scholarly journals Contribution of Target Gene Mutations and Efflux to Decreased Susceptibility of Salmonella enterica Serovar Typhimurium to Fluoroquinolones and Other Antimicrobials

2006 ◽  
Vol 51 (2) ◽  
pp. 535-542 ◽  
Author(s):  
Sheng Chen ◽  
Shenghui Cui ◽  
Patrick F. McDermott ◽  
Shaohua Zhao ◽  
David G. White ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Efflux Pump ◽  
Critical Role ◽  
Gene Mutations ◽  
Efflux Pumps ◽  
Fluoroquinolone Resistance ◽  
Naturally Occurring ◽  
Serovar Typhimurium ◽  
Resistant Mutants ◽  
Increased Susceptibility

ABSTRACT The mechanisms involved in fluoroquinolone resistance in Salmonella enterica include target alterations and overexpression of efflux pumps. The present study evaluated the role of known and putative multidrug resistance efflux pumps and mutations in topoisomerase genes among laboratory-selected and naturally occurring fluoroquinolone-resistant Salmonella enterica serovar Typhimurium strains. Strains with ciprofloxacin MICs of 0.25, 4, 32, and 256 μg/ml were derived in vitro using serovar Typhimurium S21. These mutants also showed decreased susceptibility or resistance to many nonfluoroquinolone antimicrobials, including tetracycline, chloramphenicol, and several β-lactams. The expression of efflux pump genes acrA, acrB, acrE, acrF, emrB, emrD, and mdlB were substantially increased (≥2-fold) among the fluoroquinolone-resistant mutants. Increased expression was also observed, but to a lesser extent, with three other putative efflux pumps: mdtB (yegN), mdtC (yegO), and emrA among mutants with ciprofloxacin MICs of ≥32 μg/ml. Deletion of acrAB or tolC in S21 and its fluoroquinolone-resistant mutants resulted in increased susceptibility to fluoroquinolones and other tested antimicrobials. In naturally occurring fluoroquinolone-resistant serovar Typhimurium strains, deletion of acrAB or tolC increased fluoroquinolone susceptibility 4-fold, whereas replacement of gyrA double mutations (S83F D87N) with wild-type gyrA increased susceptibility >500-fold. These results indicate that a combination of topoisomerase gene mutations, as well as enhanced antimicrobial efflux, plays a critical role in the development of fluoroquinolone resistance in both laboratory-derived and naturally occurring quinolone-resistant serovar Typhimurium strains.

scholarly journals Overexpression of Efflux Pumps, Mutations in the Pumps’ Regulators, Chromosomal Mutations, and AAC(6′)-Ib-cr Are Associated With Fluoroquinolone Resistance in Diverse Sequence Types of Neonatal Septicaemic Acinetobacter baumannii: A 7-Year Single Center Study

2021 ◽  
Vol 12 ◽  
Author(s):  
Subhasree Roy ◽  
Somdatta Chatterjee ◽  
Amrita Bhattacharjee ◽  
Pinaki Chattopadhyay ◽  
Bijan Saha ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Efflux Pump ◽  
Critical Role ◽  
Holistic Approach ◽  
Efflux Pumps ◽  
Resistance Mechanisms ◽  
Fluoroquinolone Resistance ◽  
Active Efflux ◽  
Chromosomal Mutations ◽  
Sequence Types

