Expression Of marA Research Articles

Complete genome of the extensively antibiotic-resistant GC1 Acinetobacter baumannii isolate MRSN 56 reveals a novel route to fluoroquinolone resistance.

ObjectivesTo examine the causes of antibiotic resistance in the extensively resistant global clone 1 (GC1) Acinetobacter baumannii isolate MRSN 56 recovered at a US military treatment facility.MethodsMRSN 56 was sequenced using MinION (Oxford Nanopore) and the reads combined with available Illumina MiSeq data using Unicycler. Acquired resistance genes were identified using ABRicate and their environment examined. ISAba1 and ISAba125 copies were located.ResultsMRSN 56 is ST1IP:ST231Ox:KL1:OCL1 and the complete genome includes four small plasmids, none of which carry resistance genes. The acquired resistance genes were found at four locations in the chromosome in addition to AbaR28 (aphA1, aacC1, aadA1, sul1) in comM. Tn2006 (oxa23, carbapenem resistance) was both in AbaR4 and alone elsewhere. Two copies of Tn7 (dfrA1, sat, aadA1) were identified. One was associated with a 22 852 bp adjacent segment [tetA(B), sul2] derived from the AbGRI1 island, and this novel configuration was designated Tn7+. Tn7+ was incorporated in the position preferred by Tn7, downstream of glmS, by transposition using a sequence in AbGRI1 resembling the Tn7 terminal inverted repeats. Tn7 was found at a secondary site. Fluoroquinolone resistance appears to involve a mutation in gyrA combined with inactivation by ISAba1 of the marR gene in the mar operon and constitutive expression of marA from the promoter internal to ISAba1.ConclusionsMRSN 56 represents a new sublineage of GC1 lineage 1 with novel features that had not been detected previously. The involvement of the mar operon in fluoroquinolone resistance has not been noted previously.

Carbapenem non-susceptibility associated with global transcriptional regulator MarA in Escherichia coli

Background: The AcrAB-TolC system of Escherichia coli is a multidrug efflux system pumping out a wide variety of substrates. Its expression is regulated by an intricate balance between local (AcrR and AcrS) and global regulators (MarA, SoxS and Rob). Global transcriptional regulators are known to be involved in stress response in bacteria. MarA along activating its own transcription; it activates the transcription of other genes including acrAB, tolC and micF. As carbapenem resistance is an increasing trend in hospital acquired infections. The present study was carried out to observe transcriptional response of marA, soxS and rob against concentration dependent carbapenem stress. Methods and materials: In the present study hospital isolates of Escherichia coli were tested for transcriptional expression of AcrAB-TolC and OmpF. Transcriptional expression of marA, soxS and rob with and without concentration gradient carbapenem stress were determined. Sequencing and cloning of marA, soxS and rob was also performed. Results: Twelve isolates with over-expressed AcrAB-TolC and downregulated OmpF were selected for further study. Among them, overexpression of marA against meropenem was observed in seven isolates. Antimicrobial susceptibility pattern of the clones showed that the zone of inhibition formed by the transformants (mar) was decreased as compared with that of parent strain (DH5α) as well as with the plasmid without gene of interest. Further, MIC range of the clones revealed two-fold or more increase in the inhibitory concentration against ertapenem and imipenem was noted for clone of mar while comparing with the parent strain (DH5α) Further, when the DNA sequences of marAwere compared with the reference strain of E. coli ATCC 25922, it displayed nucleotide alterations at many locations. Four-point mutation and 2 deletion mutations in marA sequence was observed. Conclusion: This study demonstrates the role of the global regulators marA in triggering the overexpression of AcrAB efflux pump system conferring resistance towards carbapenems. MarA plays a major role in efflux activity under carbapenem stress. Regulation of Rob under meropenem stress shows an antibiotic specific response and SoxS deviation in transcriptional expression amid particular concentration, needs further investigation.

