In the sequenced genome of Salmonella enterica serovar Typhimurium strain LT2, an open reading frame (STM0580) coding for a putative regulatory protein of the TetR family is found upstream of the ramA gene. Overexpression of ramA results in increased expression of the AcrAB efflux pump and, consequently, multidrug resistance (MDR) in several bacterial species. The inactivation of the putative regulatory protein gene upstream of ramA in a susceptible serovar Typhimurium strain resulted in an MDR phenotype with fourfold increases in the MICs of unrelated antibiotics, such as quinolones/fluoroquinolones, phenicols, and tetracycline. The inactivation of this gene also resulted in a fourfold increase in the expression of ramA and a fourfold increase in the expression of the AcrAB efflux pump. These results indicated that the gene encodes a local repressor of ramA and was thus named ramR. In contrast, the inactivation of marR, marA, soxR, and soxS did not affect the susceptibilities of the strain. In quinolone- or fluoroquinolone-resistant strains of serovar Typhimurium overexpressing AcrAB, several point mutations which resulted in amino acid changes or an in-frame shift were identified in ramR; in addition, mutations interrupting ramR with an IS1 element were identified in high-level fluoroquinolone-resistant serovar Typhimurium DT204 strains. One serovar Typhimurium DT104 isolate had a 2-nucleotide deletion in the putative RamR binding site found upstream of ramA. These mutations were confirmed to play a role in the MDR phenotype by complementing the isolates with an intact ramR gene or by inactivating their respective ramA gene. No mutations in the mar or sox region were found in the strains studied. In conclusion, mutations in ramR appear to play a major role in the upregulation of RamA and AcrAB and, consequently, in the efflux-mediated MDR phenotype of serovar Typhimurium.
Escherichia coli and Salmonella enterica serovar Typhimurium have evolved genetic systems, such as the soxR/S and marA regulons, to detoxify reactive oxygen species, like superoxide, which are formed as by-products of metabolism. Superoxide also serves as a microbicidal effector mechanism of the host's phagocytes. Here, we investigate whether regulatory genes other than soxR/S and marA are active in response to oxidative stress in Salmonella and may function as virulence determinants. We identified a bacterial gene, which was designated ramA (342 bp) and mapped at 13.1 min on the Salmonella chromosome, that, when overexpressed on a plasmid in E. coli or Salmonella, confers a pleiotropic phenotype characterized by increased resistance to the redox-cycling agent menadione and to multiple unrelated antibiotics. The ramA gene is present in Salmonella serovars but is absent in E. coli. The gene product displays 37 to 52% homology to the transcriptional activators soxR/S and marA and 80 to 100% identity to a multidrug resistance gene in Klebsiella pneumoniae and Salmonella enterica serovar Paratyphi A. Although a ramA soxR/S double null mutant is highly susceptible to intracellular superoxide generated by menadione and displays decreased Mn-superoxide dismutase activity, intracellular survival of this mutant within macrophage-like RAW 264.7 cells and in vivo replication in the spleens in Ityr mice are not affected. We concluded that despite its role in the protective response of the bacteria to oxidative stress in vitro, the newly identified ramA gene, together with soxR/S, does not play a role in initial replication of Salmonella in the organs of mice.
Salmonella enterica serovar Typhimurium has at least nine
multidrug efflux pumps. Among these pumps, AcrAB is effective in generating
drug resistance and has wide substrate specificity. Here we report that
indole, bile, and an Escherichia coli conditioned medium induced the
AcrAB pump in Salmonella through a specific regulator, RamA. The
RamA-binding sites were located in the upstream regions of acrAB and
tolC. RamA was required for indole induction of acrAB. Other
regulators of acrAB such as MarA, SoxS, Rob, SdiA, and AcrR did not
contribute to acrAB induction by indole in Salmonella.
