølle

    Full Text Available Plasmids are a highly effective means with which genetic traits that influence human health, such as virulence and antibiotic resistance, are disseminated through bacterial populations. The IncX-family is a hitherto sparsely populated group of plasmids that are able to thrive within Enterobacteriaceae. In this study, a replicon-centric screening method was used to locate strains from wastewater sludge containing plasmids belonging to the IncX-family. A transposon aided plasmid capture method was then employed to transport IncX-plasmids from their original hosts (and co-hosted plasmids into a laboratory strain (Escherichia coli Genehogs® for further study. The nucleotide sequences of the three newly isolated IncX-plasmids (pLN126_33, pMO17_54, pMO440_54 and the hitherto un-sequenced type-plasmid R485 revealed a remarkable occurrence of whole or partial gene cassettes that promote biofilm-formation in Klebsiella pneumonia or E. coli, in all four instances. Two of the plasmids (R485 and pLN126_33 were shown to directly induce biofilm formation in a crystal violet retention assay in E. coli. Sequence comparison revealed that all plasmid-borne forms of the type 3 fimbriae encoding gene cassette mrkABCDF were variations of a composite transposon Tn6011 first described in the E. coli IncX plasmid pOLA52. In conclusion, IncX-plasmids isolated from Enterobacteriaceae over almost 40 years and on three different continents have all been shown to carry a type 3 fimbriae gene cassette mrkABCDF stemming from pathogenic K. pneumoniae. Apart from contributing general knowledge about IncX-plasmids, this study also suggests an apparent ubiquity of a mobile form of an important virulence factor and is an illuminating example of the recruitment, evolution and dissemination of genetic traits through plasmid-mediated horizontal gene transfer.

  6. Sequencing of IncX-plasmids suggests ubiquity of mobile forms of a biofilm-promoting gene cassette recruited from Klebsiella pneumoniae.

    Science.gov (United States)

    Burmølle, Mette; Norman, Anders; Sørensen, Søren J; Hansen, Lars Hestbjerg

    2012-01-01

    Plasmids are a highly effective means with which genetic traits that influence human health, such as virulence and antibiotic resistance, are disseminated through bacterial populations. The IncX-family is a hitherto sparsely populated group of plasmids that are able to thrive within Enterobacteriaceae. In this study, a replicon-centric screening method was used to locate strains from wastewater sludge containing plasmids belonging to the IncX-family. A transposon aided plasmid capture method was then employed to transport IncX-plasmids from their original hosts (and co-hosted plasmids) into a laboratory strain (Escherichia coli Genehogs®) for further study. The nucleotide sequences of the three newly isolated IncX-plasmids (pLN126_33, pMO17_54, pMO440_54) and the hitherto un-sequenced type-plasmid R485 revealed a remarkable occurrence of whole or partial gene cassettes that promote biofilm-formation in Klebsiella pneumonia or E. coli, in all four instances. Two of the plasmids (R485 and pLN126_33) were shown to directly induce biofilm formation in a crystal violet retention assay in E. coli. Sequence comparison revealed that all plasmid-borne forms of the type 3 fimbriae encoding gene cassette mrkABCDF were variations of a composite transposon Tn6011 first described in the E. coli IncX plasmid pOLA52. In conclusion, IncX-plasmids isolated from Enterobacteriaceae over almost 40 years and on three different continents have all been shown to carry a type 3 fimbriae gene cassette mrkABCDF stemming from pathogenic K. pneumoniae. Apart from contributing general knowledge about IncX-plasmids, this study also suggests an apparent ubiquity of a mobile form of an important virulence factor and is an illuminating example of the recruitment, evolution and dissemination of genetic traits through plasmid-mediated horizontal gene transfer.

  7. High-frequency conjugal plasmid transfer from gram-negative Escherichia coli to various gram-positive coryneform bacteria.

    Science.gov (United States)

    Schäfer, A; Kalinowski, J; Simon, R; Seep-Feldhaus, A H; Pühler, A

    1990-03-01

    We report on the mobilization of shuttle plasmids from gram-negative Escherichia coli to gram-positive corynebacteria mediated by P-type transfer functions. Introduction of plasmids into corynebacteria was markedly enhanced after heat treatment of the recipient cells. High-frequency plasmid transfer was also observed when the restriction system of the recipient was mutated. On the basis of our data, we conclude that efficient DNA transfer from gram-negative to gram-positive bacteria, at least to coryneform bacteria, is conceivable in certain natural ecosystems.

  8. Plasmids in the driving seat: The regulatory RNA Rcd gives plasmid ColE1 control over division and growth of its E. coli host.

    Science.gov (United States)

    Gaimster, Hannah; Summers, David

    2015-03-01

    Regulation by non-coding RNAs was found to be widespread among plasmids and other mobile elements of bacteria well before its ubiquity in the eukaryotic world was suspected. As an increasing number of examples was characterised, a common mechanism began to emerge. Non-coding RNAs, such as CopA and Sok from plasmid R1, or RNAI from ColE1, exerted regulation by refolding the secondary structures of their target RNAs or modifying their translation. One regulatory RNA that seemed to swim against the tide was Rcd, encoded within the multimer resolution site of ColE1. Required for high fidelity maintenance of the plasmid in recombination-proficient hosts, Rcd was found to have a protein target, elevating indole production by stimulating tryptophanase. Rcd production is up-regulated in dimer-containing cells and the consequent increase in indole is part of the response to the rapid accumulation of dimers by over-replication (known as the dimer catastrophe). It is proposed that indole simultaneously inhibits cell division and plasmid replication, stopping the catastrophe and allowing time for the resolution of dimers to monomers. The idea of a plasmid-mediated cell division checkpoint, proposed but then discarded in the 1980s, appears to be enjoying a revival. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Broad-Host-Range IncP-1 plasmids and their resistance potential

    Directory of Open Access Journals (Sweden)

    Magdalena ePopowska

    2013-03-01

    Full Text Available The plasmids of the incompatibility group IncP-1, also called IncP, as extrachromosomal genetic elements can transfer and replicate virtually in all Gram-negative bacteria. They are composed of backbone genes that encode a variety of essential functions and accessory genes that have implications for human health and environmental bioremediation. Broad-host-range IncP plasmids are known to spread genes between distinct phylogenetic groups of bacteria. These genes often code for resistances to a broad spectrum of antibiotics, heavy metals and quaternary ammonium compounds used as disinfectants. The backbone of these plasmids carries modules that enable them to effectively replicate, move to a new host via conjugative transfer and to be stably maintained in bacterial cells. The adaptive, resistance and virulence genes are mainly located on mobile genetic elements integrated between the functional plasmid backbone modules. Environmental studies have demonstrated the wide distribution of IncP-like replicons in manure, soils and wastewater treatment plants. They also are present in strains of pathogenic or opportunistic bacteria, which can be a cause for concern, because they may encode multiresistance. Their broad distribution suggests that IncP plasmids play a crucial role in bacterial adaptation by utilizing horizontal gene transfer. This review summarizes the variety of genetic information and physiological functions carried by IncP plasmids, which can contribute to the spread of antibiotic and heavy metal resistance while also mediating the process of bioremediation of pollutants. Due to the location of the resistance genes on plasmids with a broad host range and the presence of transposons carrying these genes it seems that the spread of these genes would be possible and quite hazardous in infection control. Future studies are required to determine the level of risk of the spread of resistance genes located on these plasmids.

  10. Detection and genetic features of MCR-1-producing plasmid in human Escherichia coli infection in South Korea.

    Science.gov (United States)

    Kim, Eun Sil; Chong, Yong Pil; Park, Su-Jin; Kim, Mi-Na; Kim, Sung-Han; Lee, Sang-Oh; Choi, Sang-Ho; Woo, Jun Hee; Jeong, Jin-Yong; Kim, Yang Soo

    2017-10-01

    The plasmid-mediated colistin resistance gene, mcr-1, was identified for the first time from a hospitalized patient in South Korea. The mcr-1 gene was successfully transferred to E. coli J53 recipient and conferred resistance to colistin in the recipient. The mcr-1-harboring plasmid possessed a typical IncI2 group and did not have the mcr-1-associated ISApl1 element. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Plasmid typing of Shigella sonnei epidemic strains and molecular relationship of their R-plasmids.

    Science.gov (United States)

    Mendoza, M C; Gonzalez, A J; Mendez, F J; Hardisson, C

    1988-06-01

    We conducted a surveillance program on epidemic and/or endemic Shigella strains in Asturias (Spain), their frequency and dispersion in our community, and their R-plasmids. We analyzed initial isolates of Shigella sonnei from two epidemic outbreaks using antibiotic resistance patterns and plasmid profile analysis as epidemiological markers. We found that the 2 outbreaks were caused by different S. sonnei strains, which respectively carried one and two R-plasmids together with other plasmids. The molecular relationship among these and three other R-plasmids from two S. sonnei strains isolated during a previous outbreak, were studied by restriction enzyme analysis and DNA-DNA hybridizations. We were able to establish different levels of relationship among the six R-plasmids.

  12. antimicrobial susceptibility and plasmids from escherichia coli ...

    African Journals Online (AJOL)

    2001-10-10

    Oct 10, 2001 ... transmission to humans of E. coli containing antibiotic resistance plasmids ... resistant micro-organisms, which may in turn transfer resistance to .... cells were washed with sterile normal saline to remove leached. Я-lactamase ...

  13. Plasmid and chromosome segregation in prokaryotes

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Bugge Jensen, Rasmus; Gerdes, Kenn

    2000-01-01

    Recent major advances in the understanding of prokaryotic DNA segregation have been achieved by using fluorescence microscopy to visualize the localization of cellular components. Plasmids and bacterial chromosomes are partitioned in a highly dynamic fashion, suggesting the presence of a mitotic......-like apparatus in prokaryotes. The identification of chromosomal homologues of the well-characterized plasmid partitioning genes indicates that there could be a general mechanism of bacterial DNA partitioning. Udgivelsesdato: July 1...

  14. Signal transduction immunohistochemistry - Methods and protocols

    Directory of Open Access Journals (Sweden)

    CarloAlberto Redi

    2011-11-01

    Full Text Available Alexander E. Kalyuzhny statement that immunohistochemical detection of labile, low abundance and short-lived signal transduction molecules appears to be a very challenging task actually captures the same reader’s feeling. Each of us daily using immunohistochemical protocols to reveal targets either useful for research or diagnostic aims will surely wonder by which tricky techniques it is possible to overcome the preservation and unmasking of those labile antigens involved in signal transduction. Well, by seventheen chapters grouped in five parts Prof. Alexander E. Kalyuzhny is presenting an invaluable technical and methodological source of hints to satisfy our needs: to overcome troubleshottings if we are already in the field or to orientate those entering the field....

  15. Energy Harvesting By Optimized Piezo Transduction Mechanism

    CERN Document Server

    Boban, Bijo; Satheesh, U; Devaprakasam, D

    2014-01-01

    We report generation of electrical energy from nonlinear mechanical noises available in the ambient environment using optimized piezo transduction mechanisms. Obtaining energy from an ambient vibration has been attractive for remotely installed standalone microsystems and devices. The mechanical noises in the ambient environment can be converted to electrical energy by a piezo strip based on the principle of piezoelectric effect. In this work, we have designed and developed a standalone energy harvesting module based on piezo transduction mechanisms. Using this designed module we harvested noise energy and stored electrical energy in a capacitor. Using NI-PXI workstation with a LabVIEW programming, the output voltage of the piezo strip and voltage of the capacitor were measured and monitored. In this paper we discuss about the design, development, implementation, performance and characteristics of the energy harvesting module.

  16. Curing of plasmid pXO1 from Bacillus anthracis using plasmid incompatibility.

    Directory of Open Access Journals (Sweden)

    Xiankai Liu

    Full Text Available The large plasmid pXO1 encoding the anthrax toxin is important for the virulence of Bacillus anthracis. It is essential to cure pXO1 from B. anthracis to evaluate its role in the pathogenesis of anthrax infection. Because conventional methods for curing plasmids (e.g., curing agents or growth at elevated temperatures can induce mutations in the host chromosomal DNA, we developed a specific and reliable method to eliminate pXO1 from B. anthracis using plasmid incompatibility. Three putative replication origins of pXO1 were inserted into a temperature-sensitive plasmid to generate three incompatible plasmids. One of the three plasmids successfully eliminated the large plasmid pXO1 from B. anthracis vaccine strain A16R and wild type strain A16. These findings provided additional information about the replication/partitioning of pXO1 and demonstrated that introducing a small incompatible plasmid can generate plasmid-cured strains of B. anthracis without inducing spontaneous mutations in the host chromosome.