Artesunate interrupts the self-transcriptional activation of MarA to inhibit RND family pumps of Escherichia coli

Resistance-Nodulation-Division (RND) family pumps are responsible for producing multidrug resistance in Escherichia coli; however, there has been little study of targeted inhibitors of RNDs. In the present study, we investigated the inhibition of RND pumps by artesunate (AS) in E. coli, and further investigated the mechanism with respect to MarA, a regulator of RNDs. Although AS had no direct antibacterial effect, it showed a synergistic effect in combination with β-lactams against E. coli ATCC35218 in vitro and in vivo, suggesting it possesses antibacterial enhancement activity. Notably, AS, alone or in combination with β-lactams, downregulated the mRNA expression levels of marA, soxS, and rob, known as the marA-soxS-rob regulon, which then decreased the expression levels of RNDs, thereby increased ampicillin accumulation within ATCC35218. Using gene-deletion strains, we found that the antibacterial sensitization effect of AS persisted in wildtype bacteria, but was completely lost in the strain lacking marA, and decreased in the strain lacking soxS or rob, suggesting marA plays a crucial role in the sensitization of AS. Critically, we showed that AS inhibited the binding of MarA to the promoter of marA itself, not acrB, resulting in decreased mRNA expression of both acrB and marA. Mechanistically, we found AS directly bound to the central cavity of MarA through the R59 and K62 residues, and thus altered the charge distribution of MarA to interrupt the recognition between MarA and its promoter. We concluded that AS interrupts the self-transcriptional activation of MarA, thereby inhibits MarA-dependent mRNA expression of marA, acrAB, and tolC, and also certain other RNDs and regulatory genes related to MarA. Therefore, AS is a novel inhibitor of RND pumps that acts on the regulator MarA.

Molecular epidemiology and mechanisms of tigecycline resistance in carbapenem-resistant Klebsiella pneumoniae isolates.

BackgroundThe emergence and transmission of tigecycline‐ and carbapenem‐resistant Klebsiella pneumoniae (TCRKP) have become a major concern to public health globally. Here, we investigated the molecular epidemiology and mechanisms of tigecycline resistance in carbapenem‐resistant K pneumoniae (CRKP) isolates.MethodsForty‐five non‐duplicate CRKP isolates were collected from January 2017 to June 2019. We performed antimicrobial susceptibility tests, multilocus sequence typing (MLST), and pulsed‐field gel electrophoresis (PFGE). PCR and DNA sequencing were performed for the detection and mutation analysis of acrR, oqxR, ramR, rpsJ, tet(A), and tet(X) genes, which are related to tigecycline resistance. The expression levels of efflux pump genes acrB and oqxB and their regulator genes rarA, ramA, soxS, and marA were assessed by quantitative real‐time PCR.ResultsThe resistance rate to tigecycline in CRKP isolates was 37.8% (17/45). K pneumoniae ST307 was a predominant clone type (70.6%, 12/17) among the TCRKP isolates. The expression levels of acrB (P < .001) and marA (P = .009) were significantly higher in the tigecycline‐resistant group than in the tigecycline‐intermediate and tigecycline‐susceptible groups. Increased expression of acrB was associated with marA expression (r = 0.59, P = .013).ConclusionsWe found that the activated MarA‐induced overexpression of AcrAB efflux pump plays an important role in the emergence of tigecycline resistance in CRKP isolates.

MarA, RamA, and SoxS as Mediators of the Stress Response: Survival at a Cost

To survive and adapt to changing environments, bacteria have evolved mechanisms to express appropriate genes at appropriate times. Exposure to antimicrobials triggers a global stress response in Enterobacteriaceae, underpinned by activation of a family of transcriptional regulators, including MarA, RamA, and SoxS. These control a program of altered gene expression allowing a rapid and measured response to improve fitness in the presence of toxic drugs. Increased expression of marA, ramA, and soxS up regulates efflux activity to allow detoxification of the cell. However, this also results in trade-offs in other phenotypes, such as impaired growth rates, biofilm formation and virulence. Here, we review the current knowledge regarding the trade-offs that exist between drug survival and other phenotypes that result from induction of marA, ramA, and soxS. Additionally, we present some new findings linking expression of these regulators and biofilm formation in Enterobacteriaceae, thereby demonstrating the interconnected nature of regulatory networks within the cell and explaining how trade-offs can exist between important phenotypes. This has important implications for our understanding of how bacterial virulence and biofilms can be influenced by exposure to antimicrobials.