Indole activated ramA transcription, and overproduction of RamA
caused increased acrAB expression. In contrast, induction of
ramA was not required for induction of acrAB by bile. Cholic
acid binds to RamA, and we suggest that bile acts by altering pre-existing
RamA. This points to two different AcrAB regulatory modes through RamA. Our
results suggest that RamA controls the Salmonella AcrAB-TolC
multidrug efflux system through dual regulatory modes in response to
Active efflux pump is a primary fluoroquinolone resistant mechanism of clinical isolates of Salmonella enterica serovar Typhimurium. RamA is an essential element in producing multidrug resistant (MDR) S.enterica serovar Typhimurium. The aim of the present study was to elucidate the roles of RamA on the development of ciprofloxacin, the first choice for the treatment of salmonellosis, resistance in S. enterica serovar Typhimurium. Spontaneous mutants were selected via several passages of S. enterica serovar Typhimurium CVCC541 susceptible strain (ST) on M-H agar with increasing concentrations of ciprofloxacin (CIP). Accumulation of ciprofloxacin was tested by the modified fluorometric method. The expression levels of MDR efflux pumps were determined by real time RT-PCR. In ST and its spontaneous mutants, the ramA gene was inactivated by insertion of the kan gene and compensated on a recombinant plasmid pGEXΦ(gst-ramA). The mutant prevention concentration (MPC) and mutant frequencies of ciprofloxacin against ST and a spontaneous mutant in the presence, absence and overexpression of RamA were tested. Four spontaneous mutants (SI1-SI4) were obtained. The SI1 (CIP MICs, 0.1 mg/L) without any target site mutation in its quinolone resistant determining regions (QRDRs) and SI3 (CIP MICs, 16 mg/L) harboring the Ser83→Phe mutation in its QRDR of GyrA strains exhibited reduced susceptibility and resistance to multidrugs, respectively. In SI1, RamA was the main factor that controlled the susceptibility to ciprofloxacin by activating MdtK as well as increasing the expression level of acrAB. In SI3, RamA played predominant role in ciprofloxacin resistance via increasing the expression level of acrAB. Likewise, the deficiency of RamA decreased the MPCs and mutant frequencies of ST and SI2 to ciprofloxacin. In conclusion, the expression of RamA promoted the development of ciprofloxacin resistant mutants of S. enterica serovar Typhimurium. The inhibition of RamA could decrease the appearance of the ciprofloxacin resistant mutants.
The transcriptomes of Salmonella enterica serovar Typhimurium SL1344 lacking a functional ramA or ramR or with plasmid-mediated high-level overexpression of ramA were compared to those of the wild-type parental strain. Inactivation of ramA led to increased expression of 14 SPI-1 genes and decreased expression of three SPI-2 genes, and it altered expression of ribosomal biosynthetic genes and several amino acid biosynthetic pathways. Furthermore, disruption of ramA led to decreased survival within RAW 264.7 mouse macrophages and attenuation within the BALB/c ByJ mouse model. Highly overexpressed ramA led to increased expression of genes encoding multidrug resistance (MDR) efflux pumps, including acrAB, acrEF, and tolC. Decreased expression of 34 Salmonella pathogenicity island (SPI) 1 and 2 genes, decreased SipC production, decreased adhesion to and survival within macrophages, and decreased colonization of Caenorhabditis elegans were also seen. Disruption of ramR led to the increased expression of ramA, acrAB, and tolC, but not to the same level as when ramA was overexpressed on a plasmid. Inactivation of ramR had a more limited effect on pathogenicity gene expression. In silico analysis of a suggested RamA-binding consensus sequence identified target genes, including ramR, acrA, tolC, sipABC, and ssrA. This study demonstrates that the regulation of a mechanism of MDR and expression of virulence genes show considerable overlap, and we postulate that such a mechanism is dependent on transcriptional activator concentration and promoter sensitivity. However, we have no evidence to support the hypothesis that increased MDR via RamA regulation of AcrAB-TolC gives rise to a hypervirulent strain.