  17. Effects of pergolide mesylate on transduction efficiency of PEP-1-catalase protein

    Energy Technology Data Exchange (ETDEWEB)

    Sohn, Eun Jeong; Kim, Dae Won; Kim, Young Nam; Kim, So Mi [Department of Biomedical Science and Research Institute of Bioscience and Biotechnology, Hallym University, Chunchon 200-702 (Korea, Republic of); Lim, Soon Sung [Department of Food Science and Nutrition and RIC Center, Hallym University, Chunchon 200-702 (Korea, Republic of); Kang, Tae-Cheon [Department of Anatomy and Neurobiology, College of Medicine, Hallym University, Chunchon 200-702 (Korea, Republic of); Kwon, Hyeok Yil [Department of Physiology, College of Medicine, Hallym University, Chunchon 200-702 (Korea, Republic of); Kim, Duk-Soo [Department of Anatomy, College of Medicine, Soonchunhyang University, Cheonan-Si 330-090 (Korea, Republic of); Cho, Sung-Woo [Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul 138-736 (Korea, Republic of); Han, Kyu Hyung; Park, Jinseu; Eum, Won Sik [Department of Biomedical Science and Research Institute of Bioscience and Biotechnology, Hallym University, Chunchon 200-702 (Korea, Republic of); Hwang, Hyun Sook, E-mail: wazzup@hallym.ac.kr [Department of Biomedical Science and Research Institute of Bioscience and Biotechnology, Hallym University, Chunchon 200-702 (Korea, Republic of); Choi, Soo Young, E-mail: sychoi@hallym.ac.kr [Department of Biomedical Science and Research Institute of Bioscience and Biotechnology, Hallym University, Chunchon 200-702 (Korea, Republic of)

    2011-03-18

    Research highlights: {yields} We studied effects of pergolide mesylate (PM) on in vitro and in vivo transduction of PEP-1-catalase. {yields} PEP-1-catatase inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation. {yields} PM enhanced the transduction of PEP-1-catalase into HaCaT cells and skin tissue. {yields} PM increased anti-inflammatory activity of PEP-1-catalase. {yields} PM stimulated therapeutic action of anti-oxidant enzyme catalase in oxidative-related diseases. -- Abstract: The low transduction efficiency of various proteins is an obstacle to their therapeutic application. However, protein transduction domains (PTDs) are well-known for a highly effective tool for exogenous protein delivery to cells. We examined the effects of pergolide mesylate (PM) on the transduction of PEP-1-catalase into HaCaT human keratinocytes and mice skin and on the anti-inflammatory activity of PEP-1-catatase against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation using Western blot and histological analysis. PM enhanced the time- and dose-dependent transduction of PEP-1-catalase into HaCaT cells without affecting the cellular toxicity. In a mouse edema model, PEP-1-catalase inhibited the increased expressions of inflammatory mediators and cytokines such as cyclooxygenase-2, inducible nitric oxide synthase, interleukin-6 and -1{beta}, and tumor necrosis factor-{alpha} induced by TPA. On the other hand, PM alone failed to exert any significant anti-inflammatory effects. However, the anti-inflammatory effect of co-treatment with PEP-1-catalase and PM was more potent than that of PEP-1-catalase alone. Our results indicate that PM may enhance the delivery of PTDs fusion therapeutic proteins to target cells and tissues and has potential to increase their therapeutic effects of such drugs against various diseases.

  18. Ephrin A2 receptor targeting does not increase adenoviral pancreatic cancer transduction in vivo

    Institute of Scientific and Technical Information of China (English)

    Michael A van Geer; Conny T Bakker; Naoya Koizumi; Hiroyuki Mizuguchi; John G Wesseling; Ronald PJ Oude Elferink; Piter J Bosma

    2009-01-01

    AIM:To generate an adenoviral vector specifically targeting the EphA2 receptor (EphA2R) highly expressed on pancreatic cancer cells in vivo.METHODS:YSA,a small peptide ligand that binds the EphA2R with high affinity,was inserted into the HI loop of the adenovirus serotype 5 fiber knob.To further increase the specificity of this vector,binding sites for native adenoviral receptors,the coxsackie and adenovirus receptor (CAR) and integrin,were ablated from the viral capsid.The ablated retargeted adenoviral vector was produced on 293T cells.Specific targeting of this novel adenoviral vector to pancreatic cancer was investigated on established human pancreatic cancer cell lines.Upon demonstrating specific in vitro targeting,in vivo targeting to subcutaneous growing human pancreatic cancer was tested by intravenous and intraperitoneal administration of the ablated adenoviral vector.RESULTS:Ablation of native cellular binding sites reduced adenoviral transduction at least 100-fold.Insertion of the YSA peptide in the HI loop restored adenoviral transduction of EphA2R-expressing cells but not of cells lacking this receptor.YSA-mediated transduction was inhibited by addition of synthetic YSA peptide.The transduction specificity of the ablated retargeted vector towards human pancreatic cancer cells was enhanced almost 10-fold in vitro.In a subsequent in vivo study in a nude (nu/nu) mouse model however,no increased adenoviral targeting to subcutaneously growing human pancreas cancer nodules was seen upon injection into the tail vein,nor upon injection into the peritoneum.CONCLUSION:Targeting the EphA2 receptor increases specificity of adenoviral transduction of human pancreatic cancer cells in vitro but fails to enhance pancreatic cancer transduction in vivo.

  19. Multiple plasmid interference - Pledging allegiance to my enemy's enemy.

    Science.gov (United States)

    Gama, João Alves; Zilhão, Rita; Dionisio, Francisco

    2017-08-24

    As shown in the previous article, two distinct conjugative plasmids sometimes interact within bacterial cells, implicating changes of transfer rates. In most cases of interactions within bacteria, the transfer of one of the plasmids decreases. Less frequently, the transfer rate of one of the plasmids increases. Here we analyse what happens if three distinct conjugative plasmids colonize the same bacterial cell. Our aim is to understand how interactions between two plasmids affect the transfer rate of the third plasmid. After showing that plasmids interact in 59 out of 84 possible interactions we show that, with some exceptions, if the transfer rate of a plasmid decreases in the presence of a second plasmid, a decrease is also observed in the presence of a third plasmid. Moreover, if the conjugation rate of a plasmid increases in the presence of another, an increase is also observed if there is a third plasmid in the cell. Both types of interactions are mostly independent of the third plasmid's identity, even if sometimes the third plasmid quantitatively distorts the interaction of the other two plasmids. There is a bias towards negative intensifying interactions, which provide good news concerning the spread conjugative plasmids encoding antibiotic-resistance genes and virulence factors. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Optical racetrack resonator transduction of nanomechanical cantilevers.

    Science.gov (United States)

    Sauer, V T K; Diao, Z; Freeman, M R; Hiebert, W K

    2014-02-07

    Optomechanical transduction has demonstrated its supremacy in probing nanomechanical displacements. In order to apply nano-optomechanical systems (NOMS) as force and mass sensors, knowledge about the transduction responsivity (i.e. the change in measured optical transmission with nanomechanical displacement) and its tradeoffs with system design is paramount. We compare the measured responsivities of NOMS devices with varying length, optomechanical coupling strength gom, and optical cavity properties. Cantilever beams 1.5 to 5 μm long are fabricated 70 to 160 nm from a racetrack resonator optical cavity and their thermomechanical (TM) noise signals are measured. We derive a generic expression for the transduction responsivity of the NOMS in terms of optical and mechanical system parameters such as finesse, optomechanical coupling constant, and interaction length. The form of the expression holds direct insight as to how these parameters affect the responsivity. With this expression, we obtain the optomechanical coupling constants using only measurements of the TM noise power spectra and optical cavity transmission slopes. All optical pump/probe operation is also demonstrated in our side-coupled cantilever-racetrack NOMS. Finally, to assess potential operation in a gas sensing environment, the TM noise signal of a device is measured at atmospheric pressure.

  1. Characterization of an IncA/C Multidrug Resistance Plasmid in Vibrio alginolyticus.

    Science.gov (United States)

    Ye, Lianwei; Li, Ruichao; Lin, Dachuan; Zhou, Yuanjie; Fu, Aisi; Ding, Qiong; Chan, Edward Wai Chi; Yao, Wen; Chen, Sheng

    2016-05-01

    Cephalosporin-resistant Vibrio alginolyticus was first isolated from food products, with β-lactamases encoded by blaPER-1, blaVEB-1, and blaCMY-2 being the major mechanisms mediating their cephalosporin resistance. The complete sequence of a multidrug resistance plasmid, pVAS3-1, harboring the blaCMY-2 and qnrVC4 genes was decoded in this study. Its backbone exhibited genetic homology to known IncA/C plasmids recoverable from members of the family Enterobacteriaceae, suggesting its possible origin in Enterobacteriaceae. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  2. Conditional Dicer substrate formation via shape and sequence transduction with small conditional RNAs.

    Science.gov (United States)

    Hochrein, Lisa M; Schwarzkopf, Maayan; Shahgholi, Mona; Yin, Peng; Pierce, Niles A

    2013-11-20

    RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) enables knockdown of a gene of choice, executing the logical operation: silence gene Y. The fact that the siRNA is constitutively active is a significant limitation, making it difficult to confine knockdown to a specific locus and time. To achieve spatiotemporal control over silencing, we seek to engineer small conditional RNAs (scRNAs) that mediate 'conditional RNAi' corresponding to the logical operation: if gene X is transcribed, silence independent gene Y. By appropriately selecting gene X, knockdown of gene Y could then be restricted in a tissue- and time-specific manner. To implement the logic of conditional RNAi, our approach is to engineer scRNAs that, upon binding to mRNA 'detection target' X, perform shape and sequence transduction to form a Dicer substrate targeting independent mRNA 'silencing target' Y, with subsequent Dicer processing yielding an siRNA targeting mRNA Y for destruction. Toward this end, here we design and experimentally validate diverse scRNA mechanisms for conditional Dicer substrate formation. Test tube studies demonstrate strong OFF/ON conditional response, with at least an order of magnitude increase in Dicer substrate production in the presence of the cognate mRNA detection target. By appropriately dimensioning and/or chemically modifying the scRNAs, only the product of signal transduction, and not the reactants or intermediates, is efficiently processed by Dicer, yielding siRNAs. These mechanism studies explore diverse design principles for engineering scRNA signal transduction cascades including reactant stability vs metastability, catalytic vs noncatalytic transduction, pre- vs post-transcriptional transduction, reactant and product molecularity, and modes of molecular self-assembly and disassembly.

  3. Historical Events That Spawned the Field of Plasmid Biology.

    Science.gov (United States)

    Kado, Clarence I

    2014-10-01

    This chapter revisits the historical development and outcome of studies focused on the transmissible, extrachromosomal genetic elements called plasmids. Early work on plasmids involved structural and genetic mapping of these molecules, followed by the development of an understanding of how plasmids replicate and segregate during cell division. The intriguing property of plasmid transmission between bacteria and between bacteria and higher cells has received considerable attention. The utilitarian aspects of plasmids are described, including examples of various plasmid vector systems. This chapter also discusses the functional attributes of plasmids needed for their persistence and survival in nature and in man-made environments. The term plasmid biology was first conceived at the Fallen Leaf Lake Conference on Promiscuous Plasmids, 1990, Lake Tahoe, California. The International Society for Plasmid Biology was established in 2004 (www.ISPB.org).

  4. Mobile insertion cassette elements found in small non-transmissible plasmids in Proteeae may explain qnrD mobilization.

    Directory of Open Access Journals (Sweden)

    Thomas Guillard

    Full Text Available qnrD is a plasmid mediated quinolone resistance gene from unknown origin, recently described in Enterobacteriaceae. It encodes a pentapeptide repeat protein 36-60% different from the other Qnr (A, B, C, S and VC. Since most qnrD-positive strains were described as strains belonging to Proteus or Providencia genera, we hypothesized that qnrD originated in Proteeae before disseminating to other enterobacterial species. We screened 317 strains of Proteeae for qnrD and its genetic support by PCR. For all the seven qnrD-positive strains (4 Proteus mirabilis, 1 Proteus vulgaris and 2 Providencia rettgeri the gene was carried onto a small non-transmissible plasmid, contrarily to other qnr genes that are usually carried onto large multi-resistant plasmids. Nucleotide sequences of the qnrD-bearing plasmids were 96% identical. Plasmids contained 3 ORFs apart from qnrD and belonged to an undescribed incompatibility group. Only one plasmid, in P. vulgaris, was slightly different with a 1,568-bp insertion between qnrD and its promoter, leading to absence of quinolone resistance. We sought for similar plasmids in 15 reference strains of Proteeae, but which were tested negative for qnrD, and found a 48% identical plasmid (pVERM in Providencia vermicola. In order to explain how qnrD could have been inserted into such native plasmid, we sought for gene mobilization structures. qnrD was found to be located within a mobile insertion cassette (mic element which sequences are similar to one mic also found in pVERM. Our conclusions are that (i the small non-transmissible qnrD-plasmids described here may result from the recombination between an as-yet-unknown progenitor of qnrD and pVERM, (ii these plasmids are maintained in Proteeae being a qnrD reservoir (iii the mic element may explain qnrD mobilization from non-transmissible plasmids to mobilizable or conjugative plasmids from other Enterobacteriaceae, (iv they can recombined with larger multiresistant plasmids

  5. Comparative analysis of two-component signal transduction systems of Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis

    NARCIS (Netherlands)

    Been, de M.W.H.J.; Francke, C.; Moezelaar, R.; Abee, T.; Siezen, R.J.

    2006-01-01

    Members of the Bacillus cereus group are ubiquitously present in the environment and can adapt to a wide range of environmental fluctuations. In bacteria, these adaptive responses are generally mediated by two-component signal transduction systems (TCSs), which consist of a histidine kinase (HK) and

  6. Comparative analysis of two-component signal transduction systems of Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis

    NARCIS (Netherlands)

    Been, M.W.H.J. de; Francke, C.; Moezelaar, R.; Abee, T.; Siezen, R.J.