Non-steroidal anti-inflammatory drugs, acetaminophen and ibuprofen, induce phenotypic antibiotic resistance in Escherichia coli: roles of marA and acrB.

The factors contributing to antibiotic resistance in bacteria are an important area of study. Sodium salicylate (NaSal), a non-steroidal anti-inflammatory drug (NSAID), increases antibiotic resistance by inducing the expression of MarA, a transcription factor, which increases the AcrAB-TolC efflux pump. MarA is a substrate of Lon protease and the Δlon strain displays a high degree of antibiotic resistance. This study was initiated to identify commonly used NSAIDs that may induce antibiotic resistance and to compare their efficacies with NaSal and acetyl salicylic acid (ASA). Quantitative real-time expression analysis revealed induction of marA and acrB by NaSal, ASA, acetaminophen (APAP) and ibuprofen. Further, dose studies demonstrated that NaSal and ASA induce resistance at ∼2 mM while APAP and ibuprofen induce resistance at ∼5-10 mM. To dissect the roles of key molecules, atomic force microscopy and functional studies were performed using WT, Δlon, ΔmarA, ΔacrB, ΔlonΔmarA and ΔlonΔacrB strains. The induction of antibiotic resistance by NaSal, ASA and APAP is relatively higher and is partly dependent on marA, whereas ibuprofen which induces lower antibiotic resistance shows complete marA dependence. Notably, NaSal, ASA, APAP and ibuprofen induce antibiotic resistance in an acrB-dependent manner. The possible significance of some NSAIDs in inducing antibiotic resistance is discussed.

Molecular mechanisms of membrane impermeability in clinical isolates of Enterobacteriaceae exposed to imipenem selective pressure

Intrinsic mechanisms leading to carbapenem-induced membrane impermeability and multidrug resistance are poorly understood in clinical isolates of Enterobacteriaceae. In this study, molecular behaviours during the establishment of membrane impermeability in members of the Enterobacteriaceae family under imipenem selective pressure were investigated. Clinical isolates (n = 22) exhibiting susceptibility to multiple antibiotics or characterised as extended-spectrum β-lactamase (ESBL)- or AmpC-producers were submitted to progressive passages on Mueller–Hinton agar plates containing subclinical concentrations of imipenem [0.5 × the minimum inhibitory concentration (MIC)]. Changes in outer membrane permeability were evaluated by determination of antimicrobial MICs, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and gene expression analysis related to membrane permeability (i.e. omp35-like, omp36-like and acrA) and regulatory mechanisms (i.e. marA and ompR) by quantitative reverse transcription PCR. Following imipenem induction, 73% of isolates showed increased carbapenem MICs by ≥2 doubling dilutions. At an early stage of treatment, imipenem modulated the expression of porins and efflux pump genes, represented by a reduction of 78% in omp36-like and a two-fold increase in acrA expression. Transcriptional factors marA and ompR were also affected by imipenem induction, increasing mRNA expression by 14- and 4-fold, respectively. High marA expression levels were associated with higher values of acrA expression. These results suggest that imipenem is an important factor in the development of an adaptive response to carbapenems by regulating key genes involved in the control of efflux pumps and porins, which could lead to a multidrug-resistant profile in clinical isolates, contributing to possible treatment failure.

Stochastic expression of a multiple antibiotic resistance activator confers transient resistance in single cells.

Transient resistance can allow microorganisms to temporarily survive lethal concentrations of antibiotics. This can be accomplished through stochastic mechanisms, where individual cells within a population display diverse phenotypes to hedge against the appearance of an antibiotic. To date, research on transient stochastic resistance has focused primarily on mechanisms where a subpopulation of cells enters a dormant, drug-tolerant state. However, a fundamental question is whether stochastic gene expression can also generate variable resistance levels among growing cells in a population. We hypothesized that stochastic expression of antibiotic-inducible resistance mechanisms might play such a role. To investigate this, we focused on a prototypical example of such a system: the multiple antibiotic resistance activator MarA. Previous studies have shown that induction of MarA can lead to a multidrug resistant phenotype at the population level. We asked whether MarA expression also has a stochastic component, even when uninduced. Time lapse microscopy showed that isogenic cells express heterogeneous, dynamic levels of MarA, which were correlated with transient antibiotic survival. This finding has important clinical implications, as stochastic expression of resistance genes may be widespread, allowing populations to hedge against the sudden appearance of an antibiotic.