Klebsiella pneumoniae is a significant human pathogen, in part due to high rates of multidrug resistance. RamA is an intrinsic regulator in K. pneumoniae established to be important for the bacterial response to antimicrobial challenge; however, little is known about its possible wider regulatory role in this organism during infection. In this work, we demonstrate that RamA is a global transcriptional regulator that significantly perturbs the transcriptional landscape of K. pneumoniae, resulting in altered microbe-drug or microbe-host response. This is largely due to the direct regulation of 68 genes associated with a myriad of cellular functions. Importantly, RamA directly binds and activates the lpxC, lpxL-2 and lpxO genes associated with lipid A biosynthesis, thus resulting in modifications within the lipid A moiety of the lipopolysaccharide. RamA-mediated alterations decrease susceptibility to colistin E, polymyxin B and human cationic antimicrobial peptide LL-37. Increased RamA levels reduce K. pneumoniae adhesion and uptake into macrophages, which is supported by in vivo infection studies, that demonstrate increased systemic dissemination of ramA overexpressing K. pneumoniae. These data establish that RamA-mediated regulation directly perturbs microbial surface properties, including lipid A biosynthesis, which facilitate evasion from the innate host response. This highlights RamA as a global regulator that confers pathoadaptive phenotypes with implications for our understanding of the pathogenesis of Enterobacter, Salmonella and Citrobacter spp. that express orthologous RamA proteins.
Bacteria can rapidly evolve under antibiotic pressure to develop resistance, which occurs when target genes mutate, or when resistance-encoding genes are transferred. Alternatively, microbes can simply alter the levels of intrinsic proteins that allow the organism to “buy” time to resist antibiotic pressure. Klebsiella pneumoniae is a pathogen that causes significant blood stream or respiratory infections, but more importantly is a bacterium that is increasingly being reported as multidrug resistant. Our data demonstrate that RamA can trigger changes on the bacterial surface that allow Klebsiella to survive both antibiotic challenge, degradation by host immune peptides and resist phagocytosis. We demonstrate that the molecular basis of increased survival of ramA overexpressing K. pneumoniae, against host-derived factors is associated with RamA-driven alterations of the lipid A moiety of Klebsiella LPS. This modification is likely to be linked to Klebsiella’s ability to resist the host response so that it remains undetected by the immune system. The relevance of our work extends beyond RamA in Klebsiella as other pathogens such as Enterobacter spp and Salmonella spp. also produce this protein. Thus our overarching conclusion is that the intrinsic regulator, RamA perturbs host-microbe and microbe-drug interactions.
The transcriptional activator RamA regulates production of the multidrug resistance efflux AcrAB–TolC system in several Enterobacteriaceae. This study investigated factors that lead to increased expression of ramA.
In order to monitor changes in ramA expression, the promoter region of ramA was fused to a gfp gene encoding an unstable green fluorescence protein (GFP) on the reporter plasmid, pMW82. The ramA reporter plasmid was transformed into Salmonella Typhimurium SL1344 and a ΔacrB mutant. The response of the reporter to subinhibitory concentrations of antibiotics, dyes, biocides, psychotropic agents and efflux inhibitors was measured during growth over a 5 h time period.
Our data revealed that the expression of ramA was increased in a ΔacrB mutant and also in the presence of the efflux inhibitors phenylalanine-arginine-β-naphthylamide, carbonyl cyanide m-chlorophenylhydrazone and 1-(1-naphthylmethyl)-piperazine. The phenothiazines chlorpromazine and thioridazine also increased ramA expression, triggering the greatest increase in GFP expression. However, inducers of Escherichia coli marA and soxS and 12 of 17 tested antibiotic substrates of AcrAB–TolC did not induce ramA expression.
This study shows that expression of ramA is not induced by most substrates of the AcrAB–TolC efflux system, but is increased by mutational inactivation of acrB or when efflux is inhibited.
antibiotic resistance; efflux inhibitors; phenothiazines
Salmonella enterica serovar Typhimurium SL1344, in which efflux pump genes (acrB, acrD, acrF, tolC) or regulatory genes thereof (marA, soxS, ramA) were inactivated, was grown in the presence of 240 antimicrobial and nonantimicrobial agents in the Biolog Phenotype MicroArray. Mutants lacking tolC, acrB, and ramA grew significantly worse than other mutants in the presence of 48 agents (some of which have not previously been identified as substrates of AcrAB-TolC) and particularly poorly in the presence of phenothiazines, which are human antipsychotics. MIC testing revealed that the phenothiazine chlorpromazine had antimicrobial activity and synergized with common antibiotics against different Salmonella serovars and SL1344. Chlorpromazine increased the intracellular accumulation of ethidium bromide, which was ablated in mutants lacking acrB, suggesting an interaction with AcrB. High-level but not low-level overexpression of ramA increased the expression of acrB; conferred resistance to chloramphenicol, tetracycline, nalidixic acid, and triclosan and organic solvent tolerance; and increased the amount of ethidium bromide accumulated. Chlorpromazine induced the modest overproduction of ramA but repressed acrB. These data suggest that phenothiazines are not efflux pump inhibitors but influence gene expression, including that of acrB, which confers the synergy with antimicrobials observed.