    2006-01-01

    Members of the Bacillus cereus group are ubiquitously present in the environment and can adapt to a wide range of environmental fluctuations. In bacteria, these adaptive responses are generally mediated by two-component signal transduction systems (TCSs), which consist of a histidine kinase (HK) and

  7. Bioaugmentation of DDT-contaminated soil by dissemination of the catabolic plasmid pDOD.

    Science.gov (United States)

    Gao, Chunming; Jin, Xiangxiang; Ren, Jingbei; Fang, Hua; Yu, Yunlong

    2015-01-01

    A plasmid transfer-mediated bioaugmentation method for the enhancement of dichlorodiphenyltrichloroethane (DDT) degradation in soil was developed using the catabolic plasmid pDOD from Sphingobacterium sp. D-6. The pDOD plasmid could be transferred to soil bacteria, such as members of Cellulomonas, to form DDT degraders and thus accelerate DDT degradation. The transfer efficiency of pDOD was affected by the donor, temperature, moisture, and soil type. Approximately 50.7% of the DDT in the contaminated field was removed 210 days after the application of Escherichia coli TG I (pDOD-gfp). The results suggested that seeding pDOD into soil is an effective bioaugmentation method for enhancing the degradation of DDT.

  8. Bacteriophage selection against a plasmid-encoded sex apparatus leads to the loss of antibiotic-resistance plasmids

    OpenAIRE

    Jalasvuori, Matti; Friman, Ville-Petri; Nieminen, Anne; Jaana K.H. Bamford; Buckling, Angus

    2011-01-01

    Antibiotic-resistance genes are often carried by conjugative plasmids, which spread within and between bacterial species. It has long been recognized that some viruses of bacteria (bacteriophage; phage) have evolved to infect and kill plasmid-harbouring cells. This raises a question: can phages cause the loss of plasmid-associated antibiotic resistance by selecting for plasmid-free bacteria, or can bacteria or plasmids evolve resistance to phages in other ways? Here, we show that multiple ant...

  9. Transposon insertion reveals pRM, a plasmid of Rickettsia monacensis.

    Science.gov (United States)

    Baldridge, Gerald D; Burkhardt, Nicole Y; Felsheim, Roderick F; Kurtti, Timothy J; Munderloh, Ulrike G

    2007-08-01

    Until the recent discovery of pRF in Rickettsia felis, the obligate intracellular bacteria of the genus Rickettsia (Rickettsiales: Rickettsiaceae) were thought not to possess plasmids. We describe pRM, a plasmid from Rickettsia monacensis, which was detected by pulsed-field gel electrophoresis and Southern blot analyses of DNA from two independent R. monacensis populations transformed by transposon-mediated insertion of coupled green fluorescent protein and chloramphenicol acetyltransferase marker genes into pRM. Two-dimensional electrophoresis showed that pRM was present in rickettsial cells as circular and linear isomers. The 23,486-nucleotide (31.8% G/C) pRM plasmid was cloned from the transformant populations by chloramphenicol marker rescue of restriction enzyme-digested transformant DNA fragments and PCR using primers derived from sequences of overlapping restriction fragments. The plasmid was sequenced. Based on BLAST searches of the GenBank database, pRM contained 23 predicted genes or pseudogenes and was remarkably similar to the larger pRF plasmid. Two of the 23 genes were unique to pRM and pRF among sequenced rickettsial genomes, and 4 of the genes shared by pRM and pRF were otherwise found only on chromosomes of R. felis or the ancestral group rickettsiae R. bellii and R. canadensis. We obtained pulsed-field gel electrophoresis and Southern blot evidence for a plasmid in R. amblyommii isolate WB-8-2 that contained genes conserved between pRM and pRF. The pRM plasmid may provide a basis for the development of a rickettsial transformation vector.

  10. Construction of disarmed Ti plasmids transferable between Escherichia coli and Agrobacterium species.

    Science.gov (United States)

    Kiyokawa, Kazuya; Yamamoto, Shinji; Sakuma, Kei; Tanaka, Katsuyuki; Moriguchi, Kazuki; Suzuki, Katsunori

    2009-04-01

    Agrobacterium-mediated plant transformation has been used widely, but there are plants that are recalcitrant to this type of transformation. This transformation method uses bacterial strains harboring a modified tumor-inducing (Ti) plasmid that lacks the transfer DNA (T-DNA) region (disarmed Ti plasmid). It is desirable to develop strains that can broaden the host range. A large number of Agrobacterium strains have not been tested yet to determine whether they can be used in transformation. In order to improve the disarming method and to obtain strains disarmed and ready for the plant transformation test, we developed a simple scheme to make certain Ti plasmids disarmed and simultaneously maintainable in Escherichia coli and mobilizable between E. coli and Agrobacterium. To establish the scheme in nopaline-type Ti plasmids, a neighboring segment to the left of the left border sequence, a neighboring segment to the right of the right border sequence of pTi-SAKURA, a cassette harboring the pSC101 replication gene between these two segments, the broad-host-range IncP-type oriT, and the gentamicin resistance gene were inserted into a suicide-type sacB-containing vector. Replacement of T-DNA with the cassette in pTiC58 and pTi-SAKURA occurred at a high frequency and with high accuracy when the tool plasmid was used. We confirmed that there was stable maintenance of the modified Ti plasmids in E. coli strain S17-1lambdapir and conjugal transfer from E. coli to Ti-less Agrobacterium strains and that the reconstituted Agrobacterium strains were competent to transfer DNA into plant cells. As the modified plasmid delivery system was simple and efficient, conversion of strains to the disarmed type was easy and should be applicable in studies to screen for useful strains.

  11. NO, nitrotyrosine, and cyclic GMP in signal transduction

    Science.gov (United States)

    Hanafy, K. A.; Krumenacker, J. S.; Murad, F.

    2001-01-01

    Over the past 25 years, the role of nitric oxide (NO) in biology has evolved from being recognized as an environmental pollutant to an endogenously produced substance involved in cell communication and signal transduction. NO is produced by a family of enzymes called nitric oxide synthases (NOSs), which can be stimulated by a variety of factors that mediate responses to various stimuli. NO can initiate its biological effects through activation of the heterodimeric enzyme, soluble guanylyl cyclase (sGC), or through several other chemical reactions. Activation of sGC results in the production of 3',5'-cyclic guanosine monophosphate (cGMP), an intracellular second messenger signaling molecule, which can subsequently mediate such diverse physiological events such as vasodilatation and immunomodulation. Chemically reactive NO can affect physiological changes through modifications to cellular proteins, one of which is tyrosine nitration. The demonstration that NO is involved in so many biological pathways indicates the importance of this endogenously produced substance, and suggests that there is much more to be discovered about its role in biology in years to come.

  12. Identification of intracellular domains in the growth hormone receptor involved in signal transduction

    DEFF Research Database (Denmark)

    Billestrup, N; Allevato, G; Norstedt, G

    1994-01-01

    The growth hormone (GH) receptor belongs to the GH/prolactin/cytokine super-family of receptors. The signal transduction mechanism utilized by this class of receptors remains largely unknown. In order to identify functional domains in the intracellular region of the GH receptor we generated...... a number of GH receptor mutants and analyzed their function after transfection into various cell lines. A truncated GH receptor missing 184 amino acids at the C-terminus was unable to mediate GH effects on transcription of the Spi 2.1 and insulin genes. However, this mutant was fully active in mediating GH...

  13. Identification of putative DnaN-binding motifs in plasmid replication initiation proteins.

    Science.gov (United States)

    Dalrymple, Brian P; Kongsuwan, Kritaya; Wijffels, Gene

    2007-01-01

    Recently the plasmid RK2 replication initiation protein, TrfA, has been shown to bind to the beta subunit of DNA Polymerase III (DnaN) via a short pentapeptide with the consensus QL[S/D]LF. A second consensus peptide, the hexapeptide QLxLxL, has also been demonstrated to mediate binding to DnaN. Here we describe the results of a comprehensive survey of replication initiation proteins encoded by bacterial plasmids to identify putative DnaN-binding sites. Both pentapeptide and hexapeptide motifs have been identified in a number of families of replication initiation proteins. The distribution of sites is sporadic and closely related families of proteins may differ in the presence, location, or type of putative DnaN-binding motif. Neither motif has been identified in replication initiation proteins encoded by plasmids that replicate via rolling circles or strand displacement. The results suggest that the recruitment of DnaN to the origin of replication of a replisome by plasmid replication initiation proteins is not generally required for plasmid replication, but that in some cases it may be beneficial for efficiency of replication initiation.

  14. Optimization of a plasmid electroporation protocol for Aeromonas salmonicida subsp. salmonicida.

    Science.gov (United States)

    Dallaire-Dufresne, Stéphanie; Emond-Rheault, Jean-Guillaume; Attéré, Sabrina A; Tanaka, Katherine H; Trudel, Mélanie V; Frenette, Michel; Charette, Steve J

    2014-03-01

    Aeromonas salmonicida subsp. salmonicida is a major fish pathogen. Molecular tools are required to study the virulence and genomic stability of this bacterium. An efficient electroporation-mediated transformation protocol for A. salmonicida subsp. salmonicida would make genetic studies faster and easier. In the present study, we designed the 4.1-kb pSDD1 plasmid as a tool for optimizing an electroporation protocol for A. salmonicida subsp. salmonicida. We systematically tested the electroporation conditions to develop a protocol that generates the maximum number of transformants. Under these optimal conditions (25 kV/cm, 200 Ω, 25 μF), we achieved an electroporation efficiency of up to 1×10(5) CFU/μg DNA. The electroporation protocol was also tested using another plasmid of 10.6-kb and three different strains of A. salmonicida subsp. salmonicida. The strains displayed significant differences in their electro-transformation competencies. Strain 01-B526 was the easiest to electroporate, especially with the pSDD1 plasmid. This plasmid was stably maintained in the 01-B526 transformants, as were the native plasmids, but could be easily cured by removing the selection conditions. This is the first efficient electroporation protocol reported for A. salmonicida subsp. salmonicida, and offers new possibilities for studying this bacterium.

  15. Temperature Depended Role of Shigella flexneri Invasion Plasmid on the Interaction with Acanthamoeba castellanii

    Directory of Open Access Journals (Sweden)

    Amir Saeed

    2012-01-01

    Full Text Available Shigella flexneri is a Gram-negative bacterium causing the diarrhoeal disease shigellosis in humans. The virulence genes required for invasion are clustered on a large 220 kb plasmid encoding type three secretion system (TTSS apparatus and virulence factors such as adhesions and invasion plasmid antigens (Ipa. The bacterium is transmitted by contaminated food, water, or from person to person. Acanthamoebae are free-living amoebae (FLA which are found in diverse environments and isolated from various water sources. Different bacteria interact differently with FLA since Francisella tularensis, Vibrio cholerae, Shigella sonnei, and S. dysenteriae are able to grow inside A. castellanii. In contrast, Pseudomonas aeruginosa induces both necrosis and apoptosis to kill A. castellanii. The aim of this study is to examine the role of invasion plasmid of S. flexneri on the interaction with A. castellanii at two different temperatures. A. castellanii in the absence or presence of wild type, IpaB mutant, or plasmid-cured strain S. flexneri was cultured at 30∘C and 37∘C and the interaction was analysed by viable count of both bacteria and amoebae, electron microscopy, flow cytometry, and statistical analysis. The outcome of the interaction was depended on the temperature since the growth of A. castellanii was inhibited at 30∘C, and A. castellanii was killed by invasion plasmid mediated necrosis at 37∘C.

  16. plasmid mediated resistance in multidrug resistant bacteria isolated ...

    African Journals Online (AJOL)

    User

    3Department of Paediatrics, Ahmadu Bello University Teaching Hospital, Zaria ... The knowledge of the epidemiological and antimicrobial pattern of common pathogens that cause septicaemia is useful for ..... commonly prescribed antibiotics in the locality investigated. ... to monitor and restrict the use and sale of antibiotics.

  17. Enhancement of plasmid-mediated stable gene expression by ...

    African Journals Online (AJOL)

    ARL

    2012-06-12

    GenBank accession number J02442) and inserted 3′ end of the .... The expressions of these two interest proteins were driven by the same promoter of hEF1α. .... expression at the posttranscriptional level in neural cells: implications.

  18. Molecular characterization of encoding plasmid-mediated ESBLs and AmpC β-lactamases genes in Citrobacter freundii%质粒介导产ESBLs与AmpCβ-内酰胺酶基因弗氏柠檬酸杆菌的研究

    Institute of Scientific and Technical Information of China (English)

    陈坚; 余方友

    2011-01-01

    OBJECTIVE To investigate the resistant mechanism of Citrobacter freundii which coexisted with ESBLs and AmpC genes. METHODS A multi-resistant Citrobacter freundii was isolated from hospital by VITEK-60 system. The detection of ESBLs was performed by the CLSI-recommended confirmatory test, Cefoxitin three-dimentional test was presented to identify AmpC β-lactamases, the mimimal inhibitative concentration (MIC) was determined by E-test. Polymerase chain reaction(PCR) and sequencing were carried out for analyzing the encoding genes of β-lactamases. Conjugation study was performed to determine whether resistant genes were likely transferred by plasmid. RESULTS The isolate was highly resistant to ceftazidime (MICs,96 jug/ml), cefotaxime (MlCs,96 μg/ml), cefoxitin(MICs.256 μg/ml) , aztreonam(MICs, 192 fig/ml) , ampicillin(MICs,>256 μg/ml) and sinomin compositea(MICs,>32 fig/ml). The clinical isolate produced AmpCs and ESBLs. The presence of blacrx-M-3 and blaCMY-2 of clinical isolate were identified by PCR and sequenced. Those genes of clinical isolate could be transferred to Escherichia coli J53 through conjugation. CONCLUSION Citrobacter freundii carries ESBLs and AmpC β-lactamases genes synchronously, which were mediated by plasmid.%目的 研究一株同时产ESBLs和AmpC β-内酰胺酶弗氏柠檬酸杆菌(CFR)的耐药机制.方法 2008年1月从临床尿液标本中分离出多药耐药弗氏柠檬酸杆菌1株,采用双纸片扩散法检测产ESBLs,头孢西丁三维试验检测AmpC酶,E-test法测定抗菌药物最低抑菌浓度(MIC),聚合酶链反应(PCR)检测产ESBss和AmpC酶基因,DNA测序决定基因型;接合试验测定耐药基因的转移性.结果 临床分离出一株同时产ESBLs和AmpCβ-内酰胺酶的多药耐药弗氏柠檬酸杆菌,对头孢他啶、头孢噻肟、头孢西丁、氨曲南、氨苄西林、磺胺甲噁唑/甲氧苄啶的MIC分别为96、96、256、192、>256、>32 μg/ml,PCR扩增及测序

  19. Electromechanical Energy Transduction for Hybrid Vehicles

    Science.gov (United States)

    Reddy Vanja, Sridhar; Kelly, Michael W.; Caruso, A. N.

