Antibiotics do not induce expression of acrAB directly but via a RamA-dependent pathway.

The aim of this study was to determine if acrAB induction in Salmonella Typhimurium relies solely on RamA or if other transcriptional activator pathways are also involved, and to better understand the kinetics of induction of both acrAB and ramA. We evaluated the expression of acrAB in S. Typhimurium in response to a variety of compounds that are known to induce the expression of one or more of the transcriptional activators, MarA, SoxS, RamA, and Rob. We utilized green fluorescent protein (GFP) transcriptional reporter fusions to investigate the changes in the expression of acrAB, ramA, marA, and soxS following exposure to sub-inhibitory concentrations of antimicrobial compounds. Of the compounds tested, 13 induce acrAB expression in S. Typhimurium via RamA, MarA, SoxS, and Rob-dependent pathways. None of the tested antibiotics induced acrAB expression, and compounds that induced acrAB expression also induced a general stress response. The results from this study show that the majority of compounds tested induced acrAB via the RamA-dependent pathway. However, none of the antibiotic substrates of the AcrB efflux pump directly increased the expression of AcrAB either directly or indirectly via the induction of one of the transcriptional activators. Using a dual GFP/RFP reporter, we investigated the kinetics of the induction of ramA and acrAB simultaneously and found that acrAB gene expression was transient compared to ramA gene expression. ramA gene expression increased with time and would remain high or decrease slowly over the course of the experiment indicating that RamA exerts a wider global effect and is not limited to efflux regulation alone.

Exposure of Escherichia coli to antibiotic-efflux pump inhibitor combinations in a pharmacokinetic model: impact on bacterial clearance and drug resistance.

BACKGROUND: Efflux pump inhibitors (EPIs) offer an attractive therapeutic option when combined with existing classes. However, their optimal dosing strategies are unknown. METHODS: MICs of ciprofloxacin (CIP)+/-chlorpromazine, phenylalanine-arginine ß naphthylamide (PAßN) and a developmental molecule MBX-4191 were determined and the pharmacodynamics (PD) was studied in an in vitro model employing Escherichia coli MG1655 and its isogenic MarR mutant (I1147). Exposure ranging experiments were performed initially then fractionation. Changes in bacterial load and population profiles were assessed. Strains recovered after EPI simulations were studied by WGS. RESULTS: The CIPMICs for E. coli MG1655 and I1147 were 0.08 and 0.03 mg/L. Chlorpromazine at a concentration of 60 mg/L, PAßN concentrations of 30 mg/L and MBX-4191 concentrations of 0.5-1.0 mg/L reduced CIP MICs for I1147 and enhanced bacterial killing. Using CIP at an AUC of 1.2 mg·h/L, chlorpromazine AUC was best related to reduction in bacterial load at 24 h, however, when the time drug concentration was greater than 25 mg/L (T > 25 mg/L) chlorpromazine was also strongly related to the effect. For PaßN with CIP AUC, 0.6 mg·h/L PaßN AUC was best related to a reduction in bacterial load. MBX-4191T > 0.5-0.75 mg·h/L was best related to reduction in bacterial load. Changes in population profiles were not seen in experiments of ciprofloxacin + EPIs. WGS of recovered strains from simulations with all three EPIs showed mutations in gyrA, gyrB or marR. CONCLUSIONS: AUC was the pharmacodynamic driver for chlorpromazine and PAßN while T > threshold was the driver for MBX-4191 and important in the activity of chlorpromazine and PAßN. Changes in population profiles did not occur with combinations of ciprofloxacin + EPIs, however, mutations in gyrA, gyrB and marR were detected.

Absence, loss-of-function, or inhibition of Escherichia coli AcrB does not increase expression of other efflux pump genes supporting the discovery of AcrB inhibitors as antibiotic adjuvants.