Multidrug resistance (MDR) in Enterobacter aerogenes can be mediated by induction of MarA, which is triggered by certain antibiotics and phenolic compounds. In this study, we identified the gene encoding RamA, a 113-amino-acid regulatory protein belonging to the AraC-XylS transcriptional activator family, in the Enterobacter aerogenes ATCC 13048 type strain and in a clinical multiresistant isolate. Overexpression of RamA induced an MDR phenotype in drug-susceptible Escherichia coli JM109 and E. aerogenes ATCC 13048, as demonstrated by 2- to 16-fold-increased resistance to β-lactams, tetracycline, chloramphenicol, and quinolones, a decrease in porin production, and increased production of AcrA, a component of the AcrAB-TolC drug efflux pump. We show that RamA enhances the transcription of the marRAB operon but is also able to induce an MDR phenotype in a mar-deleted strain. We demonstrate here that RamA is a transcriptional activator of the Mar regulon and is also a self-governing activator of the MDR cascade.
Overexpression of ramA has been implicated in resistance to multiple drugs in several enterobacterial pathogens. In the present study, Salmonella Typhimurium strain LTL with constitutive expression of ramA was compared to its ramA-deletion mutant by employing both DNA microarrays and phenotype microarrays (PM). The mutant strain with the disruption of ramA showed differential expression of at least 33 genes involved in 11 functional groups. The study confirmed at the transcriptional level that the constitutive expression of ramA was directly associated with increased expression of multidrug efflux pump AcrAB-TolC and decreased expression of porin protein OmpF, thereby conferring multiple drug resistance phenotype. Compared to the parent strain constitutively expressing ramA, the ramA mutant had increased susceptibility to over 70 antimicrobials and toxic compounds. The PM analysis also uncovered that the ramA mutant was better in utilization of 10 carbon sources and 5 phosphorus sources. This study suggested that the constitutive expression of ramA locus regulate not only multidrug efflux pump and accessory genes but also genes involved in carbon metabolic pathways.
The transcriptional activator RamA is involved in multidrug resistance (MDR) by increasing expression of the AcrAB-TolC RND-type efflux system in several pathogenic Enterobacteriaceae. In Salmonella enterica serovar Typhimurium (S. Typhimurium), ramA expression is negatively regulated at the local level by RamR, a transcriptional repressor of the TetR family. We here studied the DNA-binding activity of the RamR repressor with the ramA promoter (PramA). As determined by high-resolution footprinting, the 28-bp-long RamR binding site covers essential features of PramA, including the −10 conserved region, the transcriptional start site of ramA, and two 7-bp inverted repeats. Based on the RamR footprint and on electrophoretic mobility shift assays (EMSAs), we propose that RamR interacts with PramA as a dimer of dimers, in a fashion that is structurally similar to the QacR-DNA binding model. Surface plasmon resonance (SPR) measurements indicated that RamR has a 3-fold-lower affinity (KD [equilibrium dissociation constant] = 191 nM) for the 2-bp-deleted PramA of an MDR S. Typhimurium clinical isolate than for the wild-type PramA (KD = 66 nM). These results confirm the direct regulatory role of RamR in the repression of ramA transcription and precisely define how an alteration of its binding site can give rise to an MDR phenotype.
RamA regulates the AcrAB-TolC multidrug efflux system. Using Salmonella Typhimurium, we investigated the stability of RamA and its impact on antibiotic resistance.
To detect RamA, we introduced ramA::3XFLAG::aph into plasmid pACYC184 and transformed this into Salmonella Typhimurium SL1344ramA::cat and lon::aph mutants. An N-terminus-deleted mutant [pACYC184ramA(Δ2-21)::3XFLAG::aph] in which the first 20 amino acids of RamA were deleted was also constructed. To determine the abundance and half-life of FLAG-tagged RamA, we induced RamA with chlorpromazine (50 mg/L) and carried out western blotting using anti-FLAG antibody. Susceptibility to antibiotics and phenotypic characterization of the lon mutant was also carried out.