    2010-03-01

    Hybrid vehicle technology seeks to reduce the total energy consumption used for vehicle locomotion by recovering and reutilizing kinetic energy that is otherwise unrecovered or dissipated in conventional vehicle deceleration. The goal of the work is to determine the transduction mechanisms that work towards a Carnot efficiency without considering constraints or limitations placed by cost or materials. Specifically, this talk will present ideal thermodynamic models of energy exchange between mechanical, electrostatic, electromechanical and electrochemical devices with a goal of projecting an ideal hybrid vehicle.

  20. Complete plasmid sequence carrying type IV-like and type VII secretion systems from an atypical mycobacteria strain.

    Science.gov (United States)

    Morgado, Sergio Mascarenhas; Marín, Michel Abanto; Freitas, Fernanda S; Fonseca, Erica Lourenço; Vicente, Ana Carolina Paulo

    2017-07-01

    The genus Mycobacterium is highly diverse and ubiquitous in nature, comprehending fast- and slow-growing species with distinct impact in public health. The plasmid-mediated horizontal gene transfer represents one of the major events in bacteria evolution. Here, we report the complete sequence of a 160,489 bp circular plasmid (pCBMA213_2) from an atypical and fast-growing environmental mycobacteria. This is a unique plasmid, in comparison with the characterised mycobacteria plasmids, harboring a type IV-like and ESX-P2 type VII secretion systems. pCBMA213_2 can be further explored for evolutionary and conjugation studies as well as a tool to manipulate DNA within this bacteria genus.

  1. Plasmid and Host Strain Characteristics of Escherichia coli Resistant to Extended-Spectrum Cephalosporins in the Norwegian Broiler Production.

    Science.gov (United States)

    Mo, Solveig Sølverød; Slettemeås, Jannice Schau; Berg, Einar Sverre; Norström, Madelaine; Sunde, Marianne

    2016-01-01

    Escherichia coli resistant to extended-spectrum cephalosporins have been detected in the Norwegian broiler production, despite the fact that antimicrobial agents are rarely used. The genetic mechanism responsible for cephalosporin resistance is mainly attributed to the presence of the blaCMY-2 gene encoding a plasmid-mediated AmpC-beta-lactamase (pAmpC). The aim of this study was to characterize and compare blaCMY-2 containing Escherichia coli isolated from the intestinal flora of broilers and retail chicken meat (fillets) to identify possible successful clones and/or resistance plasmids widespread in the Norwegian broiler production. Methods used included PCR based phylotyping, conjugation experiments, plasmid replicon typing, pulsed-field gel electrophoresis, multiple locus variable-number tandem-repeats analysis and whole genome sequencing. The nucleotide sequence of an IncK plasmid carrying blaCMY-2 was determined. Intestinal isolates displayed a higher degree of genetic diversity than meat isolates. A cluster of genetically related isolates belonging to ST38, phylogroup D, carrying blaCMY-2 containing IncK plasmids was identified. Furthermore, genes encoding plasmid stability systems (relBE/stbDE and pndAC) were identified on the IncK plasmid. Single nucleotide polymorphism (SNP) analysis of a subset of isolates confirmed a close genetic relationship within the two most prevalent STs. The IncK plasmids within these two STs also shared a high degree of similarity. Cephalosporin-resistant E. coli with the same genetic characteristics have been identified in the broiler production in other European countries, and the IncK plasmid characterized in this study showed close homology to a plasmid isolated from retail chicken meat in the Netherlands. The results indicate that both clonal expansion and horizontal transfer of blaCMY-2 containing plasmids contribute to dissemination of cephalosporin resistant E. coli in the broiler production. The presence of plasmid

  2. Conjugative transfer of an IncA/C plasmid-borne blaCMY-2 gene through genetic re-arrangements with an IncX1 plasmid.

    Science.gov (United States)

    Wiesner, Magdalena; Fernández-Mora, Marcos; Cevallos, Miguel A; Zavala-Alvarado, Crispín; Zaidi, Mussaret B; Calva, Edmundo; Silva, Claudia

    2013-11-21

    Our observation that in the Mexican Salmonella Typhimurium population none of the ST19 and ST213 strains harbored both the Salmonella virulence plasmid (pSTV) and the prevalent IncA/C plasmid (pA/C) led us to hypothesize that restriction to horizontal transfer of these plasmids existed. We designed a conjugation scheme using ST213 strain YU39 as donor of the blaCMY-2 gene (conferring resistance to ceftriaxone; CRO) carried by pA/C, and two E. coli lab strains (DH5α and HB101) and two Typhimurium ST19 strains (SO1 and LT2) carrying pSTV as recipients. The aim of this study was to determine if the genetic background of the different recipient strains affected the transfer frequencies of pA/C. YU39 was able to transfer CRO resistance, via a novel conjugative mechanism, to all the recipient strains although at low frequencies (10-7 to 10-10). The presence of pSTV in the recipients had little effect on the conjugation frequency. The analysis of the transconjugants showed that three different phenomena were occurring associated to the transfer of blaCMY-2: 1) the co-integration of pA/C and pX1; 2) the transposition of the CMY region from pA/C to pX1; or 3) the rearrangement of pA/C. In addition, the co-lateral mobilization of a small (5 kb) ColE1-like plasmid was observed. The transconjugant plasmids involving pX1 re-arrangements (either via co-integration or ISEcp1-mediated transposition) obtained the capacity to conjugate at very high levels, similar to those found for pX1 (10-1). Two versions of the region containing blaCMY-2 were found to transpose to pX1: the large version was inserted into an intergenic region located where the "genetic load" operons are frequently inserted into pX1, while the short version was inserted into the stbDE operon involved in plasmid addiction system. This is the first study to report the acquisition of an extended spectrum cephalosporin (ESC)-resistance gene by an IncX1 plasmid. We showed that the transfer of the YU39 blaCMY-2 gene

  3. Distribution of small native plasmids in Streptococcus pyogenes in India.

    Science.gov (United States)

    Bergmann, René; Nerlich, Andreas; Chhatwal, Gursharan S; Nitsche-Schmitz, D Patric

    2014-05-01

    Complete characterization of a Streptococcus pyogenes population from a defined geographic region comprises information on the plasmids that circulate in these bacteria. Therefore, we determined the distribution of small plasmids (pyogenes isolates from India, where diversity of strains and incidence rates of S. pyogenes infections are high. The collection comprised 77 emm-types. For plasmid detection and discrimination, we developed PCRs for different plasmid replication initiation protein genes, the putative repressor gene copG and bacteriocin genes dysA and scnM57. Plasmid distribution was limited to 13 emm-types. Co-detection analysis using aforementioned PCRs revealed four distinct plasmid sub-types, two of which were previously unknown. Representative plasmids pA852 and pA996 of the two uncharacterized plasmid sub-types were sequenced. These two plasmids could be assigned to the pMV158 and the pC194/pUB110 family of rolling-circle plasmids, respectively. The majority of small plasmids found in India belonged to the two newly characterized sub-types, with pA852- and pA996-like plasmids amounting to 42% and 22% of all detected plasmids, respectively. None of the detected plasmids coded for a known antibiotic resistance gene. Instead, all of the four plasmid sub-types carried known or potential bacteriocin genes. These genes may have influence on the evolutionary success of certain S. pyogenes genotypes. Notably, pA852-like plasmids were found in all isolates of the most prevalent emm-type 11.0. Together, a priori fitness of this genotype and increased fitness due to the acquired plasmids may have rendered type emm11.0 successful and caused the prevalence of pA852-like plasmids in India.

  4. Conjugative multi-resistant plasmids in Haihe River and their impacts on the abundance and spatial distribution of antibiotic resistance genes.

    Science.gov (United States)

    Dang, Bingjun; Mao, Daqing; Xu, Yan; Luo, Yi

    2017-03-15

    In this study, five classes of antibiotic resistance genes (ARGs) were quantified in sediment samples of Haihe River, China, with abundance ranging from 1.39 × 10(4) to 1.58 × 10(10) copies/g dry weight. Meanwhile, antibiotic resistant conjugative plasmids were also isolated from these samples through filter mating assays. In total, 202 transconjugants were isolated and tested for their antibiotic resistance phenotypes, among which 26 different types of conjugative plasmids were observed. The majority of these plasmids showed a multi-resistant phenotype and the most prevalent resistance was tetracycline resistance and sulfonamide resistance. Furthermore, we tested the transfer frequencies of these plasmids, determined their genotypes and then compared the plasmid-borne ARGs with their corresponding abundance in Haihe River. Most of the isolated plasmids exhibited high transfer frequencies to the recipient strain Escherichia coli J53. Plasmids isolated from the urban areas of Haihe River have higher transfer frequencies than the rural areas. Results from comprehensive analysis of plasmid genotypes, ARG abundance and plasmid sequencing confirmed that most of the plasmid-borne ARGs were the dominant genes in the Haihe River. Therefore, conjugative plasmids isolated from the Haihe River plays a crucial role in the dissemination, abundance and spatial distribution of ARGs in Haihe River, especially some unfrequent ARGs like blaGES-1. This study will help to increase the knowledge on the conjugative plasmid-mediated ARG propagation in the environment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Involvement of the p38 mitogen-activated protein kinase signal transduction pathway in burns-induced lung injury

    Institute of Scientific and Technical Information of China (English)

    CHEN Xu-lin; XIA Zhao-fan; WEI Duo; WANG Yong-jie; WANG Chang-rong

    2005-01-01

    @@ Acute lung injury (ALI) is a leading complication in extensively burned patients, especially those with inhalation injury.1 It can cause hypoxia resulting in injury of remote organs and dysfunction. P38 mitogen-activated protein kinase (p38 MAPK) is a stress activated protein kinase in the MAPK family.2 Most of the previous studies have demonstrated that p38 MAPK signal transduction pathway mediated ALI in rats with acute severe pancreatitis, sepsis etc.3-5 However, there is little information regarding the role of p38 MAPK signal transduction pathway in ALI after severe burn trauma.

  6. STUDY REGARDING EFFICIENCY OF INDUCED GENETIC TRANSFORMATION IN BACILLUS LICHENIFORMIS WITH PLASMID DNA

    Directory of Open Access Journals (Sweden)

    VINTILĂ T.

    2007-01-01

    Full Text Available A strain of Bacillus licheniformis was subject to genetic transformation with plasmidvectors (pLC1 and pNC61, using electroporation technique, protoplasttransformation and bivalent cations (CaCl2 mediated transformation. In the case oftransformation by electroporation of Bacillus licheniformis B40, the highest numberof transformed colonies (3 were obtained only after a 1,79 KV electric shock, for 2,2milliseconds. Using this transformation technique we have obtained six kanamycinresistant transformants. The frequency of Bacillus licheniformis B40 protoplaststransformation using pLC1 and pNC61 plasmid vectors is approximately 10% (TF =10%. As a result of pLC1 plasmid integration in Bacillus licheniformis protoplasts,six kanamycin resistant transformants were obtained. The pNC61 plasmid, whichconfers trimethoprim resistance, does not integrate in receiver cells by protoplasttransformation. The direct genetic transformation in the presence of bivalent cations(CaCl2, mediated by pLC1 and pNC61 plasmid vectors, produce a lowtransformation frequency. Using this technique, we have obtained three trimethoprimresistant colonies and four kanamycin resistant colonies. The chemical way oftransformation is the only technique, which realizes the integration of pNC61 in B.licheniformis B40 cells.

  7. Automated modelling of signal transduction networks

    Directory of Open Access Journals (Sweden)

    Aach John

    2002-11-01

    Full Text Available Abstract Background Intracellular signal transduction is achieved by networks of proteins and small molecules that transmit information from the cell surface to the nucleus, where they ultimately effect transcriptional changes. Understanding the mechanisms cells use to accomplish this important process requires a detailed molecular description of the networks involved. Results We have developed a computational approach for generating static models of signal transduction networks which utilizes protein-interaction maps generated from large-scale two-hybrid screens and expression profiles from DNA microarrays. Networks are determined entirely by integrating protein-protein interaction data with microarray expression data, without prior knowledge of any pathway intermediates. In effect, this is equivalent to extracting subnetworks of the protein interaction dataset whose members have the most correlated expression profiles. Conclusion We show that our technique accurately reconstructs MAP Kinase signaling networks in Saccharomyces cerevisiae. This approach should enhance our ability to model signaling networks and to discover new components of known networks. More generally, it provides a method for synthesizing molecular data, either individual transcript abundance measurements or pairwise protein interactions, into higher level structures, such as pathways and networks.