This study investigates susceptibility toward three fluoroquinolones (ciprofloxacin, levofloxacin, moxifloxacin), multiple fluoroquinolone-resistance mechanisms, and epidemiological relationship of neonatal septicaemic Acinetobacter baumannii. Previous studies on fluoroquinolone resistance in A. baumannii focused primarily on ciprofloxacin susceptibility and assessed a particular mechanism of resistance; a more holistic approach was taken here. Epidemiological relationship was evaluated by Multi Locus Sequence Typing. Minimum Inhibitory Concentrations of fluoroquinolones was determined with and without efflux pump inhibitors. Overexpression of efflux pumps, resistance-nodulation-cell-division (RND)-type, and multidrug and toxic compound extrusion (MATE)-type efflux pumps were evaluated by reverse transcriptase-qPCR. Mutations within regulatory proteins (AdeRS, AdeN, and AdeL) of RND-pumps were examined. Chromosomal mutations, presence of qnr and aac(6′)-Ib-cr were investigated. A. baumannii were highly diverse as 24 sequence-types with seven novel STs (ST-1440/ST-1441/ST-1481/ST-1482/ST-1483/ST-1484/ST-1486) were identified among 47 A. baumannii. High resistance to ciprofloxacin (96%), levofloxacin (92%), and particularly moxifloxacin (90%) was observed, with multiple mechanisms being active. Resistance to 4th generation fluoroquinolone (moxifloxacin) in neonatal isolates is worrisome. Mutations within GyrA (S83L) and ParC (S80L) were detected in more than 90% of fluoroquinolone-resistant A. baumannii (FQRAB) spread across 10 different clonal complexes (CC1/CC2/CC10/CC25/CC32/CC126/CC149/CC216/CC218/CC513). Efflux-based FQ resistance was found in 65% of FQRAB with ≥2 different active pumps in 38% of strains. Overexpression of adeB was highest (2.2−34-folds) followed by adeJ, adeG, and abeM. Amino acid changes in the regulators (AdeRS/AdeN/AdeL) either as single or multiple substitutions substantiated the overexpression of the pumps. Diverse mutations within AdeRS were detected among different CCs whereas mutations within AdeN linked to CC10 and CC32. Chromosomal mutations and active efflux pumps were detected simultaneously among 64% of FQRAB. Presence of aac(6′)-Ib-cr was also high (74% of FQRAB) but qnrS were absent. As most FQRABs had chromosomal mutations, this was considered predominant, however, isolates where pumps were also active had higher MIC values, establishing the critical role of the efflux pumps. The high variability of FQ susceptibility among FQRAB, possessing the same set of mutations in gyrA, parC, and efflux pump regulators, was also noted. This reveals the complexity of interpreting the interplay of multiple resistance mechanisms in A. baumannii.

scholarly journals Ciprofloxacin-Resistant Salmonella enterica Serovar Typhimurium Strains Are Difficult To Select in the Absence of AcrB and TolC

2006 ◽  
Vol 50 (1) ◽  
pp. 38-42 ◽  
Author(s):  
Vito Ricci ◽  
Peter Tzakas ◽  
Anthony Buckley ◽  
Nick C. Coldham ◽  
Laura J. V. Piddock
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Efflux Pump ◽  
Multiple Antibiotic Resistance ◽  
Efflux System ◽  
Efflux Pump Inhibitor ◽  
Serovar Typhimurium ◽  
Resistant Mutants ◽  
Selection Of ◽  
Selection Of Resistance

ABSTRACT It has been proposed that lack of a functional efflux system(s) will lead to a lower frequency of selection of resistance to fluoroquinolones and other antibiotics. We constructed five strains of Salmonella enterica serovar Typhimurium SL1344 that lacked efflux gene components of resistance nodulation cell division pumps (acrB, acrD, acrF, acrBacrF, and tolC) plus three strains that lack genes that effect efflux gene expression (marA, soxS, and ramA) and a hypermutable strain (mutS::aph). Strains were exposed to ciprofloxacin at 2× the MIC in agar, in the presence and absence of Phe-Arg-β-naphthylamide, an efflux pump inhibitor. Mutants were selected from all strains except those lacking acrB, tolC, or acrBacrF. For strains from which mutants were selected, there were no significant differences between the frequencies of resistance. Except for mutants of the ramA::aph strain, two phenotypes arose: resistance to quinolones only and multiple antibiotic resistance (MAR). ramA::aph mutants were resistant to quinolones only, suggesting a role for ramA in MAR in S. enterica serovar Typhimurium. Phe-Arg-β-naphthylamide (20 μg/ml) had no effect on the frequencies of resistance or ciprofloxacin MICs. In conclusion, functional AcrB and TolC in S. enterica serovar Typhimurium are important for the selection of ciprofloxacin-resistant mutants.