We show that on removal of chlorpromazine, a known inducer of ramA, the abundance of RamA decreased to pre-induced levels. However, in cells lacking functional Lon, we found that the RamA protein was not degraded. We also demonstrated that the 21 amino acid residues of the RamA N-terminus are required for recognition by the Lon protease. Antimicrobial susceptibility and phenotypic tests showed that the lon mutant was more susceptible to fluoroquinolone antibiotics, was filamentous when observed by microscopy and grew poorly, but showed no difference in motility or the ability to form a biofilm. There was also no difference in the ability of the lon mutant to invade human intestinal cells (INT-407).
In summary, we show that the ATP-dependent Lon protease plays an important role in regulating the expression of RamA and therefore multidrug resistance via AcrAB-TolC in Salmonella Typhimurium.
Salmonella; transcription factors; proteolysis
Tigecycline resistance has been attributed to ramA overexpression and subsequent acrA upregulation. The ramA locus, originally identified in Klebsiella pneumoniae, has homologues in Enterobacter and Salmonella spp. In this study, we identify in silico that the ramR binding site is also present in Citrobacter spp. and that Enterobacter, Citrobacter and Klebsiella spp. share key regulatory elements in the control of the romA–ramA locus. RACE (rapid amplification of cDNA ends) mapping indicated that there are two promoters from which romA–ramA expression can be regulated in K. pneumoniae. Correspondingly, electrophoretic binding studies clearly showed that purified RamA and RamR proteins bind to both of these promoters. Hence, there appear to be two RamR binding sites within the Klebsiella romA–ramA locus. Like MarA, RamA binds the promoter region, implying that it might be subject to autoregulation. We have identified changes within ramR in geographically distinct clinical isolates of K. pneumoniae. Intriguingly, levels of romA and ramA expression were not uniformly affected by changes within the ramR gene, thereby supporting the dual promoter finding. Furthermore, a subset of strains sustained no changes within the ramR gene but which still overexpressed the romA–ramA genes, strongly suggesting that a secondary regulator may control ramA expression.
Klebsiella pneumoniae; romA; ramA; ramR; acrA; Tigecycline
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.
Control of membrane permeability is a key step in regulating the intracellular concentration of antibiotics. Efflux pumps confer innate resistance to a wide range of toxic compounds such as antibiotics, dyes, detergents, and disinfectants in members of the Enterobacteriaceae. The AcrAB-TolC efflux pump is involved in multidrug resistance in Enterobacter cloacae. However, the underlying mechanism that regulates the system in this microorganism remains unknown. In Escherichia coli, the transcription of acrAB is upregulated under global stress conditions by proteins such as MarA, SoxS, and Rob. In the present study, two clinical isolates of E. cloacae, EcDC64 (a multidrug-resistant strain overexpressing the AcrAB-TolC efflux pump) and Jc194 (a strain with a basal AcrAB-TolC expression level), were used to determine whether similar global stress responses operate in E. cloacae and also to establish the molecular mechanisms underlying this response. A decrease in susceptibility to erythromycin, tetracycline, telithromycin, ciprofloxacin, and chloramphenicol was observed in clinical isolate Jc194 and, to a lesser extent in EcDC64, in the presence of salicylate, decanoate, tetracycline, and paraquat. Increased expression of the acrAB promoter in the presence of the above-described conditions was observed by flow cytometry and reverse transcription-PCR, by using a reporter fusion protein (green fluorescent protein). The expression level of the AcrAB promoter decreased in E. cloacae EcDC64 derivates deficient in SoxS, RobA, and RamA. Accordingly, the expression level of the AcrAB promoter was higher in E. cloacae Jc194 strains overproducing SoxS, RobA, and RamA. Overall, the data showed that SoxS, RobA, and RamA regulators were associated with the upregulation of acrAB, thus conferring antimicrobial resistance as well as a stress response in E. cloacae. In summary, the regulatory proteins SoxS, RobA, and RamA were cloned and sequenced for the first time in this species. The involvement of these proteins in conferring antimicrobial resistance through upregulation of acrAB was demonstrated in E. cloacae.