  8. Identification of plasmid-mediated AmpC gene, blaDHA-1 from clinical isolates of Escherichia coli and Klebsiella pneumoniae%从大肠埃希菌和肺炎克雷伯菌中检出质粒介导的AmpC DHA-1型β内酰胺酶基因

    Institute of Scientific and Technical Information of China (English)

    王煜; 李振华

    2008-01-01

    目的 了解产质粒介导AmpC酶大肠埃希菌和肺炎克雷伯菌的耐药性和基因型.方法 收集2002年1月-2004年5月间我院呼吸科临床标本中分离的大肠埃希菌和肺炎克雷伯菌共110株,用酶提取物三维试验检测AmpC酶;用等电聚焦电泳、耐药质粒电转化试验、聚合酶链反应(PCR)及测序确定AmpC酶基因型.结果 大肠埃希菌和肺炎克雷伯菌中AmpC酶检出率分剐为9.30%和4.48%.药敏试验显示产酶株对头孢西丁全部耐药,对第三代头孢菌素、酶抑制剂、氨曲南、阿米卡星及环丙沙星均有不同程度耐药,对头孢吡肟及亚胺培南较敏感.7株产AmpC酶菌株中有5株通过电转化试验可将头孢西丁耐药性传递给受体菌,经PCR扩增和测序证实为质粒介导DHA-1型AmpC酶.结论 我院临床分离的大肠埃希菌和肺炎克雷伯菌中已经出现产质粒介导AmpC酶菌株,其耐药性能够水平传播,给临床抗感染治疗带来重大威胁.%[Objective]To investigate the susceptibility and genotype characteristics of Escherichia coli and Klebsiella pneumoniae producing plasmid-mediated AmpC β-lactamase. [Methods]A total of 110 strains of Escherichia coli and Klebsiella pneumoniae were collected from the patients hospitalized in our respiratory ward from January 2002 to May 2004. The isohtes harboring AmpC β-lactamase were detected by three-dimensional test, isoelectric focusing analysis, electroporation and PCR, the PCR products were sequenced subsequently. [Results]AmpC enzyme was detected in 9.30% of Escherichia coli and 4.48 % of Klebsiella pneumoniae. The susceptibility test showed 7 isolates producing plnsmid-mediated AmpC β-lactamase were all resistant to cefoxitin, part of these strains were resistant to the third-generation Cephalosparins, β-lactamase combined with the β-lactamase inhibitors, Aztreonam,Amikacin and Cipmfloxacin, most of them were susceptible to cefeime and imipenem. 3 strains of Klebsiella

  9. Engineering key components in a synthetic eukaryotic signal transduction pathway

    OpenAIRE

    Antunes, Mauricio S; Kevin J Morey; Tewari-Singh, Neera; Bowen, Tessa A.; Smith, J. Jeff; Webb, Colleen T.; Hellinga, Homme W.; Medford, June I.

    2009-01-01

    Signal transduction underlies how living organisms detect and respond to stimuli. A goal of synthetic biology is to rewire natural signal transduction systems. Bacteria, yeast, and plants sense environmental aspects through conserved histidine kinase (HK) signal transduction systems. HK protein components are typically comprised of multiple, relatively modular, and conserved domains. Phosphate transfer between these components may exhibit considerable cross talk between the otherwise apparent...

  10. Engineering fusogenic molecules to achieve targeted transduction of enveloped lentiviral vectors

    Directory of Open Access Journals (Sweden)

    Wang Pin

    2009-06-01

    Full Text Available Abstract Background Lentiviral vectors with broad tropism are one of the most promising gene delivery systems capable of efficiently delivering genes of interest into both dividing and non-dividing cells while maintaining long-term transgene expression. However, there are needs for developing lentiviral vectors with the capability to deliver genes to specific cell types, thus reducing the "off-target" effect of gene therapy. In the present study, we investigated the possibility of engineering the fusion-active domain of a fusogenic molecule (FM with the aim to improve targeted transduction of lentiviral vectors co-displaying an anti-CD20 antibody (αCD20 and a FM. Results Specific mutations were introduced into the fusion domain of a binding-deficient Sindbis virus glycoprotein to generate several mutant FMs. Lentiviral vectors incorporated with αCD20 and one of the engineered FMs were successfully produced and demonstrated to be able to preferentially deliver genes to CD-20-expressing cells. Lentiviral vectors bearing engineered FMs exhibited 8 to 17-fold enhanced transduction towards target cells as compared to the parental FM. Different levels of enhancement were observed for the different engineered FMs. A pH-dependent study of vector transduction showed that the broader pH range of the engineered FM is a possible mechanism for the resulted increase in transduction efficiency. Conclusion The fusion domain of Sindbis virus glycoprotein is amenable for engineering and the engineered proteins provide elevated capacity to mediate lentiviral vectors for targeted transduction. Our data suggests that application of such an engineering strategy can optimize the two-molecular targeting method of lentiviral vectors for gene delivery to predetermined cells.

  11. Study on drug resistance and plasmid-mediated resistance mechanisms of ciprofloxacin-resistant Pseudomonas aeruginosa%铜绿假单胞菌耐药性及质粒介导的耐环丙沙星分子机制研究

    Institute of Scientific and Technical Information of China (English)

    陈茶; 黄彬; 陈利达; 吴强贵; 陈树林; 李有强; 林冬玲; 张妮

    2012-01-01

    OBJECTIVE To study the drug resistance and detect plasmid-mediated ciprofloxacin-resistant genes in clinical isolates of Pseudomonas aeruginosa , and study the molecular resistant mechanisms. METHODS The clinical isolates were identified by the automatic VITEK 2 system and the antimicrobial susceptibility was tested by K-B disc diffusion. The quinolone-resistant genes mediated by plasmids including qnrA, qnrB, qnrC, qnrD, qnrS, qepA and aac(6')-Ibcr were analyzed by polymerase chain reaction. RESULTS The resistance rates of 423 isolates of P. aeruginosa to ciprofloxacin and levofloxacin were 23. 2%, the resistance rates to the first and third generation of cephalosporins were higher than 49. 2% except 22. 7% to ceftazidime, the resistance rates to aminoglycoside antibiotics such as gentamicin, tobramycin, and amikacin were 17. 5%, 17. 5% and 13. 0%, respectively; the drug resistance rates to penicillins were higher than 40. 4% except 26. 2% to piperacillin, there was large difference in the resistant rate to β-lactamase inhibitor combinations, piperacillin/tazobactam (17. 0%) and ampicillin/sulbactam (98. 6%); the resistance rates to imipenem and meropenem were 24. 6% and 26. 0%, respectively; of 127 strains of ciprofloxacin-resistant P. aeruginosa isolates, the drug resistance increased significantly, the resistance rate to levofloxacin was 86. 6%, the resistance rate to the third generation of cephalosporin rose above 61. 4% , the resistance rate to the fourth generation of cephalosporin cefepime rose from 20.3% to 62. 2%; the resistance rate to β-lactamase inhibitor complexes increased from 17. 0% to 49. 6% or above, and the resistance rates to aminoglycosides (gentamicin, tobramycin) rose to 64. 6% or above, the resistance rate to amikacin increased from 13. 0% to 48. 8% ; there were no qnrS and qnrC that were detected in ciprofloxacin-resistant P. aeruginosa, the positive rates of qnrA, qnrB, qnrD, qepA and aac(6')-Ib-cr gene were 31. 2% , 87. 5% , 15. 6

  12. 一株耐碳青霉烯类的阴沟肠杆菌的KPC酶检测%Detection of plasmid-mediated carbapenem-hydrolyzing β-lactamase KPC-2 in a strain of carbapenem-resistant Enterobacter cloacae

    Institute of Scientific and Technical Information of China (English)

    蔡加昌; 周宏伟; 陈功祥; 张嵘

    2008-01-01

    Objective To investigate the mechanism of carbapenem resistance in Enterobacter cloacae.Methods A carbapenem-resistant strain of E.cloacae (strain ZY1465)was isolated.Antibiotic susceptibilities were determined by agar dilution method.Conjugation experiments were carried out in mixed broth cultures.Plasmid DNA preparations were obtained by using an alkalinelysis technique and were digested by various endonucleases;The crude β-lactamase extracts of E.cloacae and E.coli transconjugant were subjected to analytical isoelectric focusing(IEF).Specific PCR amplification and DNA sequence analysis were preformed to confirm the β-lactamase type.Outer membrane proteins(OMPs)were isolated and examined by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Results The E. cloacae isolate showed resistance against carbapenems.The MICs of imipenem and meropenem were both 32 μg/ml.The isolate was also resistant strongly against penicillins,cephalosporins,cefoxitin,aztreonam,quinolones,and aminoglycosides.Conjugation studies with E.coli resulted in the transfer of reduced carbapenem susceptibility from E.cloacae isolate.Plasmid restriction analysis showed identical restriction profiles between the transconjugants of E. cloacae ZY1465 and Serratia marcescens ZN008.Isoelectric focusing demonstrated six β-lactamases,with the isoelectrie points(pls)of5.4,6.7,7.3,7.8,7.9,and 8.6,in E.cloacae ZY1465.and only one β-lactamase with the pI of 6.7 in transconiugant.Specific PCR amplification and DNA sequence analysis confirmed that E.cloacae ZY1465 harbored TEM-1,KPC-2,DHA-1,CTX-M-14,CTX-M-3 and chromosomal AmpC(not detected in IEF)genes.Urea-SDS-PAGE analysis of OMPs showed that E. cloacae ZY1465 lacked an OMP of approximately 38 000 Da which was present in E. cloacae ATCC13047.Conclusion It is the first detection of plasmid-mediated carbapenemhydrolyzing β-lactamase KPC-2 in a clinical isolate of E.cloacae from China.Production of multiple β-lactamases,especially KPC-2 and

  13. Quantifying efficient information transduction of biochemical signaling cascades

    CERN Document Server

    Tsuruyama, Tatsuaki

    2016-01-01

    Cells can be considered as systems that utilize changes in thermodynamic entropy as information. Therefore, they serve as useful models for investigating the relationships between entropy production and information transmission, i.e., signal transduction. Based on the hypothesis that cells apply a chemical reaction cascade for the most efficient transduction of information, we adopted a coding design that minimizes the number of bits per concentration of molecules that are employed for information transduction. As a result, the average rate of entropy production is uniform across all cycles in a cascade reaction. Thus, the entropy production rate can be a valuable measure for the quantification of intracellular signal transduction.

  14. Stress responses and replication of plasmids in bacterial cells

    Directory of Open Access Journals (Sweden)

    Wegrzyn Alicja

    2002-05-01

    Full Text Available Abstract Plasmids, DNA (or rarely RNA molecules which replicate in cells autonomously (independently of chromosomes as non-essential genetic elements, play important roles for microbes grown under specific environmental conditions as well as in scientific laboratories and in biotechnology. For example, bacterial plasmids are excellent models in studies on regulation of DNA replication, and their derivatives are the most commonly used vectors in genetic engineering. Detailed mechanisms of replication initiation, which is the crucial process for efficient maintenance of plasmids in cells, have been elucidated for several plasmids. However, to understand plasmid biology, it is necessary to understand regulation of plasmid DNA replication in response to different environmental conditions in which host cells exist. Knowledge of such regulatory processes is also very important for those who use plasmids as expression vectors to produce large amounts of recombinant proteins. Variable conditions in large-scale fermentations must influence replication of plasmid DNA in cells, thus affecting the efficiency of recombinant gene expression significantly. Contrary to extensively investigated biochemistry of plasmid replication, molecular mechanisms of regulation of plasmid DNA replication in response to various environmental stress conditions are relatively poorly understood. There are, however, recently published studies that add significant data to our knowledge on relations between cellular stress responses and control of plasmid DNA replication. In this review we focus on plasmids derived from bacteriophage λ that are among the best investigated replicons. Nevertheless, recent results of studies on other plasmids are also discussed shortly.

  15. Molecular mechanism of cellular reception of ionizing radiation and of activation of signal transduction pathway

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Keiji [Nagasaki Univ. (Japan). Faculty of Pharmaceutical Sciences

    1997-03-01

    The author reviewed what in cells receives ionizing radiation as a stress and which signal transduction pathway is activated to induce the stress reaction in the following order: Activation of protein kinase C (PKC) pathway by radiation, activation of MAP kinase superfamily by radiation, induction of p53 function by radiation, and radiation exposure and stress reaction pathway. Conclusion was as follows: Cellular receptors to radiation can be cell membrane and DNA. Membrane reception of radiation induces activation of tyrosine kinase and sphingomyelinase, which resulting in activation of PKC- and MAP kinase-mediated signal transduction. The signal generated in the nucleus participates in regulation of cell cycle and in DNA repair. Therefore, it seems that irradiation of ionizing radiation gives energy to various cellular receptor sites as well as DNA, which generate various independent signals to be transduced and accumulated in the nucleus, and leading to cellular response. (K.H.). 63 refs.

  16. Structural insight into partner specificity and phosphoryl transfer in two-component signal transduction.

    Science.gov (United States)

    Casino, Patricia; Rubio, Vicente; Marina, Alberto

    2009-10-16

    The chief mechanism used by bacteria for sensing their environment is based on two conserved proteins: a sensor histidine kinase (HK) and an effector response regulator (RR). The signal transduction process involves highly conserved domains of both proteins that mediate autokinase, phosphotransfer, and phosphatase activities whose output is a finely tuned RR phosphorylation level. Here, we report the structure of the complex between the entire cytoplasmic portion of Thermotoga maritima class I HK853 and its cognate, RR468, as well as the structure of the isolated RR468, both free and BeF(3)(-) bound. Our results provide insight into partner specificity in two-component systems, recognition of the phosphorylation state of each partner, and the catalytic mechanism of the phosphatase reaction. Biochemical analysis shows that the HK853-catalyzed autokinase reaction proceeds by a cis autophosphorylation mechanism within the HK subunit. The results suggest a model for the signal transduction mechanism in two-component systems.

