scholarly journals Promoter deletions ofKlebsiella pneumoniaecarbapenemase (KPC)-encoding genes (blaKPC-2) and efflux pump (AcrAB) on β-lactam susceptibility in KPC-producingEnterobacteriaceae

2013 ◽  
Vol 348 (2) ◽  
pp. 120-126 ◽  
Author(s):  
Gomattie D. Seecoomar ◽  
Brenda C. Marmol ◽  
Dong H. Kwon
Keyword(s):  
Efflux Pump ◽  
Encoding Genes ◽  
Promoter Deletions

scholarly journals Cloning, Sequencing, and Characterization of the SdeAB Multidrug Efflux Pump of Serratia marcescens

2005 ◽  
Vol 49 (4) ◽  
pp. 1495-1501 ◽  
Author(s):  
Ayush Kumar ◽  
Elizabeth A. Worobec
Keyword(s):  
Serratia Marcescens ◽  
Type Strain ◽  
Wild Type Strain ◽  
Genomic Library ◽  
Efflux Pump ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Diverse Range ◽  
Mutant Strains ◽  
Encoding Genes

ABSTRACT Serratia marcescens is an important nosocomial agent known for causing various infections in immunocompromised individuals. Resistance of this organism to a broad spectrum of antibiotics makes the treatment of infections very difficult. This study was undertaken to identify multidrug resistance efflux pumps in S. marcescens. Three mutant strains of S. marcescens were isolated in vitro by the serial passaging of a wild-type strain in culture medium supplemented with ciprofloxacin, norfloxacin, or ofloxacin. Fluoroquinolone accumulation assays were performed to detect the presence of a proton gradient-dependent efflux mechanism. Two of the mutant strains were found to be effluxing norfloxacin, ciprofloxacin, and ofloxacin, while the third was found to efflux only ofloxacin. A genomic library of S. marcescens wild-type strain UOC-67 was constructed and screened for RND pump-encoding genes by using DNA probes for two putative RND pump-encoding genes. Two different loci were identified: sdeAB, encoding an MFP and an RND pump, and sdeCDE, encoding an MFP and two different RND pumps. Northern blot analysis revealed overexpression of sdeB in two mutant strains effluxing fluoroquinolones. Analysis of the sdeAB and sdeCDE loci in Escherichia coli strain AG102MB, deficient in the RND pump (AcrB), revealed that gene products of sdeAB are responsible for the efflux of a diverse range of substrates that includes ciprofloxacin, norfloxacin, ofloxacin, chloramphenicol, sodium dodecyl sulfate, ethidium bromide, and n-hexane, while those of sdeCDE did not result in any change in susceptibilities to any of these agents.

scholarly journals The Odyssey of the Ancestral Escherich Strain through Culture Collections: an Example of Allopatric Diversification

mSphere ◽  
2018 ◽  
Vol 3 (1) ◽  
Author(s):  
M. Desroches ◽  
G. Royer ◽  
D. Roche ◽  
M. Mercier-Darty ◽  
D. Vallenet ◽  
...  
Keyword(s):  
Experimental Evolution ◽  
Efflux Pump ◽  
Strain History ◽  
Culture Collections ◽  
E Coli ◽  
Molecular Features ◽  
Natural Isolates ◽  
History Of ◽  
Encoding Genes

Mutator phenotypes have been described in laboratory-evolved bacteria, as well as in natural isolates. Several genes can be impacted, each of them being associated with a typical mutational spectrum. By studying one of the oldest strains available, the ancestral Escherich strain, we were able to identify its mutator status leading to tremendous genetic diversity among the isolates from various collections and allowing us to reconstruct the phylogeographic history of the strain. This mutator phenotype was probably acquired during the storage of the strain, promoting adaptation to a specific environment. Other mutations inrpoSand efflux pump- and porin-encoding genes highlight the acclimatization of the strain through self-preservation and nutritional competence regulation. This strain history can be viewed as unintentional experimental evolution in culture collections all over the word since 1885, mimicking the long-term experimental evolution ofE. coliof Lenski et al. (O. Tenaillon, J. E. Barrick, N. Ribeck, D. E. Deatherage, J. L. Blanchard, A. Dasgupta, G. C. Wu, S. Wielgoss, S. Cruveiller, C. Médigue, D. Schneider, and R. E. Lenski, Nature 536:165–170, 2016, https://doi.org/10.1038/nature18959) that shares numerous molecular features.

scholarly journals Characterization of emeA, anorA Homolog and Multidrug Resistance Efflux Pump, inEnterococcus faecalis

2001 ◽  
Vol 45 (12) ◽  
pp. 3574-3579 ◽  
Author(s):  
Brandie M. Jonas ◽  
Barbara E. Murray ◽  
George M. Weinstock
Keyword(s):  
Drug Resistance ◽  
Multidrug Resistance ◽  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Efflux Pump ◽  
Wild Type ◽  
Type Gene ◽  
Encoding Genes