RamA is a transcription factor involved in regulating multidrug resistance in Salmonella enterica serovar Typhimurium SL1344. Green fluorescent protein (GFP) reporter fusions were exploited to investigate the regulation of RamA expression by RamR. We show that RamR represses the ramA promoter by binding to a palindromic sequence and describe a superrepressor RamR mutant that binds to the ramA promoter sequence more efficiently, thus exhibiting a ramA inactivated phenotype.
Mechanisms of antibiotic resistance were examined in nalidixic acid-resistant Salmonella enterica serovar Enteritidis field isolates displaying decreased susceptibility to ciprofloxacin and in in vitro-derived ciprofloxacin-resistant mutants (104-cip and 5408-cip). All field isolates harbored a single gyrA mutation (D87Y). Deletion of acrB and complementation with wild-type gyrA increased quinolone susceptibility. Selection for ciprofloxacin resistance was associated with the development of an additional gyrA (S83F) mutation in 104-cip, novel gyrB (E466D) and parE (V461G) mutations in 5408-cip, overexpression of acrB and decreased susceptibility to nonquinolone antibiotics in both mutants, and decreased OmpF production and altered lipopolysaccharide in 104-cip. Complementation of mutated gyrA and gyrB with wild-type alleles restored susceptibility to quinolones in 104-cip and significantly decreased the ciprofloxacin MIC in 5408-cip. Complementation of parE had no effect on quinolone MICs. Deletion of acrB restored susceptibility to ciprofloxacin and other antibiotics tested. Both soxS and marA were overexpressed in 104-cip, and ramA was overexpressed in 5408-cip. Inactivation of each of these global regulators lowered ciprofloxacin MICs, decreased expression of acrB, and restored susceptibility to other antibiotics. Mutations were found in soxR (R20H) and in soxS (E52K) in 104-cip and in ramR (G25A) in 5408-cip. In conclusion, both efflux activity and a single gyrA mutation contribute to nalidixic acid resistance and reduced ciprofloxacin sensitivity. Ciprofloxacin resistance and decreased susceptibility to multiple antibiotics can result from different genetic events leading to development of target gene mutations, increased efflux activity resulting from differential expression of global regulators associated with mutations in their regulatory genes, and possible altered membrane permeability.
A genomic library from a strain of Salmonella enterica serovar Paratyphi B that exhibits multiple drug resistance (MDR) was constructed in Escherichia coli. Two of the recombinant plasmids, pNOR5 and pNOR5, conferred resistance only to fluoroquinolones in E. coli, whereas the third, pNCTR4, conferred the MDR phenotype. Sequence and subcloning analysis showed that it is the presence of RecA on the first two plasmids which confers resistance to fluoroquinolones in E. coli. A similar analysis established that the MDR phenotype conferred by pNCTR4 is due to a gene, rma (resistance to multiple antibiotics), which encodes a 13.5-kDa polypeptide. The derived sequence for Rma exhibits a high degree of similarity to those of a group of MarA-like activators that confer MDR in E. coli. A MalE-Rma fusion protein was purified to near homogeneity and was shown to interact with a DNA fragment carrying a MarA operator sequence. Furthermore, overexpression of rma in E. coli caused changes in the outer membrane protein profile that were similar to those reported for MarA. These results suggest that Rma might act as a transcriptional activator of the marA regulon.
The relationship between efflux system overexpression and cross-resistance to cefoxitin, quinolones, and chloramphenicol has recently been reported in Klebsiella pneumoniae. In 3 previously published clinical isolates and 17 in vitro mutants selected with cefoxitin or fluoroquinolones, mutations in the potential regulator genes of the AcrAB efflux pump (acrR, ramR, ramA, marR, marA, soxR, soxS, and rob) were searched, and their impacts on efflux-related antibiotic cross-resistance were assessed. All mutants but 1, and 2 clinical isolates, overexpressed acrB. No mutation was detected in the regulator genes studied among the clinical isolates and 8 of the mutants. For the 9 remaining mutants, a mutation was found in the ramR gene in 8 of them and in the soxR gene in the last one, resulting in overexpression of ramA and soxS, respectively. Transformation of the ramR mutants and the soxR mutant with the wild-type ramR and soxR genes, respectively, abolished overexpression of acrB and ramA in the ramR mutants and of soxS in the soxR mutant, as well as antibiotic cross-resistance. Resistance due to efflux system overexpression was demonstrated for 4 new antibiotics: cefuroxime, cefotaxime, ceftazidime, and ertapenem. This study shows that the ramR and soxR genes control the expression of efflux systems in K. pneumoniae and suggests the existence of efflux pumps other than AcrAB and of other loci involved in the regulation of AcrAB expression.