  17. Receptor component protein (RCP): a member of a multi-protein complex required for G-protein-coupled signal transduction.

    Science.gov (United States)

    Prado, M A; Evans-Bain, B; Dickerson, I M

    2002-08-01

    The calcitonin-gene-related peptide (CGRP) receptor component protein (RCP) is a 148-amino-acid intracellular protein that is required for G-protein-coupled signal transduction at receptors for the neuropeptide CGRP. RCP works in conjunction with two other proteins to constitute a functional CGRP receptor: calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying protein 1 (RAMP1). CRLR has the stereotypical seven-transmembrane topology of a G-protein-coupled receptor; it requires RAMP1 for trafficking to the cell surface and for ligand specificity, and requires RCP for coupling to the cellular signal transduction pathway. We have made cell lines that expressed an antisense construct of RCP and determined that CGRP-mediated signal transduction was reduced, while CGRP binding was unaffected. Furthermore, signalling at two other endogenous G-protein-coupled receptors was unaffected, suggesting that RCP was specific for a limited subset of receptors.

  18. Endogenous mutagenesis in recombinant sulfolobus plasmids.

    Science.gov (United States)

    Sakofsky, Cynthia J; Grogan, Dennis W

    2013-06-01

    Low rates of replication errors in chromosomal genes of Sulfolobus spp. demonstrate that these extreme thermoacidophiles can maintain genome integrity in environments with high temperature and low pH. In contrast to this genetic stability, we observed unusually frequent mutation of the β-D-glycosidase gene (lacS) of a shuttle plasmid (pJlacS) propagated in Sulfolobus acidocaldarius. The resulting Lac(-) mutants also grew faster than the Lac(+) parent, thereby amplifying the impact of the frequent lacS mutations on the population. We developed a mutant accumulation assay and corrections for the effects of copy number and differential growth for this system; the resulting measurements and calculations yielded a corrected rate of 5.1 × 10(-4) mutational events at the lacS gene per plasmid replication. Analysis of independent lacS mutants revealed three types of mutations: (i) G · C-to-A · T transitions, (ii) slipped-strand events, and (iii) deletions. These mutations were frequent in plasmid-borne lacS expressed at a high level but not in single-copy lacS in the chromosome or at lower levels of expression in a plasmid. Substitution mutations arose at only two of 12 potential priming sites of the DNA primase of the pRN1 replicon, but nearly all these mutations created nonsense (chain termination) codons. The spontaneous mutation rate of plasmid-borne lacS was 175-fold higher under high-expression than under low-expression conditions. The results suggest that important DNA repair or replication fidelity functions are impaired or overwhelmed in pJlacS, with results analogous to those of the "transcription-associated mutagenesis" seen in bacteria and eukaryotes.

  19. Genetic characterization of mcr-1-bearing plasmids to depict molecular mechanisms underlying dissemination of the colistin resistance determinant.

    Science.gov (United States)

    Li, Ruichao; Xie, Miaomiao; Zhang, Jinfei; Yang, Zhiqiang; Liu, Lizhang; Liu, Xiaobo; Zheng, Zhiwei; Chan, Edward Wai-Chi; Chen, Sheng

    2017-02-01

    To analyse and compare mcr-1-bearing plasmids from animal Escherichia coli isolates, and to investigate potential mechanisms underlying dissemination of mcr-1. Ninety-seven ESBL-producing E. coli strains isolated from pig farms in China were screened for the mcr-1 gene. Fifteen mcr-1-positive strains were subjected to molecular characterization and bioinformatic analysis of the mcr-1-bearing plasmids that they harboured. Three major types of mcr-1-bearing plasmids were recovered: IncX4 (∼33 kb), IncI2 (∼60 kb) and IncHI2 (∼216-280 kb), among which the IncX4 and IncI2 plasmids were found to harbour the mcr-1 gene only, whereas multiple resistance elements including blaCTX-M, blaCMY, blaTEM, fosA, qnrS, floR and oqxAB were detected, in various combinations, alongside mcr-1 in the IncHI2 plasmids. The profiles of mcr-1-bearing plasmids in the test strains were highly variable, with coexistence of two mcr-1-bearing plasmids being common. However, the MIC of colistin was not affected by the number of mcr-1-carrying plasmids harboured. Comparative analysis of the plasmids showed that they contained an mcr-1 gene cassette with varied structures (mcr-1-orf, ISApl1-mcr-1-orf and Tn6330), with the IncHI2 type being the most active in acquiring foreign resistance genes. A novel transposon, Tn6330, with the structure ISApl1-mcr-1-orf-ISApl1 was found to be the key element mediating translocation of mcr-1 into various plasmid backbones through formation of a circular intermediate. The mcr-1 gene can be disseminated via multiple mobile elements including Tn6330, its circular intermediate and plasmids harbouring such elements. It is often co-transmitted with other resistance determinants through IncHI2 plasmids. The functional mechanism of Tn6330, a typical composite transposon harbouring mcr-1, should be further investigated. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For

  20. Characteristics of plasmids in multi-drug-resistant Enterobacteriaceae isolated during prospective surveillance of a newly opened hospital in Iraq.

    Directory of Open Access Journals (Sweden)

    Xiao-Zhe Huang

    Full Text Available BACKGROUND: Gram-negative multidrug-resistant (MDR bacteria are major causes of nosocomial infections, and antibiotic resistance in these organisms is often plasmid mediated. Data are scarce pertaining to molecular mechanisms of antibiotic resistance in resource constrained areas such as Iraq. METHODOLOGY/PRINCIPAL FINDINGS: In this study, all MDR Enterobacteriaceae (n = 38 and randomly selected non-MDR counterparts (n = 41 isolated from patients, healthcare workers and environmental surfaces in a newly opened hospital in Iraq were investigated to characterize plasmids found in these isolates and determine their contribution to antibiotic resistance. Our results demonstrated that MDR E. coli and K. pneumoniae isolates harbored significantly more (≥ 3 plasmids compared to their non-MDR counterparts, which carried ≤ 2 plasmids (p<0.01. Various large plasmids (~52 to 100 kb from representative isolates were confirmed to contain multiple resistance genes by DNA microarray analysis. Aminoglycoside (acc, aadA, aph, strA/B, and ksgA, β-lactam (bla(TEM1, bla(AMPC, bla(CTX-M-15, bla(OXA-1, bla(VIM-2 and bla(SHV, sulfamethoxazole/trimethoprim (sul/dfr, tetracycline (tet and chloramphenicol (cat resistance genes were detected on these plasmids. Additionally, multiple plasmids carrying multiple antibiotic resistance genes were found in the same host strain. Genetic transfer-associated genes were identified on the plasmids from both MDR and non-MDR isolates. Seven plasmid replicon types (FII, FIA, FIB, B/O, K, I1 and N were detected in the isolates, while globally disseminated IncA/C and IncHI1 plasmids were not detected in these isolates. CONCLUSIONS/SIGNIFICANCE: This is the first report of the characteristics of the plasmids found in Enterobacteriaceae isolated following the opening of a new hospital in Iraq. The information provided here furthers our understanding of the mechanisms of drug resistance in this specific region and their evolutionary

  1. Genetics of auditory mechano-electrical transduction.

    Science.gov (United States)

    Michalski, Nicolas; Petit, Christine

    2015-01-01

    The hair bundles of cochlear hair cells play a central role in the auditory mechano-electrical transduction (MET) process. The identification of MET components and of associated molecular complexes by biochemical approaches is impeded by the very small number of hair cells within the cochlea. In contrast, human and mouse genetics have proven to be particularly powerful. The study of inherited forms of deafness led to the discovery of several essential proteins of the MET machinery, which are currently used as entry points to decipher the associated molecular networks. Notably, MET relies not only on the MET machinery but also on several elements ensuring the proper sound-induced oscillation of the hair bundle or the ionic environment necessary to drive the MET current. Here, we review the most significant advances in the molecular bases of the MET process that emerged from the genetics of hearing.

  2. Nonequilibrium phase transitions in biomolecular signal transduction

    Science.gov (United States)

    Smith, Eric; Krishnamurthy, Supriya; Fontana, Walter; Krakauer, David

    2011-11-01

    We study a mechanism for reliable switching in biomolecular signal-transduction cascades. Steady bistable states are created by system-size cooperative effects in populations of proteins, in spite of the fact that the phosphorylation-state transitions of any molecule, by means of which the switch is implemented, are highly stochastic. The emergence of switching is a nonequilibrium phase transition in an energetically driven, dissipative system described by a master equation. We use operator and functional integral methods from reaction-diffusion theory to solve for the phase structure, noise spectrum, and escape trajectories and first-passage times of a class of minimal models of switches, showing how all critical properties for switch behavior can be computed within a unified framework.

  3. Plasmid DNA entry into postmitotic nuclei of primary rat myotubes.

    OpenAIRE

    Dowty, M E; Williams, P.; G. Zhang; Hagstrom, J E; Wolff, J A

    1995-01-01

    These studies were initiated to elucidate the mechanism of DNA nuclear transport in mammalian cells. Biotin- or gold-labeled plasmid and plasmid DNA expression vectors for Escherichia coli beta-galactosidase or firefly luciferase were microinjected into the cytoplasm of primary rat myotubes in culture. Plasmid DNA was expressed in up to 70% of the injected myotubes, which indicates that it entered intact, postmitotic nuclei. The nuclear transport of plasmid DNA occurred through the nuclear po...

  4. Replication of plasmids in gram-negative bacteria.

    OpenAIRE

    1989-01-01

    Replication of plasmid deoxyribonucleic acid (DNA) is dependent on three stages: initiation, elongation, and termination. The first stage, initiation, depends on plasmid-encoded properties such as the replication origin and, in most cases, the replication initiation protein (Rep protein). In recent years the understanding of initiation and regulation of plasmid replication in Escherichia coli has increased considerably, but it is only for the ColE1-type plasmids that significant biochemical d...

  5. Striatal Signal Transduction and Drug Addiction

    Science.gov (United States)

    Philibin, Scott D.; Hernandez, Adan; Self, David W.; Bibb, James A.

    2011-01-01

    Drug addiction is a severe neuropsychiatric disorder characterized by loss of control over motivated behavior. The need for effective treatments mandates a greater understanding of the causes and identification of new therapeutic targets for drug development. Drugs of abuse subjugate normal reward-related behavior to uncontrollable drug-seeking and -taking. Contributions of brain reward circuitry are being mapped with increasing precision. The role of synaptic plasticity in addiction and underlying molecular mechanisms contributing to the formation of the addicted state are being delineated. Thus we may now consider the role of striatal signal transduction in addiction from a more integrative neurobiological perspective. Drugs of abuse alter dopaminergic and glutamatergic neurotransmission in medium spiny neurons of the striatum. Dopamine receptors important for reward serve as principle targets of drugs abuse, which interact with glutamate receptor signaling critical for reward learning. Complex networks of intracellular signal transduction mechanisms underlying these receptors are strongly stimulated by addictive drugs. Through these mechanisms, repeated drug exposure alters functional and structural neuroplasticity, resulting in transition to the addicted biological state and behavioral outcomes that typify addiction. Ca2+ and cAMP represent key second messengers that initiate signaling cascades, which regulate synaptic strength and neuronal excitability. Protein phosphorylation and dephosphorylation are fundamental mechanisms underlying synaptic plasticity that are dysregulated by drugs of abuse. Increased understanding of the regulatory mechanisms by which protein kinases and phosphatases exert their effects during normal reward learning and the addiction process may lead to novel targets and pharmacotherapeutics with increased efficacy in promoting abstinence and decreased side effects, such as interference with natural reward, for drug addiction. PMID

  6. Striatal signal transduction and drug addiction

    Directory of Open Access Journals (Sweden)

    Scott D. Philibin

    2011-09-01

    Full Text Available Drug addiction is a severe neuropsychiatric disorder characterized by loss of control over motivated behavior. The need for effective treatments mandates a greater understanding of the causes and identification of new therapeutic targets for drug development. Drugs of abuse subjugate normal reward-related behavior to uncontrollable drug-seeking and -taking. Contributions of brain reward circuitry are being mapped with increasing precision. The role of synaptic plasticity in addiction and underlying molecular mechanisms contributing to the formation of the addicted state are being delineated. Thus we may now consider the role of striatal signal transduction in addiction from a more integrative neurobiological perspective. Drugs of abuse alter dopaminergic and glutamatergic neurotransmission in medium spiny neurons of the striatum. Dopamine receptors important for reward serve as principle targets of drugs abuse, which interact with glutamate receptor signaling critical for reward learning. Complex networks of intracellular signal transduction mechanisms underlying these receptors are strongly stimulated by addictive drugs. Through these mechanisms, repeated drug exposure alters functional and structural neuroplasticity, resulting in transition to the addicted biological state and behavioral outcomes that typify addiction. Ca2+ and cAMP represent key second messengers that initiate signaling cascades, which regulate synaptic strength and neuronal excitability. Protein phosphorylation and dephosphorylation are fundamental mechanisms underlying synaptic plasticity that are dysregulated by drugs of abuse. Increased understanding of the regulatory mechanisms by which protein kinases and phosphatases exert their effects during normal reward learning and the addiction process may lead to novel targets and pharmacotherapeutics with increased efficacy in promoting abstinence and decreased side effects, such as interference with natural reward, for drug

  7. 深圳市菌痢流行分子特征与对质粒介导的喹诺酮耐药机制%The molecular characteristics of bacillary dysentery epidemic and the antibiotic resistance mechanisms of plasmid mediated quinolones in Shenzhen