ABSTRACT We hypothesized that multidrug resistance efflux pumps (MDRs) may be contributing to the drug resistance of enterococci. We recently identified potential MDR-encoding genes in the Enterococcus faecalis V583 genome. Among the putative MDRs, we found a gene that encodes a NorA homolog and have characterized this enterococcal MDR in the present study. A mutant from which the enterococcal NorA homolog has been deleted has reduced resistance to several NorA substrates. Complementation of the deletion mutant with the wild-type gene verified the involvement of this enterococcal gene in resistance to ethidium bromide (EtBr) and norfloxacin. Known MDR inhibitors (reserpine, lansoprazole, and verapamil) inhibit the efflux of EtBr and norfloxacin in wild-type strain OG1RF. A fluorescence assay with EtBr allowed us to quantitate the efflux capability of the enterococcal NorA pump. On the basis of these results, we have named this enterococcal gene emeA (enterococcal multidrug resistance efflux).

scholarly journals Activity of imipenem/relebactam on Klebsiella pneumoniae with different mechanisms of imipenem non-susceptibility

2021 ◽  
Author(s):  
Mervat El-Sayed Mashaly ◽  
Ghada El-Saeed Mashaly
Keyword(s):  
Gene Expression ◽  
Klebsiella Pneumoniae ◽  
Efflux Pump ◽  
Molecular Profile ◽  
Efflux Pump Inhibitor ◽  
Extended Spectrum Beta Lactamase ◽  
Reverse Transcription Pcr ◽  
Treat Ment ◽  
Encoding Genes

Background and Objectives: Imipenem/relebactam (IMP/R) is a newly FDA approved β-lactam/β-lactamase inhibitor combination. Relebactam ability to restore IMP activity could differ according to the cause of imipenem non-susceptibility. Therefore, we investigated the in-vitro activity of IMP/R against Klebsiella pneumoniae with different mechanisms of imi- penem non-susceptibility. Materials and Methods: Imipenem-nonsusceptible (IMP-NS) K. pneumoniae isolates were collected and characterized for β-lactamase encoding genes by multiplex PCR. For IMP-NS carbapenemase-negative isolates, study of Ompk35 & Ompk36 gene expression was performed by reverse transcription-PCR while efflux pump activity was studied by minimum inhibitory concentration (MIC) reduction assay using efflux pump inhibitor. Susceptibility testing of K. pneumoniae to IMP and IMP/R were achieved by broth microdilution (BMD) method. Results: During the study period, 140 isolates of IMP-NS K. pneumoniae were collected. BMD method showed that relebac- tam restored IMP susceptibility in 100%, 60% and 49% of isolates that only harbor AmpC, extended spectrum beta lactamase (ESBL) and carbapenemases, respectively. IMP/R was most potent against all bla KPC and 50% of bla _producing isolates. No demonstrable activity of IMP/R against K. pneumoniae harboring metallo-β-lactamases (MBLs). Out of 18 isolates with IMP non-suceptibility due to porins loss with overproduction of ESBL and/or AmpC, 14 (77.7%) isolates were IMP/R sus- ceptible. IMP/R showed no activity against isolates with only efflux pump hyperactivity. Conclusion: Relebactam could restore IPM activity in KPC or AmpC-producing IMP/NS K. pneumoniae but with no ac- tivity against MBL- producing isolates. Relebactam activity against isolates harbouring-bla OXA-48 or with altered Ompk35 & Ompk36 gene expression and efflux pump hyperactivity need further studies. Therefore, using IMP/R antibiotic in the treat- ment of infections caused by IMP/NS K. pneumoniae should be based on its molecular profile of IMP resistance to optimize the utility of IMP/R.

scholarly journals Triclosan Can Select for an AdeIJK-Overexpressing Mutant of Acinetobacter baumannii ATCC 17978 That Displays Reduced Susceptibility to Multiple Antibiotics

2014 ◽  
Vol 58 (11) ◽  
pp. 6424-6431 ◽  
Author(s):  
Dinesh M. Fernando ◽  
Wayne Xu ◽  
Peter C. Loewen ◽  
George G. Zhanel ◽  
Ayush Kumar
Keyword(s):  
Acinetobacter Baumannii ◽  
Efflux Pump ◽  
Resistant Mutant ◽  
Dilution Method ◽  
Gene Encoding ◽  
Content Type ◽  
Resistance Nodulation Division ◽  
Triclosan Resistance ◽  
Encoding Genes ◽  
Multiple Antibiotics