OBJECTIVES: To determine whether expression of efflux pumps and antibiotic susceptibility are altered in Escherichia coli in response to efflux inhibition. METHODS: The promoter regions of nine efflux pump genes (acrAB, acrD, acrEF, emrAB, macAB, cusCFBA, mdtK, mdtABC, mdfA) were fused to gfp in pMW82 and fluorescence from each reporter construct was used as a measure of the transcriptional response to conditions in which AcrB was inhibited, absent or made non-functional. Expression was also determined by RT-qPCR. Drug susceptibility of efflux pump mutants with missense mutations known or predicted to cause loss of function of the encoded efflux pump was investigated. RESULTS: Data from the GFP reporter constructs revealed that no increased expression of the tested efflux pump genes was observed when AcrB was absent, made non-functional, or inhibited by an efflux pump inhibitor/competitive substrate, such as PAßN or chlorpromazine. This was confirmed by RT-qPCR for PAßN and chlorpromazine; however, a small but significant increase in macB gene expression was seen when acrB is deleted. Efflux inhibitors only synergized with antibiotics in the presence of a functional AcrB. When AcrB was absent or non-functional, there was no impact on MICs when other efflux pumps were also made non-functional. CONCLUSIONS: Absence, loss-of-function, or inhibition of E. coli AcrB did not significantly increase expression of other efflux pump genes, which suggests there is no compensatory mechanism to overcome efflux inhibition and supports the discovery of inhibitors of AcrB as antibiotic adjuvants.

EnvR is a potent repressor of acrAB transcription in Salmonella.

BACKGROUND: Resistance nodulation division (RND) family efflux pumps, including the major pump AcrAB-TolC, are important mediators of intrinsic and evolved antibiotic resistance. Expression of these pumps is carefully controlled by a network of regulators that respond to different environmental cues. EnvR is a TetR family transcriptional regulator encoded upstream of the RND efflux pump acrEF. METHODS: Binding of EnvR protein upstream of acrAB was determined by electrophoretic mobility shift assays and the phenotypic consequence of envR overexpression on antimicrobial susceptibility, biofilm motility and invasion of eukaryotic cells in vitro was measured. Additionally, the global transcriptome of clinical Salmonella isolates overexpressing envR was determined by RNA-Seq. RESULTS: EnvR bound to the promoter region upstream of the genes coding for the major efflux pump AcrAB in Salmonella, inhibiting transcription and preventing production of AcrAB protein. The phenotype conferred by overexpression of envR mimicked deletion of acrB as it conferred multidrug susceptibility, decreased motility and decreased invasion into intestinal cells in vitro. Importantly, we demonstrate the clinical relevance of this regulatory mechanism because RNA-Seq revealed that a drug-susceptible clinical isolate of Salmonella had low acrB expression even though expression of its major regulator RamA was very high; this was caused by very high EnvR expression. CONCLUSIONS: In summary, we show that EnvR is a potent repressor of acrAB transcription in Salmonella, and can override binding by RamA so preventing MDR to clinically useful drugs. Finding novel tools to increase EnvR expression may form the basis of a new way to prevent or treat MDR infections.

Time dependent asymptotic analysis of the gene regulatory network of the AcrAB-TolC efflux pump system in gram-negative bacteria.

Efflux pumps are a mechanism of intrinsic and evolved resistance in bacteria. If an efflux pump can expel an antibiotic so that its concentration within the cell is below a killing threshold the bacteria are resistant to the antibiotic. Efflux pumps may be specific or they may pump various different substances. This is why many efflux pumps confer multi drug resistance (MDR). In particular over expression of the AcrAB-TolC efflux pump system confers MDR in both Salmonella and Escherichia coli. We consider the complex gene regulation network that controls expression of genes central to controlling the efflux associated genes acrAB and acrEF in Salmonella. We present the first mathematical model of this gene regulatory network in the form of a system of ordinary differential equations. Using a time dependent asymptotic analysis, we examine in detail the behaviour of the efflux system on various different timescales. Asymptotic approximations of the steady states provide an analytical comparison of targets for efflux inhibition.

Overexpression of RamA, Which Regulates Production of the Multidrug Resistance Efflux Pump AcrAB-TolC, Increases Mutation Rate and Influences Drug Resistance Phenotype.