scholarly journals Efflux-Mediated Resistance to Tigecycline (GAR-936) in Pseudomonas aeruginosa PAO1

2003 ◽  
Vol 47 (3) ◽  
pp. 972-978 ◽  
Author(s):  
Charles R. Dean ◽  
Melissa A. Visalli ◽  
Steven J. Projan ◽  
Phaik-Eng Sum ◽  
Patricia A. Bradford
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Repressor Gene ◽  
Mutant Strains ◽  
Resistant Strains ◽  
Resistant Mutants ◽  
Outer Membrane Channel ◽  
Increased Susceptibility

ABSTRACT Pseudomonas aeruginosa strains are less susceptible to tigecycline (previously GAR-936; MIC, 8 μg/ml) than many other bacteria (P. J. Petersen, N. V. Jacobus, W. J. Weiss, P. E. Sum, and R. T. Testa, Antimicrob. Agents Chemother. 43:738-744, 1999). To elucidate the mechanism of resistance to tigecycline, P. aeruginosa PAO1 strains defective in the MexAB-OprM and/or MexXY (OprM) efflux pumps were tested for susceptibility to tigecycline. Increased susceptibility to tigecycline (MIC, 0.5 to 1 μg/ml) was specifically associated with loss of MexXY. Transcription of mexX and mexY was also responsive to exposure of cells to tigecycline. To test for the emergence of compensatory efflux pumps in the absence of MexXY-OprM, mutants lacking MexXY-OprM were plated on medium containing tigecycline at 4 or 6 μg/ml. Resistant mutants were readily recovered, and these also had decreased susceptibility to several other antibiotics, suggesting efflux pump recruitment. One representative carbenicillin-resistant strain overexpressed OprM, the outer membrane channel component of the MexAB-OprM efflux pump. The mexAB-oprM repressor gene, mexR, from this strain contained a 15-bp in-frame deletion. Two representative chloramphenicol-resistant strains showed expression of an outer membrane protein slightly larger than OprM. The mexCD-OprJ repressor gene, nfxB, from these mutants contained a 327-bp in-frame deletion and an IS element insertion, respectively. Together, these data indicated drug efflux mediated by MexCD-OprJ. The MICs of the narrower-spectrum semisynthetic tetracyclines doxycycline and minocycline increased more substantially than did those of tigecycline and other glycylcyclines against the MexAB-OprM- and MexCD-OprJ-overexpressing mutant strains. This suggests that glycylcyclines, although they are subject to efflux from P. aeruginosa, are generally inferior substrates for P. aeruginosa efflux pumps than are narrower-spectrum tetracyclines.

scholarly journals Beyond Antimicrobial Resistance: Evidence for a Distinct Role of the AcrD Efflux Pump inSalmonellaBiology

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Michelle M. C. Buckner ◽  
Jessica M. A. Blair ◽  
Roberto M. La Ragione ◽  
Jane Newcombe ◽  
Daniel J. Dwyer ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Stress Responses ◽  
Salmonella Enterica ◽  
Efflux Pump ◽  
Tricarboxylic Acid Cycle ◽  
Efflux Pumps ◽  
Wild Type ◽  
Swarming Motility ◽  
Serovar Typhimurium