ABSTRACTIn order to determine if triclosan can select for mutants ofAcinetobacter baumanniiATCC 17978 that display reduced susceptibilities to antibiotics, we isolated a triclosan-resistant mutant,A. baumanniiAB042, by serial passaging ofA. baumanniiATCC 17978 in growth medium supplemented with triclosan. The antimicrobial susceptibility of AB042 was analyzed by the 2-fold serial dilution method. Expression of five different resistance-nodulation-division (RND) pump-encoding genes (adeB,adeG,adeJ,A1S_2818, andA1S_3217), two outer membrane porin-encoding genes (carOandoprD), and the MATE family pump-encoding geneabeMwas analyzed using quantitative reverse transcriptase (qRT) PCR.A. baumanniiAB042 exhibited elevated resistance to multiple antibiotics, including piperacillin-tazobactam, doxycycline, moxifloxacin, ceftriaxone, cefepime, meropenem, doripenem, ertapenem, ciprofloxacin, aztreonam, tigecycline, and trimethoprim-sulfamethoxazole, in addition to triclosan. Genome sequencing ofA. baumanniiAB042 revealed a116G→V mutation infabI, the gene encoding the target enzyme for triclosan. Expression analysis of efflux pumps showed overexpression of the AdeIJK pump, and sequencing ofadeN, the gene that encodes the repressor of theadeIJKoperon, revealed a 73-bp deletion which would cause a premature termination of translation, resulting in an inactive truncated AdeN protein. This work shows that triclosan can select for mutants ofA. baumanniithat display reduced susceptibilities to multiple antibiotics from chemically distinct classes in addition to triclosan resistance. This multidrug resistance can be explained by the overexpression of the AdeIJK efflux pump.

scholarly journals The prevalence and mechanism of triclosan resistance in Escherichia coli isolated from urine samples in Wenzhou, China

2020 ◽  
Author(s):  
Weiliang Zeng ◽  
Wenya Xu ◽  
Ye Xu ◽  
Wenli Liao ◽  
Yajie Zhao ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Urine Samples ◽  
Cross Resistance ◽  
Chain Reaction ◽  
E Coli ◽  
Resistant Strains ◽  
Polymerase Chain ◽  
Triclosan Resistance ◽  
Encoding Genes

Abstract Background: The widespread application of triclosan contributes to its residual deposition in urine, which provides an environment of long-term exposure to triclosan for the intestinal Escherichia coli. We determined the triclosan and antibiotic resistance characteristics of E. coli strains isolated from urine samples and further investigated the resistance mechanism and molecular epidemic characteristics of triclosan-resistant E. coli isolates. Methods: A total of 200 non-repetitive E. coli strains were isolated from urine samples and then identified. The minimum inhibitory concentrations (MICs) of triclosan and antibiotics, fabI mutation, efflux pump activity, the expression of 14 efflux pump encoding genes, and epidemiological characteristics were determined by the agar dilution method, polymerase chain reaction (PCR), carbonyl cyanide 3-chlorophenylhydrazone (CCCP) inhibition test, quantitative real-time polymerase chain reaction (RT-qPCR), multilocus sequence typing (MLST), and pulse-field gel electrophoresis (PFGE) for all triclosan-resistant isolates. Furthermore, we also investigated the effect of triclosan exposure in vitro on antibiotic susceptibility and the efflux pump encoding gene expressions of triclosan-susceptible strains via serial passage experiments. Results: Of the 200 E. coli isolates, 2.5% (n = 5) were found to be resistant to triclosan, and multidrug resistance (MDR) and cross-resistance phenotypes were noted for these triclosan-resistant strains. The triclosan-sensitive strains also exhibited MDR phenotypes, probably because of the high resistance rate to AMP, CIP, LVX, and GEN. Gly79Ala and Ala69Thr amino acid changes were observed in the triclosan-resistant strains, but these changes may not mediate resistance of E. coli to triclosan, because mutations of these two amino acids has also been detected in triclosan-susceptible strains. Moreover, except for DC8603, all other strains enhanced the efflux pumps activity. As compared with ATCC 25922, except for fabI, increased expressions were noted for all efflux pump encoding genes such as ydcV, ydcU, ydcS, ydcT, cysP, yihV, acrB, acrD, and mdfA among the studied strains with varying PFGE patterns and STs types. Unexpectedly, 5 susceptible E. coli isolates showed rapidly increasing triclosan resistance after exposure to triclosan in vitro for only 12 days, while MDR or cross-resistance phenotypes and the overexpression of efflux pump genes were recorded among these triclosan-induced resistant isolates. Conclusions: This is the first study to report that short-term triclosan exposure in vitro increases triclosan resistance in susceptible E. coli isolates. After acquiring resistance, these strains may present MDR or cross-resistance phenotypes. Moreover, triclosan resistance mainly involves the overexpression of fabI and efflux pumps in E. coli isolates.