In Enterobacteriales, the AcrAB-TolC efflux pump exports substrates, including antimicrobials, from the cell. Overexpression of AcrAB-TolC can occur after exposure to fluoroquinolones, leading to multidrug resistance. The expression of AcrAB-TolC in Salmonella is primarily regulated by the transcriptional activator RamA. However, other transcriptional activators, such as MarA, SoxRS, and Rob, can influence AcrAB-TolC expression. This study determined whether the overproduction or absence of RamA influences the mutation rate or the phenotype of mutants selected in Salmonella enterica serovar Typhimurium SL1344 after ciprofloxacin exposure. The absence of RamA (SL1344 ramA::aph) resulted in mutation frequencies/rates similar to those of wild-type Salmonella Typhimurium SL1344. However, the overproduction of RamA (SL1344 ramR::aph) and, consequently, AcrB resulted in a significantly higher mutation frequency and rate than for wild-type Salmonella Typhimurium SL1344. Whole-genome sequencing revealed that in addition to selecting gyrA mutants resistant to quinolones, SL1344 and SL1344 ramA::aph also produced multidrug-resistant (MDR) mutants, associated with mutations in soxR Conversely, mutations in SL1344 ramR::aph occurred in gyrA only. Although transcriptional regulators such as SoxRS are believed to play a minor role in AcrAB-TolC regulation under antibiotic selective pressure, we show that soxR mutants can be selected after exposure to ciprofloxacin, including when RamA is absent. This demonstrates that under selective pressure, Salmonella can respond to increased efflux pump expression by mutating other AcrAB-TolC regulatory genes, allowing for the evolution of MDR. Understanding how Salmonella responds to antibiotic pressure in the absence/overproduction of RamA is important if targeting transcriptional regulators to alter efflux is to be considered an avenue for future drug discovery.

Wastewater used for urban agriculture in West Africa as a reservoir for antibacterial resistance dissemination.

State of art metagenomics were used to investigate the microbial population, antibiotic resistance genes and plasmids of medical interest in wastewater used for urban agriculture in Ouagadougou (Burkina Faso). Wastewater samples were collected from three canals near agricultural fields in three neighbourhoods. Assessment of microbial population diversity revealed different microbial patterns among the different samples. Sequencing reads from the wastewaters revealed different functional specializations of microbial communities, with the predominance of carbohydrates and proteins metabolism functions. Eleven pathogen-specific and 56 orthologous virulence factor genes were detected in the wastewater samples. These virulence factors are usually found in human pathogens that cause gastroenteritis and/or diarrhoea. A wide range of antibiotic resistance genes was identified; 81 are transmissible by mobile genetic elements. These included seven different extended spectrum ß-lactamase genes encoding synthesis of four enzyme families, including two metallo-ß-lactamases (blaAIM-1 and blaGES-21). Ten different incompatibility groups of Enterobacteriaceae plasmid replicons (ColE, FIB, FIC, FII, P, Q, R, U, Y, and A/C), and 30 plasmid replicon types from Gram-positive bacteria. All are implicated in the wide distribution of antibiotic resistance genes. We conclude that wastewater used for urban agriculture in the city represents a high risk for spreading bacteria and antimicrobial resistance among humans and animals.

CsrA maximizes expression of the AcrAB multidrug resistance transporter.

Carbon Storage Regulator A (CsrA) is an RNA binding protein that acts as a global regulator of diverse genes. Using a combination of genetics and biochemistry we show that CsrA binds directly to the 5' end of the transcript encoding AcrAB. Deletion of csrA or mutagenesis of the CsrA binding sites reduced production of both AcrA and AcrB. Nucleotide substitutions at the 5' UTR of acrA mRNA that could potentially weaken the inhibitory RNA secondary structure, allow for more efficient translation of the AcrAB proteins. Given the role of AcrAB-TolC in multi-drug efflux we suggest that CsrA is a potential drug target.

Metabolic constraints on the evolution of antibiotic resistance.

Despite our continuous improvement in understanding antibiotic resistance, the interplay between natural selection of resistance mutations and the environment remains unclear. To investigate the role of bacterial metabolism in constraining the evolution of antibiotic resistance, we evolved Escherichia coli growing on glycolytic or gluconeogenic carbon sources to the selective pressure of three different antibiotics. Profiling more than 500 intracellular and extracellular putative metabolites in 190 evolved populations revealed that carbon and energy metabolism strongly constrained the evolutionary trajectories, both in terms of speed and mode of resistance acquisition. To interpret and explore the space of metabolome changes, we developed a novel constraint-based modeling approach using the concept of shadow prices. This analysis, together with genome resequencing of resistant populations, identified condition-dependent compensatory mechanisms of antibiotic resistance, such as the shift from respiratory to fermentative metabolism of glucose upon overexpression of efflux pumps. Moreover, metabolome-based predictions revealed emerging weaknesses in resistant strains, such as the hypersensitivity to fosfomycin of ampicillin-resistant strains. Overall, resolving metabolic adaptation throughout antibiotic-driven evolutionary trajectories opens new perspectives in the fight against emerging antibiotic resistance.