ABSTRACTFor over 20 years, bacterial multidrug resistance (MDR) efflux pumps have been studied because of their impact on resistance to antimicrobials. However, critical questions remain, including why produce efflux pumps under non-antimicrobial treatment conditions, and why have multiple pumps if their only purpose is antimicrobial efflux?Salmonellaspp. possess five efflux pump families, including the resistance-nodulation-division (RND) efflux pumps. Notably, the RND efflux pump AcrD has a unique substrate profile, distinct from otherSalmonellaefflux pumps. Here we show that inactivation ofacrDresults in a profoundly altered transcriptome and modulation of pathways integral toSalmonellabiology. The most significant transcriptome changes were central metabolism related, with additional changes observed in pathogenicity, environmental sensing, and stress response pathway expression. The extent of tricarboxylic acid cycle and fumarate metabolism expression changes led us to hypothesize thatacrDinactivation may result in motility defects due to perturbation of metabolite concentrations, such as fumarate, for which a role in motility has been established. Despite minimal detectable changes in flagellar gene expression, we found that anacrDmutantSalmonella entericaserovar Typhimurium isolate was significantly impaired for swarming motility, which was restored by addition of fumarate. TheacrDmutant outcompeted the wild type in fitness experiments. The results of these diverse experiments provide strong evidence that the AcrD efflux pump is not simply a redundant system providing response resilience, but also has distinct physiological functions. Together, these data indicate that the AcrD efflux pump has a significant and previously underappreciated impact on bacterial biology, despite only minor perturbations of antibiotic resistance profiles.IMPORTANCEEfflux pumps in Gram-negative bacteria are studied because of their important contributions to antimicrobial resistance. However, the role of these pumps in bacterial biology has remained surprisingly elusive. Here, we provide evidence that loss of the AcrD efflux pump significantly impacts the physiology ofSalmonella entericaserovar Typhimurium. Inactivation ofacrDled to changes in the expression of 403 genes involved in fundamental processes, including basic metabolism, virulence, and stress responses. Pathways such as these allowSalmonellato grow, survive in the environment, and cause disease. Indeed, our data show that theacrDmutant is more fit than wild-typeSalmonellaunder standard lab conditions. We hypothesized that inactivation ofacrDwould alter levels of bacterial metabolites, impacting traits such as swarming motility. We demonstrated this by exogenous addition of the metabolite fumarate, which partially restored theacrDmutant’s swarming defect. This work extends our understanding of the role of bacterial efflux pumps.

scholarly journals Role for Neutrophils in Host Immune Responses and Genetic Factors That Modulate Resistance to Salmonella enterica Serovar Typhimurium in the Inbred Mouse Strain SPRET/Ei

2010 ◽  
Vol 78 (9) ◽  
pp. 3848-3860 ◽  
Author(s):  
Lien Dejager ◽  
Iris Pinheiro ◽  
Pieter Bogaert ◽  
Liesbeth Huys ◽  
Claude Libert
Keyword(s):  
Mouse Strain ◽  
Salmonella Enterica ◽  
Genetic Factors ◽  
Inbred Mouse ◽  
Critical Role ◽  
Inbred Mouse Strain ◽  
Natural Resistance ◽  
Host Bacterium ◽  
A Genome ◽  
Serovar Typhimurium

ABSTRACT Infection with Salmonella enterica serovar Typhimurium is a complex disease in which the host-bacterium interactions are strongly influenced by genetic factors of the host. We demonstrate that SPRET/Ei, an inbred mouse strain derived from Mus spretus, is resistant to S. Typhimurium infections. The kinetics of bacterial proliferation, as well as histological examinations of tissue sections, suggest that SPRET/Ei mice can control bacterial multiplication and spreading despite significant attenuation of the cytokine response. The resistance of SPRET/Ei mice to S. Typhimurium infection is associated with increased leukocyte counts in the circulation and enhanced neutrophil influx into the peritoneum during the course of infection. A critical role of neutrophils was confirmed by neutrophil depletion: neutropenic SPRET/Ei mice were sensitive to infection with S. Typhimurium and showed much higher bacterial loads. To identify genes that modulate the natural resistance of SPRET/Ei mice to S. Typhimurium infection, we performed a genome-wide study using an interspecific backcross between C3H/HeN and SPRET/Ei mice. The results of this analysis demonstrate that at least two loci, located on chromosomes 6 and 11, affect survival following lethal infection with S. Typhimurium. These two loci contain several interesting candidate genes which may have important implications for the search for genetic factors controlling Salmonella infections in humans and for our understanding of complex host-pathogen interactions in general.