First emergence of acrAB and oqxAB mediated tigecycline resistance in clinical isolates of Klebsiella pneumoniae pre-dating the use of tigecycline in a Chinese hospital.

Tigecycline is one of the few therapeutic options for treating infections caused by some multi-drug resistant pathogens, such as Klebsiella pneumoniae. However, tigecycline-resistant K. pneumoniae has been discovered recently in China. From 2009 to 2013, nine tigecycline-resistant K. pneumoniae isolates were identified in our hospital. Six of nine strains were identified before using tigecycline. To investigate the efflux-mediated resistance mechanisms of K. pneumoniae, the expression of efflux pump genes (acrA, acrB, tolC, oqxA and oqxB) and pump regulators (acrR, marA, soxS, rarA, rob and ramA) were examined by real-time RT-PCR. Molecular typing of the tigecycline resistant strains was performed. ST11 was the predominant clone of K. pneumoniae strains, while ST1414 and ST1415 were novel STs. Efflux pump inhibitor (EPI)-carbonyl cyanide chlorophenylhydrazone (CCCP) was able to reverse the resistance patterns of 5 resistant K. pneumoniae strains. In comparison with strain A111, a tigecycline-susceptible strain (negative control), we found that the expression levels of efflux pump genes and pump regulators were higher in a majority of resistant strains. Higher expression levels of regulators rarA (2.41-fold, 9.55-fold, 28.44-fold and 18.31-fold, respectively) and pump gene oqxB (3.87-fold, 31.96-fold, 50.61-fold and 29.45-fold, respectively) were observed in four tigecycline resistant strains (A363, A361, A368, A373, respectively). Increased expression of acrB was associated with ramA and marA expression. To our knowledge, studies on tigecycline resistance mechanism in K. pneumoniae are limited especially in China. In our study, we found that both efflux pump AcrAB-TolC and OqxAB contributed to tigecycline resistance in K. pneumoniae isolates.

Expression of marA is remarkably increased from the early stage of development of fluoroquinolone-resistance in uropathogenic Escherichia coli.

Analyses of efflux pumps overexpression and mutations in quinolone resistance determining region (QRDR) in early stage of development of resistance to fluoroquinolones (FQs) are valuable to discuss countermeasures against them. We induced levofloxacin (LVFX)-resistant strains from susceptible uropathogenic Escherichia coli in vitro to analyze the mechanisms of development of FQs-resistance. 89 strains were exposed to discontinuous elevation of LVFX dose, and mRNA level of efflux pumps and their regulators as well as mutations developed in QRDR of LVFX-resistant strains were analyzed. In 5 strains, a stepwise increase in MIC to LVFX (up to >128 μg/ml)was observed. Compared to the parent strains, additional mutations in QRDR were observed in the strains developing high MIC. Remarkable increase of marA expression was observed even in the early stage of LVFX-resistance development, and it lasted until high-level resistance was developed. On the other hand, moderate increase in acrB expression but only low increase in yhiU, yhiV, mdfA, tolC and sdiA were observed. These results suggested that marA expression is a sensitive marker for early detection of development of LVFX-resistance.

Increase of multidrug efflux pump expression in fluoroquinolone-resistant Salmonella mutants induced by ciprofloxacin selective pressure

Multidrug-resistant foodborne pathogens are a leading public health concern, as antimicrobial resistance can lead to therapeutic failure. In this study, a ciprofloxacin-susceptible Salmonella Istanbul (Sal10-FC-KU12) was isolated from chicken meat obtained from a market in Korea to induce ciprofloxacin-resistant mutants (SalML, SalMM, and SalMH). Minimum inhibitory concentrations (MICs) of 12 antibiotics were measured in the presence or absence of an efflux pump inhibitor. Expression levels of efflux pump-related genes (acrB, acrF, marA, ramA, rob, and soxS) were determined using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Elevated MICs for the derived mutants were shown to result from the action of the efflux pump, with increased expression of marA, ramA, and acrB compared with the wild-type strain. The results of this study suggest that continued use of ciprofloxacin might induce the emergence of Salmonella mutants resistant not only to fluoroquinolones, but also to several other classes of antimicrobials.