AcrB drug-binding pocket substitution confers clinically relevant resistance and altered substrate specificity.

The incidence of multidrug-resistant bacterial infections is increasing globally and the need to understand the underlying mechanisms is paramount to discover new therapeutics. The efflux pumps of Gram-negative bacteria have a broad substrate range and transport antibiotics out of the bacterium, conferring intrinsic multidrug resistance (MDR). The genomes of pre- and posttherapy MDR clinical isolates of Salmonella Typhimurium from a patient that failed antibacterial therapy and died were sequenced. In the posttherapy isolate we identified a novel G288D substitution in AcrB, the resistance-nodulation division transporter in the AcrAB-TolC tripartite MDR efflux pump system. Computational structural analysis suggested that G288D in AcrB heavily affects the structure, dynamics, and hydration properties of the distal binding pocket altering specificity for antibacterial drugs. Consistent with this hypothesis, recreation of the mutation in standard Escherichia coli and Salmonella strains showed that G288D AcrB altered substrate specificity, conferring decreased susceptibility to the fluoroquinolone antibiotic ciprofloxacin by increased efflux. At the same time, the substitution increased susceptibility to other drugs by decreased efflux. Information about drug transport is vital for the discovery of new antibacterials; the finding that one amino acid change can cause resistance to some drugs, while conferring increased susceptibility to others, could provide a basis for new drug development and treatment strategies.

Expression of homologous RND efflux pump genes is dependent upon AcrB expression: implications for efflux and virulence inhibitor design.

OBJECTIVES: Enterobacteriaceae have multiple efflux pumps that confer intrinsic resistance to antibiotics. AcrB mediates clinically relevant multidrug resistance and is required for virulence and biofilm formation, making it an attractive target for the design of inhibitors. The aim of this study was to assess the viability of single transporters as a target for efflux inhibition using Salmonella Typhimurium as the model pathogen. METHODS: The expression of resistance-nodulation-division (RND) efflux pump genes in response to the inactivation of single or multiple homologues was measured using real-time RT-PCR. Phenotypes of mutants were characterized by measuring antimicrobial susceptibility, dye accumulation and the ability to cause infection in vitro. RESULTS: The expression of all RND efflux pump genes was increased when single or multiple acr genes were inactivated, suggesting a feedback mechanism that activates the transcription of homologous efflux pump genes. When two or three acr genes were inactivated, the mutants had further reduced efflux, altered susceptibility to antimicrobials (including increased susceptibility to some, but conversely and counterintuitively, decreased susceptibility to some others) and were more attenuated in the tissue culture model than mutants lacking single pumps were. CONCLUSIONS: These data indicate that it is critical to understand which pumps an inhibitor is active against and the effect of this on the expression of homologous systems. For some antimicrobials, an inhibitor with activity against multiple pumps will have a greater impact on susceptibility, but an unintended consequence of this may be decreased susceptibility to other drugs, such as aminoglycosides.

RamA, which controls expression of the MDR efflux pump AcrAB-TolC, is regulated by the Lon protease.

OBJECTIVES: RamA regulates the AcrAB-TolC multidrug efflux system. Using Salmonella Typhimurium, we investigated the stability of RamA and its impact on antibiotic resistance. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Regulation of RamA by RamR in Salmonella enterica serovar Typhimurium: isolation of a RamR superrepressor.

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.

The TCA cycle is not required for selection or survival of multidrug-resistant Salmonella.