We experimentally identified and characterized 97 novel, non-protein-coding RNA candidates (npcRNAs) from the human pathogen Salmonella enterica serovar Typhi (hereafter referred to as S. typhi). Three were specific to S. typhi, 22 were restricted to Salmonella species and 33 were differentially expressed during S. typhi growth. We also identified Salmonella Pathogenicity Island-derived npcRNAs that might be involved in regulatory mechanisms of virulence, antibiotic resistance and pathogenic specificity of S. typhi. An in-depth characterization of S. typhi StyR-3 npcRNA showed that it specifically interacts with RamR, the transcriptional repressor of the ramA gene, which is involved in the multidrug resistance (MDR) of Salmonella. StyR-3 interfered with RamR–DNA binding activity and thus potentially plays a role in regulating ramA gene expression, resulting in the MDR phenotype. Our study also revealed a large number of cis-encoded antisense npcRNA candidates, supporting previous observations of global sense–antisense regulatory networks in bacteria. Finally, at least six of the npcRNA candidates interacted with the S. typhi Hfq protein, supporting an important role of Hfq in npcRNA networks. This study points to novel functional npcRNA candidates potentially involved in various regulatory roles including the pathogenicity of S. typhi.
Transcriptional regulators, such as SoxS, RamA, MarA, and Rob, which upregulate the AcrAB efflux pump, have been shown to be associated with multidrug resistance in clinically relevant Gram-negative bacteria. In addition to the multidrug resistance phenotype, these regulators have also been shown to play a role in the cellular metabolism and possibly the virulence potential of microbial cells. As such, the increased expression of these proteins is likely to cause pleiotropic phenotypes. Klebsiella pneumoniae is a major nosocomial pathogen which can express the SoxS, MarA, Rob, and RamA proteins, and the accompanying paper shows that the increased transcription of ramA is associated with tigecycline resistance (M. Veleba and T. Schneiders, Antimicrob. Agents Chemother. 56:4466–4467, 2012). Bioinformatic analyses of the available Klebsiella genome sequences show that an additional AraC-type regulator is encoded chromosomally. In this work, we characterize this novel AraC-type regulator, hereby called RarA (Regulator of antibiotic resistance A), which is encoded in K. pneumoniae, Enterobacter sp. 638, Serratia proteamaculans 568, and Enterobacter cloacae. We show that the overexpression of rarA results in a multidrug resistance phenotype which requires a functional AcrAB efflux pump but is independent of the other AraC regulators. Quantitative real-time PCR experiments show that rarA (MGH 78578 KPN_02968) and its neighboring efflux pump operon oqxAB (KPN_02969_02970) are consistently upregulated in clinical isolates collected from various geographical locations (Chile, Turkey, and Germany). Our results suggest that rarA overexpression upregulates the oqxAB efflux pump. Additionally, it appears that oqxR, encoding a GntR-type regulator adjacent to the oqxAB operon, is able to downregulate the expression of the oqxAB efflux pump, where OqxR complementation resulted in reductions to olaquindox MICs.