Regulation of acrAB expression by cellular metabolites in Escherichia coli

Multidrug efflux pumps mediate resistance to antibiotics and other toxic compounds. We studied the role of AcrAB-TolC, the main efflux pump in Escherichia coli, in regulating gene expression. Deletion mutants, an acrABp-lacZ fusion and reverse transcription-real-time quantitative PCR experiments were used to study the role of AcrAB-TolC and metabolism in regulating gene expression of the acrAB operon and its transcriptional regulators. Deletion of the acrB gene increased the expression of the acrAB operon. A similar induction of acrAB was found when acrA or tolC was deleted, and when the pump function was inhibited using phenylalanine-arginine-β-naphthylamide. The induction of acrAB in the ΔacrB strain was totally (AcrR or SoxS) or partially (SoxR or MarA) prevented when the genes for these acrAB regulators were also deleted. The expression of soxS and marA, but not of acrR, was increased in the ΔacrB strain, which also showed altered expression of many other genes related to different cellular processes, including motility. Deletion of the metabolic genes entA and entE (enterobactin biosysnthesis), glpX (gluconeogenesis), cysH (cysteine biosynthesis) and purA (purine biosynthesis) also prevented activation of the acrAB promoter in the ΔacrB strain. Addition of the enterobactin biosynthesis intermediate metabolite 2,3-dihydroxybenzoate induced the expression of acrAB. These results together suggest a model in which the AcrAB-TolC pump effluxes cellular metabolites that are toxic and/or have a signalling role. If the pump is inactivated or inhibited, these metabolites would accumulate, inactivating AcrR and/or up-regulating soxS and marA expression, ultimately triggering the up-regulation of acrAB expression to restore homeostasis.

P63 The second nationwide surveillance of bacterial urinary pathogens for complicated urinary tract infection conducted by JSC, JAID and JSCM

There are several mechanisms of fluoroquinolone (FQ) resistance, such as mutations in the quinolone resistance-determining regions (QRDRs) of target genes and efflux pump expression. The purpose of this study was to investigate which factor plays the main role in FQ resistance in Escherichia coli causing urinary tract infections (UTIs) from a statistical analysis of our two previous works. DNA sequencing of the QRDRs of the FQ resistance-related genes gyrA and parC as well as real-time reverse transcriptase (RT)-PCR for the expression of efflux pump genes such as marA or yhiU were performed and the correlations of mutations or efflux pump gene expression with FQ minimal inhibitory concentrations (MICs) were investigated. Significant factors for high MICs of sitafloxacin (STFX), ciprofloxacin (CPFX) and levofloxacin were the mutations S83L and D87N in gyrA and S80I and E84V in parC as well as the expression of marA. Mutations in the QRDRs of gyrA or parC had a greater effect on FQ MICs than efflux pump gene expression. Based on the regression coefficient, these mutations correlated with MICs to CPFX most, and STFX had the lowest effects from these mutations among the three tested FQs. In conclusion, in E. coli causing UTIs, mutations in the QRDRs of gyrA or parC had a greater effect on FQ resistance, especially to CPFX, than efflux pump gene expression from a statistical analysis study of our two previous works. Further research into the molecular basis for FQ resistance could lead to new therapeutic strategies against FQ-resistant E. coli.

Study the Expression of marA Gene in Ciprofloxacin and Tetracycline Resistant Mutants of Esherichia coli.

MarA activates two membrane dependent mechanisms of resistance to different antibiotics, such as ciprofloxacin and tetracycline, including promotion of outflux and inhibition of influx of antibiotics. Thus, MarA causes multiple antibiotic resistance phenotype. The activation of these mechanisms needs overexpression of marA. This could happen through mutation in marR. Thus, the aim of this study was to measure marA expression in ciprofloxacin resistant E. coli gyrA mutants and clones with or without marR mutation. For this purpose, real time PCR was used to measure relative expression of marA in above mutants and clones. Results showed that two clones, C14 and C17 overexpressed marA. It is concluded that the level of marA expression is important for activation of above mechanisms.