    Institute of Scientific and Technical Information of China (English)

    蔡长争; 舒少为; 陈爱平; 黄国清; 周美容

    2016-01-01

    Objective To analyze the molecular characteristics of bacillary dysentery epidemic and the antibiotic resistance mechanisms of plasmid mediated quinolones in Shenzhen.Methods Clinical specimens were collected in 18 hospitals in Shenzhen form January 2010 to Febuary 2014 and isolation cultivation and serotype identiifcation were applied. The plasmid mediated main type of quinolones genes were detected by PCR. Minimal inhibitory concentration (MIC) was tested by Agar dilution method. Transcojugants genotype and drug resistance were tested by joint transfer experiment.Results Serological distribution: during all 126 strains ofShigella, there were 108 (87.51%) strains ofShigellalfexneri and 16 (12.70%) strains ofShigella sonnei. The superiority serotype of shigella flexneri was serumⅣ-C, with 42 strains counted for 38.89%. Commonly used antimicrobial susceptibility situation analysis: the sensitivity ofShigella lfexneri to NAl, FEP and GM were signiifcantly lower than that ofShigella sonnei, while the sensitivity ofShigella lfexneri to LEV, GIP, NOR, CAZ and AMC were signiifcantly higher than that ofShigella sonnei (P all < 0.05). Ampliifcation results and sequence analysis: 3 (2.38%) cases with qnr genes, 4 cases with aac6’ genes, and 1 case with qepA genes were checked out in 126 strainsShigella. Minimum inhibitory concentration: compared with receptor bacteria, the MIC of transconjugants on NAL, GIP, LEV, NOR, GM were improved by 2-32 times. Conclusions The main types ofShigella infection in Shenzhen areShigella lfexneri andShigellasonnei. The superiority serotype of shigella lfexneri isⅣ-C. Target gene mutation is the main cause of quinolones resistance. The quinolones resistance of different types of shigella varies signiifcantly.%目的:分析深圳市菌痢流行分子特征及对质粒介导的喹诺酮耐药机制。方法对2010年1月至2014年2月深圳市18家医院收集的临床标本进行分离培养与血清型鉴定,

  8. Surveillance on antibiotic susceptibility and plasmid-mediated resistance of Neisseria gonorrhoeae in Panyu Guangzhou%广州市番禺区淋病奈瑟球菌的耐药性及质粒介导耐药株的流行趋势

    Institute of Scientific and Technical Information of China (English)

    郭炽星; 张晖燕; 蒋敏慧; 徐碧红; 黎敬忠; 罗嘉莉

    2014-01-01

    Objective To monitor the minimum inhibitory concentration (MIC) of 5 antibiotics for Neisseria gonorrhoeae and its plasmid-mediated resistant strains,and to analyze the trend of drug resistant strains penicillinase-producing Neisseria gonorrhoeae (PPNG) and tetracycline-resistant Neisseria gonorrhoeae (TRNG).Methods Four hundred and thirty-six isolates of Neisseria gonorrhoeae were collected from Panyu Institute of Chronic Disease,Guangzhou from 2008 and 2012.The production of β-lactamase was determined by paper acidometric method.The agar dilution method was used to determine the MIC of spectinomycin,cefatriaxone,penicillin,tetracycline and ciprofloxacin.Results Out of 436 isolates,147 (33.72%) were plasmid-mediated PPNG strains and 222(50.92%) were TRNG strains.During the 5-year period,the prevalence of PPNG and TRNG ranged from 24.32% to 45.59% and from 21.31% to 67.57%,with significant differences(x2=11.659,38.464,P all<0.05).None of the strains were resistant to spectinomycin and ceftriaxone,but the ceftriaxone intermediate rate fluctuated from 26.13% to 72.13% with significant differences (x2=39.720,P<0.01),and their MIC50 and MIC90 were all in the sensitive ranges.However,the MIC50 and MIC90 of penicillin,tetracycline and ciprofloxacin changed greatly,and were significantly higher than resistant standards,with the resistance rates of 76.37% (333/ 436),88.76% (387/436) and 94.26% (411/436),respectively.Conclusions From 2008 to 2012,spectinomycin and ceftriaxone are both sensitive to Neisseria gonorrhoeae,and are recommended as the first-line antibiotics against gonorrhea.The MIC50 and MIC90 of penicillin,tetracycline and ciprofloxacin are unstable and rise above the resistant standards,indicating that it is inappropriate to use them as the first-line antibiotics in treatment of gonorrhea.%目的 监测淋病奈瑟球菌(淋球菌)对5种抗菌药物的最小抑菌浓度(MIC)和质粒介导耐药株,分析产β-内酰胺酶淋球

  9. Defining the Role of ATP Hydrolysis in Mitotic Segregation of Bacterial Plasmids

    Science.gov (United States)

    Ah-Seng, Yoan; Rech, Jérôme; Lane, David; Bouet, Jean-Yves

    2013-01-01

    Hydrolysis of ATP by partition ATPases, although considered a key step in the segregation mechanism that assures stable inheritance of plasmids, is intrinsically very weak. The cognate centromere-binding protein (CBP), together with DNA, stimulates the ATPase to hydrolyse ATP and to undertake the relocation that incites plasmid movement, apparently confirming the need for hydrolysis in partition. However, ATP-binding alone changes ATPase conformation and properties, making it difficult to rigorously distinguish the substrate and cofactor roles of ATP in vivo. We had shown that mutation of arginines R36 and R42 in the F plasmid CBP, SopB, reduces stimulation of SopA-catalyzed ATP hydrolysis without changing SopA-SopB affinity, suggesting the role of hydrolysis could be analyzed using SopA with normal conformational responses to ATP. Here, we report that strongly reducing SopB-mediated stimulation of ATP hydrolysis results in only slight destabilization of mini-F, although the instability, as well as an increase in mini-F clustering, is proportional to the ATPase deficit. Unexpectedly, the reduced stimulation also increased the frequency of SopA relocation over the nucleoid. The increase was due to drastic shortening of the period spent by SopA at nucleoid ends; average speed of migration per se was unchanged. Reduced ATP hydrolysis was also associated with pronounced deviations in positioning of mini-F, though time-averaged positions changed only modestly. Thus, by specifically targeting SopB-stimulated ATP hydrolysis our study reveals that even at levels of ATPase which reduce the efficiency of splitting clusters and the constancy of plasmid positioning, SopB still activates SopA mobility and plasmid positioning, and sustains near wild type levels of plasmid stability. PMID:24367270

  10. Protection from ischemic heart injury by a vigilant heme oxygenase-1 plasmid system.

    Science.gov (United States)

    Tang, Yao Liang; Tang, Yi; Zhang, Y Clare; Qian, Keping; Shen, Leping; Phillips, M Ian

    2004-04-01

    Although human heme oxygenase-1 (hHO-1) could provide a useful approach for cellular protection in the ischemic heart, constitutive overexpression of hHO-1 may lead to unwanted side effects. To avoid this, we designed a hypoxia-regulated hHO-1 gene therapy system that can be switched on and off. This vigilant plasmid system is composed of myosin light chain-2v promoter and a gene switch that is based on an oxygen-dependent degradation domain from the hypoxia inducible factor-1-alpha. The vector can sense ischemia and switch on the hHO-1 gene system, specifically in the heart. In an in vivo experiment, the vigilant hHO-1 plasmid or saline was injected intramyocardially into myocardial infarction mice or sham operation mice. After gene transfer, expression of hHO-1 was only detected in the ischemic heart treated with vigilant hHO-1 plasmids. Masson trichrome staining showed significantly fewer fibrotic areas in vigilant hHO-1 plasmids-treated mice compared with saline control (43.0%+/-4.8% versus 62.5%+/-3.3%, PhHO-1 expression in peri-infarct border areas, concomitant with higher Bcl-2 levels and lower Bax, Bak, and caspase 3 levels in the ischemic myocardium compared with saline control. By use of a cardiac catheter, heart from vigilant hHO-1 plasmids-treated mice showed improved recovery of contractile and diastolic performance after myocardial infarction compared with saline control. This study documents the beneficial regulation and therapeutic potential of vigilant plasmid-mediated hHO-1 gene transfer. This novel gene transfer strategy can provide cardiac-specific protection from future repeated bouts of ischemic injury.

  11. Defining the role of ATP hydrolysis in mitotic segregation of bacterial plasmids.

    Directory of Open Access Journals (Sweden)

    Yoan Ah-Seng

    Full Text Available Hydrolysis of ATP by partition ATPases, although considered a key step in the segregation mechanism that assures stable inheritance of plasmids, is intrinsically very weak. The cognate centromere-binding protein (CBP, together with DNA, stimulates the ATPase to hydrolyse ATP and to undertake the relocation that incites plasmid movement, apparently confirming the need for hydrolysis in partition. However, ATP-binding alone changes ATPase conformation and properties, making it difficult to rigorously distinguish the substrate and cofactor roles of ATP in vivo. We had shown that mutation of arginines R36 and R42 in the F plasmid CBP, SopB, reduces stimulation of SopA-catalyzed ATP hydrolysis without changing SopA-SopB affinity, suggesting the role of hydrolysis could be analyzed using SopA with normal conformational responses to ATP. Here, we report that strongly reducing SopB-mediated stimulation of ATP hydrolysis results in only slight destabilization of mini-F, although the instability, as well as an increase in mini-F clustering, is proportional to the ATPase deficit. Unexpectedly, the reduced stimulation also increased the frequency of SopA relocation over the nucleoid. The increase was due to drastic shortening of the period spent by SopA at nucleoid ends; average speed of migration per se was unchanged. Reduced ATP hydrolysis was also associated with pronounced deviations in positioning of mini-F, though time-averaged positions changed only modestly. Thus, by specifically targeting SopB-stimulated ATP hydrolysis our study reveals that even at levels of ATPase which reduce the efficiency of splitting clusters and the constancy of plasmid positioning, SopB still activates SopA mobility and plasmid positioning, and sustains near wild type levels of plasmid stability.

  12. A new and improved host-independent plasmid system for RK2-based conjugal transfer.

    Directory of Open Access Journals (Sweden)

    Trine Aakvik Strand

    Full Text Available Bacterial conjugation is a process that is mediated either by a direct cell-to-cell junction or by formation of a bridge between the cells. It is often used to transfer DNA constructs designed in Escherichia coli to recipient bacteria, yeast, plants and mammalian cells. Plasmids bearing the RK2/RP4 origin of transfer (oriT are mostly mobilized using the E. coli S17-1/SM10 donor strains, in which transfer helper functions are provided from a chromosomally integrated RP4::Mu. We have observed that large plasmids were occasionally modified after conjugal transfer when using E. coli S17-1 as a donor. All modified plasmids had increased in size, which most probably was a result of co-transfer of DNA from the chromosomally located oriT. It has earlier also been demonstrated that the bacteriophage Mu is silently transferred to recipient cells by these donor strains, and both occurrences are very likely to lead to mutations within the recipient DNA. Here we report the construction of a new biological system addressing both the above mentioned problems in which the transfer helper functions are provided by a plasmid lacking a functional oriT. This system is compatible with all other replicons commonly used in conjugation experiments and further enables the use of diverse bacterial strains as donors. Plasmids containing large inserts were successfully conjugated and the plasmid modifications observed when E. coli S17-1 was used as donor were eliminated by the use of the new host-independent vector system.

  13. An approach towards bronchoscopic-based gene therapy using electrical field accelerated plasmid droplets.

    Science.gov (United States)

    Hradetzky, D; Boehringer, S; Geiser, Th; Gazdhar, A

    2012-01-01

    Idiopathic pulmonary fibrosis (IPF) is a devastating disease affecting the distal lung, due to failure of the alveolar epithelium to heal after micro-injuries, leading to inefficient gas exchange and resulting in death. Therapeutic options are very limited. A new therapeutic approach based on gene therapy restores the self-healing process within the lung in the experimental setup. A basic requirement of this therapy is the successful transduction of genes into the alveolar epithelium in the distal part of the lung, for which a new therapeutic instrument is required. In this paper we present the concept and first experimental results of a device which uses an electrical field to accelerate the charged droplets of plasmid suspension toward the tissue and which overcomes cell membrane with its impact energy. The aim is to develop a therapeutic device capable of being integrated into minimally invasive procedures such as bronchoscopy.

  14. Signal transduction around thymic stromal lymphopoietin (TSLP in atopic asthma

    Directory of Open Access Journals (Sweden)

    Kuepper Michael

    2008-08-01

    Full Text Available Abstract Thymic stromal lymphopoietin (TSLP, a novel interleukin-7-like cytokine, triggers dendritic cell-mediated inflammatory responses ultimately executed by T helper cells of the Th2 subtype. TSLP emerged as a central player in the development of allergic symptoms, especially in the airways, and is a prime regulatory cytokine at the interface of virus- or antigen-exposed epithelial cells and dendritic cells (DCs. DCs activated by epithelium-derived TSLP can promote naïve CD4+ T cells to adopt a Th2 phenotype, which in turn recruite eosinophilic and basophilic granulocytes as well as mast cells into the airway mucosa. These different cells secrete inflammatory cytokines and chemokines operative in inducing an allergic inflammation and atopic asthma. TSLP is, thus, involved in the control of both an innate and an adaptive immune response. Since TSLP links contact of allergen with the airway epithelium to the onset and maintainance of the asthmatic syndrome, defining the signal transduction underlying TSLP expression and function is of profound interest for a better understandimg of the disease and for the development of new therapeutics.