Tigecycline is an expanded broad-spectrum antibacterial agent that is active against many clinically relevant species of bacterial pathogens, including Klebsiella pneumoniae. The majority of K. pneumoniae isolates are fully susceptible to tigecycline; however, a few strains that have decreased susceptibility have been isolated. One isolate, G340 (for which the tigecycline MIC is 4 μg/ml and which displays a multidrug resistance [MDR] phenotype), was selected for analysis of the mechanism for this decreased susceptibility by use of transposon mutagenesis with IS903φkan. A tigecycline-susceptible mutant of G340, GC7535, was obtained (tigecycline MIC, 0.25 μg/ml). Analysis of the transposon insertion mapped it to ramA, a gene that was previously identified to be involved in MDR in K. pneumoniae. For GC7535, the disruption of ramA led to a 16-fold decrease in the MIC of tigecycline and also a suppression of MDR. Trans-complementation with plasmid-borne ramA restored the original parental phenotype of decreased susceptibility to tigecycline. Northern blot analysis revealed a constitutive overexpression of ramA that correlated with an increased expression of the AcrAB transporter in G340 compared to that in tigecycline-susceptible strains. Laboratory mutants of K. pneumoniae with decreased susceptibility to tigecycline could be selected at a frequency of approximately 4 × 10−8. These results suggest that ramA is associated with decreased tigecycline susceptibility in K. pneumoniae due to its role in the expression of the AcrAB multidrug efflux pump.
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.
efflux; tigecycline; multidrug-resistant
Understanding the impact of antimicrobial use on the emergence of resistant bacteria is imperative to prevent its emergence. For instance, activation of the AcrAB efflux pumps is responsible for the emergence of antimicrobial-resistant Salmonella strains. Here, we examined the expression levels of acrB and its multiple regulator genes (RamA, SoxS, MarA, and Rob) in 17 field isolates of S. Choleraesuis by using quantitative PCR methods. The expression of acrB increased in eight of the field isolates (P < 0.05). The expression of acrB was associated with that of ramA in one isolate, soxS in one isolate, and both these genes in six isolates. Thereafter, to examine the effect of selected antimicrobials (enrofloxacin, ampicillin, oxytetracycline, kanamycin, and spectinomycin) on the expression of acrB and its regulator genes, mutants derived from five isolates of S. Choleraesuis were selected by culture on antimicrobial-containing plates. The expression of acrB and ramA was higher in the mutants selected using enrofloxacin (3.3–6.3- and 24.5–37.7-fold, respectively), ampicillin (1.8–7.7- and 16.1–55.9-fold, respectively), oxytetracycline (1.7–3.3- and 3.2–31.1-fold, respectively), and kanamycin (1.6–2.2- and 5.6–26.4-fold, respectively), which are AcrAB substrates, than in each of the parental strains (P < 0.05). In contrast, in AcrAB substrate-selected mutants, the expression of soxS, marA, and rob remained similar to that in parental strains. Of the four antimicrobials, the level of ramA expression was significantly higher in the enrofloxacin- and ampicillin-selected mutants than in the oxytetracycline- and kanamycin-selected mutants (P < 0.05), whereas the expression levels of acrB and multiple regulator genes in spectinomycin-selected mutants were similar to those in each parental strain. These data suggest that exposure to antimicrobials that are AcrAB substrates enhance the activation of the AcrAB efflux pump via RamA, but not via SoxS, MarA, or Rob in S. Choleraesuis.
AcrAB efflux pump; antimicrobial resistance; RamA; Salmonella Choleraesuis; SoxS
In order to understand the role of the mar locus in Salmonella with regard to multiple antibiotic resistance, cyclohexane resistance, and outer membrane protein F (OmpF) regulation, a marA::gfp reporter mutant was constructed in an antibiotic-sensitive Salmonella enterica serovar Typhimurium DT104 background. Salicylate induced marA, whereas a number of antibiotics, disinfectants, and various growth conditions did not. Increased antibiotic resistance was observed upon salicylate induction, although this was shown to be by both mar-dependent and mar-independent pathways. Cyclohexane resistance, however, was induced by salicylate by a mar-dependent pathway. Complementation studies with a plasmid that constitutively expressed marA confirmed the involvement of mar in Salmonella with low-level antibiotic resistance and cyclohexane resistance, although the involvement of mar in down regulation of OmpF was unclear. However, marA overexpression did increase the expression of a ca. 50-kDa protein, but its identity remains to be elucidated. Passage of the marA::gfp reporter mutant with increasing levels of tetracycline, a method reported to select for mar mutants in Escherichia coli, led to both multiple-antibiotic and cyclohexane resistance. Collectively, these data indicate that low-level antibiotic resistance, cyclohexane resistance, and modulation of OMPs in Salmonella, as in E. coli, can occur in both a mar-dependent and mar-independent manner.