Does mutation in gyrA and/or parC or efflux pump expression play the main role in fluoroquinolone resistance in Escherichia coli urinary tract infections?: A statistical analysis study

There are several mechanisms of fluoroquinolone (FQ) resistance, such as mutations in the quinolone resistance-determining regions (QRDRs) of target genes and efflux pump expression. The purpose of this study was to investigate which factor plays the main role in FQ resistance in Escherichia coli causing urinary tract infections (UTIs) from a statistical analysis of our two previous works. DNA sequencing of the QRDRs of the FQ resistance-related genes gyrA and parC as well as real-time reverse transcriptase (RT)-PCR for the expression of efflux pump genes such as marA or yhiU were performed and the correlations of mutations or efflux pump gene expression with FQ minimal inhibitory concentrations (MICs) were investigated. Significant factors for high MICs of sitafloxacin (STFX), ciprofloxacin (CPFX) and levofloxacin were the mutations S83L and D87N in gyrA and S80I and E84V in parC as well as the expression of marA. Mutations in the QRDRs of gyrA or parC had a greater effect on FQ MICs than efflux pump gene expression. Based on the regression coefficient, these mutations correlated with MICs to CPFX most, and STFX had the lowest effects from these mutations among the three tested FQs. In conclusion, in E. coli causing UTIs, mutations in the QRDRs of gyrA or parC had a greater effect on FQ resistance, especially to CPFX, than efflux pump gene expression from a statistical analysis study of our two previous works. Further research into the molecular basis for FQ resistance could lead to new therapeutic strategies against FQ-resistant E. coli.

Differential expression of the transcription factors MarA, Rob, and SoxS of Salmonella Typhimurium in response to sodium hypochlorite: down-regulation of rob by MarA and SoxS

To survive, Salmonella enterica serovar Typhimurium (S. Typhimurium) must sense signals found in phagocytic cells and modulate gene expression. In the present work, we evaluated the expression and cross-regulation of the transcription factors MarA, Rob, and SoxS in response to NaOCl. We generated strains ΔsoxS and ΔmarA, which were 20 times more sensitive to NaOCl as compared to the wild-type strain; while Δrob only 5 times. Subsequently, we determined that marA and soxS transcript and protein levels were increased while those of rob decreased in a wild-type strain treated with NaOCl. To assess if changes in S. Typhimurium after exposure to NaOCl were due to a cross-regulation, as in Escherichia coli, we evaluated the expression of marA, soxS, and rob in the different genetic backgrounds. The positive regulation observed in the wild-type strain of marA and soxS was retained in the Δrob strain. As in the wild-type strain, rob was down-regulated in the ΔmarA and ΔsoxS treated with NaOCl; however, this effect was decreased. Since rob was down-regulated by both factors, we generated a ΔmarA ΔsoxS strain finding that the negative regulation was abolished, confirming our hypothesis. Electrophoretic mobility shift assays using MarA and SoxS confirmed an interaction with the promoter of rob.

Salicylate increases the expression of marA and reduces in vitro biofilm formation in uropathogenic Escherichia coli by decreasing type 1 fimbriae expression

Escherichia coli is one of the most frequent bacteria implicated in biofilm formation, which is a dynamic process whose first step consists in bacteria adhesion to surfaces through type 1 fimbriae. Salicylate induces a number of morphological and physiological alterations in bacteria including the activation of the transcriptional regulator MarA. In this report the effects of salicylate on biofilm formation and their relationship with MarA were studied. An inverse relationship was observed between in vitro biofilm formation and salicylate concentration added to the culture medium. Salicylate increases the expression of marA and decreases the expression of fimA and fimB genes in the wild-type strain. In addition, the fimA and fimB expression was decreased in a MarR mutant in which marA was also overexpressed. In conclusion, the expression of type 1 fimbriae in presence of salicylate may be regulated by the level of marA expression through fimB regulator, albeit through neither the ompX nor the tolC genes.