scholarly journals Understanding the basis of antibiotic resistance: a platform for drug discovery

Microbiology ◽  
2014 ◽  
Vol 160 (11) ◽  
pp. 2366-2373 ◽  
Author(s):  
Laura J. V. Piddock
Keyword(s):  
Antibiotic Resistance ◽  
Salmonella Enterica ◽  
Efflux Pump ◽  
Antibiotic Resistance Genes ◽  
Multi Drug Resistance ◽  
Annual Conference ◽  
Altered Expression ◽  
Genetic Inactivation ◽  
Fluoroquinolone Antibiotics ◽  
Serovar Typhimurium

There are numerous genes in Salmonella enterica serovar Typhimurium that can confer resistance to fluoroquinolone antibiotics, including those that encode topoisomerase proteins, the primary targets of this class of drugs. However, resistance is often multifactorial in clinical isolates and it is not uncommon to also detect mutations in genes that affect the expression of proteins involved in permeability and multi-drug efflux. The latter mechanism, mediated by tripartite efflux systems, such as that formed by the AcrAB–TolC system, confers inherent resistance to many antibiotics, detergents and biocides. Genetic inactivation of efflux genes gives multi-drug hyper-susceptibility, and in the absence of an intact AcrAB–TolC system some chromosomal and transmissible antibiotic resistance genes no longer confer clinically relevant levels of resistance. Furthermore, a functional multi-drug resistance efflux pump, such as AcrAB–TolC, is required for virulence and the ability to form a biofilm. In part, this is due to altered expression of virulence and biofilm genes being sensitive to efflux status. Efflux pump expression can be increased, usually due to mutations in regulatory genes, and this confers resistance to clinically useful drugs such as fluoroquinolones and β-lactams. Here, I discuss some of the work my team has carried out characterizing the mechanisms of antibiotic resistance in Salmonella enterica serovar Typhimurium from the late 1980s to 2014. A video of this Prize Lecture, presented at the Society for General Microbiology Annual Conference 2014, can be viewed via this link: https://www.youtube.com/watch?v=MCRumMV99Yw.

scholarly journals MdsABC-Mediated Pathway for Pathogenicity in Salmonella enterica Serovar Typhimurium

2015 ◽  
Vol 83 (11) ◽  
pp. 4266-4276 ◽  
Author(s):  
Saemee Song ◽  
Boeun Lee ◽  
Ji-Hyun Yeom ◽  
Soonhye Hwang ◽  
Ilnam Kang ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Disulfide Bonds ◽  
Efflux Pump ◽  
Host Cells ◽  
Deletion Strain ◽  
Bacterial Number ◽  
Phagocytic Cells ◽  
Content Type ◽  
Serovar Typhimurium ◽  
Key Factor

ABSTRACTMdsABC is aSalmonella-specific tripartite efflux pump that has been implicated in the virulence ofSalmonella entericaserovar Typhimurium; however, little is known about the virulence factors associated with this pump. We observed MdsABC expression-dependent alterations in the degree of resistance to extracellular oxidative stress and macrophage-mediated killing. Thin-layer chromatography and tandem mass spectrometry analyses revealed that overexpression of MdsABC led to increased secretion of 1-palmitoyl-2-stearoyl-phosphatidylserine (PSPS), affecting the ability of the bacteria to invade and survive in host cells. Overexpression of MdsABC and external addition of PSPS similarly rendered themdsABCdeletion strain resistant to diamide. Diagonal gel analysis showed that PSPS treatment reduced the diamide-mediated formation of disulfide bonds, particularly in the membrane fraction of the bacteria.Salmonellainfection of macrophages induced the upregulation of MdsABC expression and led to an increase of intracellular bacterial number and host cell death, similar to the effects of MdsABC overexpression and PSPS pretreatment on themdsABCdeletion strain. Our study shows that MdsABC mediates a previously uncharacterized pathway that involves PSPS as a key factor for the survival and virulence ofS. Typhimurium in phagocytic cells.

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