OBJECTIVES: The initial aim of this study was to use a systems biology approach to analyse a ciprofloxacin-selected multidrug-resistant (MDR) Salmonella enterica serotype Typhimurium, L664. METHODS: The whole genome sequence and transcriptome of L664 were analysed. Site-directed mutagenesis to recreate each mutation was carried out, followed by phenotypic characterization and mutation frequency analysis. As a mutation in the TCA cycle was detected we tested the controversial hypothesis regarding the bacterial response to bactericidal antibiotics, put forward by Kohanski et al. (Cell 2007; 130: 797-810 and Mol Cell 2010; 37: 311-20), that exposure of bacteria to agents such as ciprofloxacin produces reactive oxygen species (ROS), which transiently increase the mutation rate giving rise to MDR bacteria. RESULTS: L664 contained a mutation in ramR that conferred MDR. A mutation in tctA affected the TCA cycle and conferred the inability to grow on minimal agar. The virulence of L664 was not attenuated. Ciprofloxacin exposure produced ROS in L664 and SL1344 (tctA::aph), but it was reduced and occurred later. There were no significant differences in the rates of killing or mutations per generation to antibiotic resistance between the strains. CONCLUSIONS: Whilst we confirm production of ROS in response to ciprofloxacin, we have no data to support the hypothesis that this leads to selection of MDR strains. Our results indicate that the mutations in tctA and glgA were random as they did not pre-exist in the parental strain, and that the mutation in tctA did not provide a survival advantage or disadvantage in the presence of antibiotic.

Exploiting the role of TolC in pathogenicity: identification of a bacteriophage for eradication of Salmonella serovars from poultry.

Using a screening procedure, three bacteriophages, ST27, ST29, and ST35, were identified with selective activity for Salmonella enterica serovar Typhimurium (SL1344) but not SL1344 tolC::aph. Overproduction of TolC led to a lower efficiency of plating (EOP), further suggesting that TolC was the target receptor. Activity against other serovars of Salmonella was observed but not against other species of Enterobacteriaceae. This study provides proof of principle that bacteriophages can be active against the outer membrane protein of tripartite resistance-nodulation-division (RND) efflux pumps and so could be used to reduce the numbers of Salmonella cells in animals reared for food production.

The O-Antigen Epitope Governs Susceptibility to Colistin in Salmonella enterica.

Group D and group B Salmonella enterica serovars differ in their susceptibility to colistin with the former frequently intrinsically resistant (MIC > 2 µg/ml); however, the mechanism has not been described. Here, we show that the O-antigen epitope in group D Salmonella governs the levels of colistin susceptibility. Substitution of the rfbJ gene in a group B Salmonella with the rfbSE genes from a group D Salmonella conferred a decrease in susceptibility to colistin. The presence of dideoxyhexose, abequose, and the deoxymannose, tyvelose, differentiate the Salmonella group B and group D O antigens, respectively. We hypothesize that the subtle difference between abequose and tyvelose hinders the colistin molecule from reaching its target. Whole-genome sequencing also revealed that increased colistin susceptibility in a group D Salmonella veterinary isolate was due to a defect in the O-antigen polymerase protein, Rfc. This study shows that two different mechanisms that influence the presence and composition of O antigens affect colistin susceptibility in Salmonella entericaIMPORTANCE Some serovars of Salmonella, namely, those belonging to group D, appear to show a degree of intrinsic resistance to colistin. This observed intrinsic colistin resistance is of concern since this last-resort drug might no longer be effective for treating severe human infections with the most common Salmonella serovar, Salmonella enterica serovar Enteritidis. Here, we show that the O-antigen epitope in group D Salmonella governs the levels of colistin susceptibility. Using whole-genome sequencing, we also revealed that increased colistin susceptibility in a group D Salmonella veterinary isolate was due to a defect in the O-antigen polymerase protein, Rfc. In summary, we show that two different mechanisms that influence the presence and composition of O antigens affect colistin susceptibility in Salmonella enterica.

New Multidrug Efflux Inhibitors for Gram-Negative Bacteria.