Efflux inhibition by selective serotonin reuptake inhibitors in Escherichia coli

To evaluate the antimicrobial and synergistic (hypothetically due to the inhibition of efflux pumps) effects of selective serotonin reuptake inhibitors (SSRIs) in Escherichia coli strains overproducing various resistance-nodulation-division (RND) efflux pumps. MICs of various SSRIs and of clinically relevant antibiotics in the presence and absence of sertraline were determined for E. coli strains overproducing the RND efflux pumps AcrAB, AcrEF, MdtEF and MexAB. The effect of sertraline on Nile red efflux was evaluated in a real-time efflux assay. Expression of marA and acrB was monitored using quantitative RT-PCR. In MIC assays there was limited synergy of sertraline with tetracycline, oxacillin, linezolid and clarithromycin, depending on the individual pump overexpressed and on whether rich or minimal medium was used. Sertraline, as the most potent SSRI with regard to bacterial growth inhibition, led to rapid dose-dependent Nile red efflux inhibition, and was also found to increase the expression of marA and acrB. A possible explanation for the discrepancy between the MIC and real-time efflux assays was that sertraline is a weak inducer of marA and acrB, thereby reducing its initial antibacterial and sensitizing effects over time. The results indicate that sertraline may be useful as a model efflux pump inhibitor for in vitro short-term experiments in E. coli, but is unlikely to be clinically useful as a co-drug against Gram-negative bacteria.

Correlation of the expression of acrB and the regulatory genes marA, soxS and ramA with antimicrobial resistance in clinical isolates of Klebsiella pneumoniae endemic to New York City

Nosocomial isolates of Klebsiella pneumoniae resistant to all commonly used antimicrobial agents have emerged in many regions of the world. It is unknown if efflux systems contribute to the multidrug resistance phenotype. The expression of genes encoding the efflux pump AcrAB and the global regulators MarA, SoxS and RamA were examined and correlated with antimicrobial resistance. Twenty isolates belonged to the two important clones representing KPC-possessing strains endemic to our region. Virtually all of these isolates had negligible or absent expression of the genes, and resistance to fluoroquinolones and aminoglycosides could be explained by alternative mechanisms. All of these isolates were susceptible to tigecycline. A group of 14 heterogeneous isolates was also examined. There was a correlation between expression of marA with expression of soxS. Only expression of soxS was significantly correlated with expression of acrB. With a background substitution in GyrA, increased expression of acrB and marA appeared to contribute to fluoroquinolone resistance in some isolates. A correlation was noted between expression of soxS and ramA (but not marA and acrB) and tigecycline MICs. Following in vitro exposure to tigecycline, resistance occurred in association with a marked increase in marA and acrB expression in isolates lacking expression of soxS and ramA. While laboratory-derived tigecycline resistance was associated with increased acrB expression, the variation in tigecycline MICs in clinical isolates was associated only with selected regulator genes. It appears that other mechanisms beyond activation of the acrAB system mediate tigecycline resistance.

Ciprofloxacin selects for multidrug resistance in Salmonella enterica serovar Typhimurium mediated by at least two different pathways

The aim of this study was to understand the role of ramA in conferring multidrug resistance (MDR) in Salmonella enterica serovar Typhimurium. Two phenotypically distinct isogenic MDR laboratory mutants derived from Salmonella Typhimurium SL1344 and three human clinical isolates from a patient that failed antibiotic therapy, including with ciprofloxacin, were investigated for the cause of MDR. MICs were determined by agar dilution and efflux activity assessed by monitoring the accumulation of Hoechst 33342. The combination of specific genes and MDR was assessed by inactivation, and complementation RT-PCR was used to assess gene expression and DNA sequencing to identify mutations within genes of interest. Mutation in ramR and the consequent over-production of ramA and acrB were revealed in one laboratory mutant selected with ciprofloxacin and this was associated with cyclohexane tolerance. Complementation of SL1344 ramR::aph with pUC19 ramR(mutant) conferred MDR and cyclohexane tolerance. However, analysis of a second ciprofloxacin-selected MDR mutant, which was susceptible to cyclohexane, revealed no mutation in ramR or altered expression of marA, soxS or rob. There was a mutation in ramR in both the pre- and post-therapy clinical isolates and no difference between the isolates in the level of expression of ramA. These data show that ciprofloxacin exposure can select for mutations within ramR and consequently mediate MDR. These data also indicate that there is at least one further unidentified gene in addition to marA, soxS, rob and ramA that confers MDR in S. enterica.