  15. Modulation of signal transduction by tea catechins and related phytochemicals

    Energy Technology Data Exchange (ETDEWEB)

    Shimizu, Masahito [Herbert Irving Comprehensive Cancer Center and Department of Medicine, Columbia University Medical Center, HHSC-1509, 701 West 168 Street, NY 10032-2704 (United States); Weinstein, I. Bernard [Herbert Irving Comprehensive Cancer Center and Department of Medicine, Columbia University Medical Center, HHSC-1509, 701 West 168 Street, NY 10032-2704 (United States)]. E-mail: ibw1@columbia.edu

    2005-12-11

    Epidemiologic studies in human populations and experimental studies in rodents provide evidence that green tea and its constituents can inhibit both the development and growth of tumors at a variety of tissue sites. In addition, EGCG, a major biologically active component of green tea, inhibits growth and induces apoptosis in a variety of cancer cell lines. The purpose of this paper is to review evidence that these effects are mediated, at least in part, through inhibition of the activity of specific receptor tyrosine kinases (RTKs) and related downstream pathways of signal transduction. We also review evidence indicating that the antitumor effects of the related polyphenolic phytochemicals resveratrol, genistein, curcumin, and capsaicin are exerted via similar mechanisms. Some of these agents (EGCG, genistein, and curcumin) appear to directly target specific RTKs, and all of these compounds cause inhibition of the activity of the transcription factors AP-1 and NF-{kappa}B, thus inhibiting cell proliferation and enhancing apoptosis. Critical areas of future investigation include: (1) identification of the direct molecular target(s) of EGCG and related polyphenolic compounds in cells; (2) the in vivo metabolism and bioavailability of these compounds; (3) the ancillary effects of these compounds on tumor-stromal interactions; (4) the development of synergistic combinations with other antitumor agents to enhance efficacy in cancer prevention and therapy, and also minimize potential toxicities.

  16. Glycation & the RAGE axis: targeting signal transduction through DIAPH1.

    Science.gov (United States)

    Shekhtman, Alexander; Ramasamy, Ravichandran; Schmidt, Ann Marie

    2017-02-01

    The consequences of chronic disease are vast and unremitting; hence, understanding the pathogenic mechanisms mediating such disorders holds promise to identify therapeutics and diminish the consequences. The ligands of the receptor for advanced glycation end products (RAGE) accumulate in chronic diseases, particularly those characterized by inflammation and metabolic dysfunction. Although first discovered and reported as a receptor for advanced glycation end products (AGEs), the expansion of the repertoire of RAGE ligands implicates the receptor in diverse milieus, such as autoimmunity, chronic inflammation, obesity, diabetes, and neurodegeneration. Areas covered: This review summarizes current knowledge regarding the ligand families of RAGE and data from human subjects and animal models on the role of the RAGE axis in chronic diseases. The recent discovery that the cytoplasmic domain of RAGE binds to the formin homology 1 (FH1) domain, DIAPH1, and that this interaction is essential for RAGE ligand-stimulated signal transduction, is discussed. Finally, we review therapeutic opportunities targeting the RAGE axis as a means to mitigate chronic diseases. Expert commentary: With the aging of the population and the epidemic of cardiometabolic disease, therapeutic strategies to target molecular pathways that contribute to the sequelae of these chronic diseases are urgently needed. In this review, we propose that the ligand/RAGE axis and its signaling nexus is a key factor in the pathogenesis of chronic disease and that therapeutic interruption of this pathway may improve quality and duration of life.

  17. Plasmid Segregation: Spatial Awareness at the Molecular Level

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Gerdes, Kenn

    2007-01-01

    In bacteria, low-copy number plasmids ensure their stable inheritance by partition loci (par), which actively distribute plasmid replicates to each side of the cell division plane. Using time-lapse fluorescence microscopic tracking of segregating plasmid molecules, a new study provides novel insi...

  18. Cloning of Two Bacteriocin Genes from a Lactococcal Bacteriocin Plasmid

    NARCIS (Netherlands)

    Belkum, Marco J. van; Hayema, Bert Jan; Geis, Arnold; Kok, Jan; Venema, Gerard

    1989-01-01

    Lactococcus lactis subsp. cremoris 9B4 plasmid p9B4-6 (60 kilobases [kb]), which specifies bacteriocin production and immunity, was analyzed with restriction endonucleases, and fragments of this plasmid were cloned into shuttle vectors based on the broad-host-range plasmid pWVO1. Two regions on p9B4

  19. Multilocus sequence typing of IncN plasmids

    DEFF Research Database (Denmark)

    García-Fernández, Aurora; Villa, Laura; Moodley, Arshnee

    2011-01-01

    categorization of IncN plasmids. METHODS: Twelve fully sequenced IncN plasmids available at GenBank were analysed in silico for selecting the loci for the IncN-specific pMLST. A total of 58 plasmids originating from different reservoirs (human, pig, poultry, cattle and horses) and geographic regions (Italy...

  20. A low-copy-number plasmid for retrieval of toxic genes from BACs and generation of conditional targeting constructs.

    Science.gov (United States)

    Na, Giyoun; Wolfe, Andrew; Ko, Chemyong; Youn, Hyesook; Lee, Young-Min; Byun, Sung June; Jeon, Iksoo; Koo, Yongbum

    2013-06-01

    Bacterial Artificial Chromosome (BAC) clones are widely used for retrieving genomic DNA sequences for gene targeting. In this study, low-copy-number plasmids pBAC-FB, pBAC-FC, and pBAC-DE, which carry the F plasmid replicon, were generated from pBACe3.6. pBAC-FB was successfully used to retrieve a sequence of a BAC that was resistant to retrieval by a high-copy-number plasmid via λ Red-mediated recombineering (gap-repair cloning). This plasmid was also used to retrieve two other genes from BAC, indicating its general usability retrieving genes from BAC. The retrieved genes were manipulated in generating targeting vectors for gene knockouts by recombineering. The functionality of the targeting vector was further validated in a targeting experiment with C57BL/6 embryonic stem cells. The low-copy-number plasmid pBAC-FB is a plasmid of choice to retrieve toxic DNA sequences from BACs and to manipulate them to generate gene-targeting constructs by recombineering.

  1. Signal transduction through the IL-4 and insulin receptor families.

    Science.gov (United States)

    Wang, L M; Keegan, A; Frankel, M; Paul, W E; Pierce, J H

    1995-07-01

    Activation of tyrosine kinase-containing receptors and intracellular tyrosine kinases by ligand stimulation is known to be crucial for mediating initial and subsequent events involved in mitogenic signal transduction. Receptors for insulin and insulin-like growth factor 1 (IGF-1) contain cytoplasmic tyrosine kinase domains that undergo autophosphorylation upon ligand stimulation. Activation of these receptors also leads to pronounced and rapid tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) in cells of connective tissue origin. A related substrate, designated 4PS, is similarly phosphorylated by insulin and IGF-1 stimulation in many hematopoietic cell types. IRS-1 and 4PS possess a number of tyrosine phosphorylation sites that are within motifs that bind specific SH2-containing molecules known to be involved in mitogenic signaling such as PI-3 kinase, SHPTP-2 (Syp) and Grb-2. Thus, they appear to act as docking substrates for a variety of signaling molecules. The majority of hematopoietic cytokines bind to receptors that do not possess intrinsic kinase activity, and these receptors have been collectively termed as members of the hematopoietin receptor superfamily. Despite their lack of tyrosine kinase domains, stimulation of these receptors has been demonstrated to activate intracellular kinases leading to tyrosine phosphorylation of multiple substrates. Recent evidence has demonstrated that activation of different members of the Janus family of tyrosine kinases is involved in mediating tyrosine phosphorylation events by specific cytokines. Stimulation of the interleukin 4 (IL-4) receptor, a member of the hematopoietin receptor superfamily, is thought to result in activation of Jak1, Jak3, and/or Fes tyrosine kinases.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Enhanced plasmid loss in bacterial populations exposed to the antimicrobial compound irgasan delivered from interpenetrating polymer network silicone hydrogels

    DEFF Research Database (Denmark)

    Riber, Leise; Burmølle, Mette; Alm, Martin;

    2016-01-01

    into liquid growth medium from irgasan-impregnated Interpenetrating Polymer Network (IPN) silicone hydrogel objects, a novel technology developed as drug-delivery platform. IPN-mediated irgasan-release was indirectly monitored as the extent of plasmid loss from bacterial cells during a 24-hour period...

  3. FCJ-124 Interactive Environments as Fields of Transduction

    Directory of Open Access Journals (Sweden)

    Cristoph Brunner

    2011-10-01

    Full Text Available This article proposes a critical inquiry of interactive environments as fields of transduction. It is argued that Gilbert Simondon’s concepts of individuation, transduction, in-formation, the preindividual, and the associated milieu enable a processual thinking of the analysis and design of interactive technologies as technogenetic emergence. These concepts offer a way for interaction design to understand interactive environments through the dynamics between fields of transduction and fields of experience in relational and affective terms. The article analyses the way in which two technological assemblages, Voz Alta and the Impossible Room, provide different experiential fields experimenting with the transductive power of digital and interactive media. We emphasise the potential for creating new modes of experience. Our aim is to underline the necessary convergences between practices of design and thought; to enable affectively engaging fields of transduction.

  4. Transductive versions of the LASSO and the Dantzig Selector

    CERN Document Server

    Alquier, Pierre

    2010-01-01

    Transductive methods are useful in prediction problems when the training dataset is composed of a large number of unlabeled observations and a smaller number of labeled observations. In this paper, we propose an approach for developing transductive prediction procedures that are able to take advantage of the sparsity in the high dimensional linear regression. More precisely, we define transductive versions of the LASSO and the Dantzig Selector . These procedures combine labeled and unlabeled observations of the training dataset to produce a prediction for the unlabeled observations. We propose an experimental study of the transductive estimators, that shows that they improve the LASSO and Dantzig Selector in many situations, and particularly in high dimensional problems when the predictors are correlated. We then provide non-asymptotic theoretical guarantees for these estimation methods. Interestingly, our theoretical results show that the Transductive LASSO and Dantzig Selector satisfy sparsity inequalities ...

  5. Bacteriophages limit the existence conditions for conjugative plasmids.

    Science.gov (United States)

    Harrison, Ellie; Wood, A Jamie; Dytham, Calvin; Pitchford, Jonathan W; Truman, Julie; Spiers, Andrew; Paterson, Steve; Brockhurst, Michael A

    2015-06-02

    Bacteriophages are a major cause of bacterial mortality and impose strong selection on natural bacterial populations, yet their effects on the dynamics of conjugative plasmids have rarely been tested. We combined experimental evolution, mathematical modeling, and individual-based simulations to explain how the ecological and population genetics effects of bacteriophages upon bacteria interact to determine the dynamics of conjugative plasmids and their persistence. The ecological effects of bacteriophages on bacteria are predicted to limit the existence conditions for conjugative plasmids, preventing persistence under weak selection for plasmid accessory traits. Experiments showed that phages drove faster extinction of plasmids in environments where the plasmid conferred no benefit, but they also revealed more complex effects of phages on plasmid dynamics under these conditions, specifically, the temporary maintenance of plasmids at fixation followed by rapid loss. We hypothesized that the population genetic effects of bacteriophages, specifically, selection for phage resistance mutations, may have caused this. Further mathematical modeling and individual-based simulations supported our hypothesis, showing that conjugative plasmids may hitchhike with phage resistance mutations in the bacterial chromosome. Conjugative plasmids are infectious loops of DNA capable of transmitting DNA between bacterial cells and between species. Because plasmids often carry extra genes that allow bacteria to live in otherwise-inhospitable environments, their dynamics are central to understanding bacterial adaptive evolution. The plasmid-bacterium interaction has typically been studied in isolation, but in natural bacterial communities, bacteriophages, viruses that infect bacteria, are ubiquitous. Using experiments, mathematical models, and computer simulations we show that bacteriophages drive plasmid dynamics through their ecological and evolutionary effects on bacteria and ultimately

  6. Bacteriophage selection against a plasmid-encoded sex apparatus leads to the loss of antibiotic-resistance plasmids.

    Science.gov (United States)

    Jalasvuori, Matti; Friman, Ville-Petri; Nieminen, Anne; Bamford, Jaana K H; Buckling, Angus

    2011-12-23

    Antibiotic-resistance genes are often carried by conjugative plasmids, which spread within and between bacterial species. It has long been recognized that some viruses of bacteria (bacteriophage; phage) have evolved to infect and kill plasmid-harbouring cells. This raises a question: can phages cause the loss of plasmid-associated antibiotic resistance by selecting for plasmid-free bacteria, or can bacteria or plasmids evolve resistance to phages in other ways? Here, we show that multiple antibiotic-resistance genes containing plasmids are stably maintained in both Escherichia coli and Salmonella enterica in the absence of phages, while plasmid-dependent phage PRD1 causes a dramatic reduction in the frequency of antibiotic-resistant bacteria. The loss of antibiotic resistance in cells initially harbouring RP4 plasmid was shown to result from evolution of phage resistance where bacterial cells expelled their plasmid (and hence the suitable receptor for phages). Phages also selected for a low frequency of plasmid-containing, phage-resistant bacteria, presumably as a result of modification of the plasmid-encoded receptor. However, these double-resistant mutants had a growth cost compared with phage-resistant but antibiotic-susceptible mutants and were unable to conjugate. These results suggest that bacteriophages could play a significant role in restricting the spread of plasmid-encoded antibiotic resistance.

  7. Characterization of blaTEM-52-carrying plasmids of extended-spectrum-β-lactamase-producing Salmonella enterica isolates from chicken meat with a common supplier in Japan.

    Science.gov (United States)

    Matsumoto, Y