scholarly journals The prevalence and mechanism of triclosan resistance in Escherichia coli isolates from urine samples in Wenzhou, China

2020 ◽  
Author(s):  
Weiliang Zeng ◽  
Wenya Xu ◽  
Ye Xu ◽  
Wenli Liao ◽  
Yajie Zhao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Efflux Pump ◽  
Cross Resistance ◽  
Chain Reaction ◽  
E Coli ◽  
Resistant Strains ◽  
Polymerase Chain ◽  
Triclosan Resistance ◽  
Encoding Genes

Abstract Background Widespread use of triclosan has been reported to cause its residue in urine, which provides an environment of long-term exposure to triclosan for intestinal Escherichia coli. We aimed to determine the triclosan and antibiotic resistance characteristics of Escherichia coli strains isolated from urine, and further investigate the resistance mechanism and molecular epidemic characteristics of triclosan resistant Escherichia coli isolates. Methods A total of 200 non-repetitive E. coli strains from urine samples were obtained and identified. The minimum inhibitory concentrations (MICs) of triclosan and antibiotics, fabI mutation, efflux pump activity, expression of 14 efflux pump encoding genes and epidemiological characteristics were detected with agar dilution method, polymerase chain reaction (PCR), carbonyl cyanide 3-chlorophenylhydrazone (CCCP) inhibition test, quantitative real-time polymerase chain reaction (RT-qPCR), multilocus sequence typing (MLST) and pulse field gel electrophoresis (PFGE) in all triclosan resistant isolates. Furthermore, we also investigated the effect of triclosan exposure in vitro on resistance in susceptible strains by serial passage experiment. Results Of 200 E. coli isolates, 2.5% (n = 5) were resistant to triclosan, multidrug resistance (MDR) and cross-resistance phenotypes were observed in these resistant strains, but not in susceptible strains. We did not observe any sense mutations within fabI gene in triclosan resistant strains. Moreover, except DC8603, all the others enhanced efflux pumps activity. Compared with ATCC 25922, except fabI, increased expression were also found in efflux pump encoding genes ydcV, ydcU, ydcS, ydcT, cysP, yihV, acrB, acrD and mdfA in studied strains with different PFGE patterns and STs types. Surprised, 5 susceptible E. coli isolates increased rapidly triclosan resistance only 4 days after exposure to subinhibitory triclosan concentration in vitro. Conclusions Our study is the first to be reported that short-term triclosan exposure in vitro increases triclosan resistance in susceptible E. coli isolates. Once strains have acquired resistance, they usually present MDR or cross-resistance phenotypes. Besides, our findings indicate that triclosan resistance were mainly involved by fabI overexpression in E. coli, and there was a close association between overexpression of efflux pumps with triclosan resistance.

scholarly journals Quantitative Contributions of Target Alteration and Decreased Drug Accumulation to Pseudomonas aeruginosa Fluoroquinolone Resistance

2012 ◽  
Vol 57 (3) ◽  
pp. 1361-1368 ◽  
Author(s):  
Sebastian Bruchmann ◽  
Andreas Dötsch ◽  
Bianka Nouri ◽  
Iris F. Chaberny ◽  
Susanne Häussler
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Fluoroquinolone Resistance ◽  
Content Type ◽  
Quinolone Antibiotics ◽  
Encoding Gene ◽  
Regulator Genes ◽  
Encoding Genes ◽  
Quantitative Contributions

ABSTRACTQuinolone antibiotics constitute a clinically successful and widely used class of broad-spectrum antibiotics; however, the emergence and spread of resistance increasingly limits the use of fluoroquinolones in the treatment and management of microbial disease. In this study, we evaluated the quantitative contributions of quinolone target alteration and efflux pump expression to fluoroquinolone resistance inPseudomonas aeruginosa. We generated isogenic mutations in hot spots of the quinolone resistance-determining regions (QRDRs) ofgyrA,gyrB, andparCand inactivated the efflux regulator genes so as to overexpress the corresponding multidrug resistance (MDR) efflux pumps. We then introduced the respective mutations into the reference strain PA14 singly and in various combinations. Whereas the combined inactivation of two efflux regulator-encoding genes did not lead to resistance levels higher than those obtained by inactivation of only one efflux regulator-encoding gene, the combination of mutations leading to increased efflux and target alteration clearly exhibited an additive effect. This combination of target alteration and overexpression of efflux pumps was commonly observed in clinicalP. aeruginosaisolates; however, these two mechanisms were frequently found not to be sufficient to explain the level of fluoroquinolone resistance. Our results suggest that there are additional mechanisms, independent of the expression of the MexAB-OprM, MexCD-OprJ, MexEF-OprN, and/or MexXY-OprM efflux pump, that increase ciprofloxacin resistance in isolates with mutations in the QRDRs.

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