Active efflux of antibiotics preventing their accumulation to toxic intracellular concentrations contributes to clinically relevant multidrug resistance. Inhibition of active efflux potentiates antibiotic activity, indicating that efflux inhibitors could be used in combination with antibiotics to reverse drug resistance. Expression of ramA by Salmonella enterica serovar Typhimurium increases in response to efflux inhibition, irrespective of the mode of inhibition. We hypothesized that measuring ramA promoter activity could act as a reporter of efflux inhibition. A rapid, inexpensive, and high-throughput green fluorescent protein (GFP) screen to identify efflux inhibitors was developed, validated, and implemented. Two chemical compound libraries were screened for compounds that increased GFP production. Fifty of the compounds in the 1,200-compound Prestwick chemical library were identified as potential efflux inhibitors, including the previously characterized efflux inhibitors mefloquine and thioridazine. There were 107 hits from a library of 47,168 proprietary compounds from L. Hoffmann La Roche; 45 were confirmed hits, and a dose response was determined. Dye efflux and accumulation assays showed that 40 Roche and three Prestwick chemical library compounds were efflux inhibitors. Most compounds had specific efflux-inhibitor-antibiotic combinations and/or species-specific synergy in antibiotic disc diffusion and checkerboard assays performed with Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Salmonella Typhimurium. These data indicate that both narrow-spectrum and broad-spectrum combinations of efflux inhibitors with antibiotics can be found. Eleven novel efflux inhibitor compounds potentiated antibiotic activities against at least one species of Gram-negative bacteria, and data revealing an E. coli mutant with loss of AcrB function suggested that these are AcrB inhibitors.IMPORTANCE Multidrug-resistant Gram-negative bacteria pose a serious threat to human and animal health. Molecules that inhibit multidrug efflux offer an alternative approach to resolving the challenges caused by antibiotic resistance, by potentiating the activity of old, licensed, and new antibiotics. We have developed, validated, and implemented a high-throughput screen and used it to identify efflux inhibitors from two compound libraries selected for their high chemical and pharmacological diversity. We found that the new high-throughput screen is a valuable tool to identify efflux inhibitors, as evidenced by the 43 new efflux inhibitors described in this study.

Chlorpromazine and Amitriptyline Are Substrates and Inhibitors of the AcrB Multidrug Efflux Pump.

Efflux is an important mechanism in Gram-negative bacteria conferring multidrug resistance. Inhibition of efflux is an encouraging strategy to restore the antibacterial activity of antibiotics. Chlorpromazine and amitriptyline have been shown to behave as efflux inhibitors. However, their mode of action is poorly understood. Exposure of Salmonella enterica serovar Typhimurium and Escherichia coli to chlorpromazine selected for mutations within genes encoding RamR and MarR, regulators of the multidrug tripartite efflux pump AcrAB-TolC. Further experiments with S. Typhimurium containing AcrB D408A (a nonfunctional efflux pump) and chlorpromazine or amitriptyline resulted in the reversion of the mutant acrB allele to the wild type. Together, this suggests these drugs are AcrB efflux substrates. Subsequent docking studies with AcrB from S. Typhimurium and E. coli, followed by molecular dynamics simulations and free energy calculations showed that chlorpromazine and amitriptyline bind at the hydrophobic trap, a preferred binding site for substrates and inhibitors within the distal binding pocket of AcrB. Based on these simulations, we suggest that chlorpromazine and amitriptyline inhibit AcrB-mediated efflux by interfering with substrate binding. Our findings provide evidence that these drugs are substrates and inhibitors of AcrB, yielding molecular details of their mechanism of action and informing drug discovery of new efflux inhibitors.IMPORTANCE Efflux pumps of the resistance nodulation-cell division (RND) superfamily are major contributors to multidrug resistance for most of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. The development of inhibitors of these pumps would be highly desirable; however, several issues have thus far hindered all efforts at designing new efflux inhibitory compounds devoid of adverse effects. An alternative route to de novo design relies on the use of marketed drugs, for which side effects on human health have been already assessed. In this work, we provide experimental evidence that the antipsychotic drugs chlorpromazine and amitriptyline are inhibitors of the AcrB transporter, the engine of the major RND efflux pumps in Escherichia coli and Salmonella enterica serovar Typhimurium. Furthermore, in silico calculations have provided a molecular-level picture of the inhibition mechanism, allowing rationalization of experimental data and paving the way for similar studies with other classes of marketed compounds.

Perturbed structural dynamics underlie inhibition and altered efflux of the multidrug resistance pump AcrB.

Resistance-nodulation-division efflux pumps play a key role in inherent and evolved multidrug resistance in bacteria. AcrB, a prototypical member of this protein family, extrudes a wide range of antimicrobial agents out of bacteria. Although high-resolution structures exist for AcrB, its conformational fluctuations and their putative role in function are largely unknown. Here, we determine these structural dynamics in the presence of substrates using hydrogen/deuterium exchange mass spectrometry, complemented by molecular dynamics simulations, and bacterial susceptibility studies. We show that an efflux pump inhibitor potentiates antibiotic activity by restraining drug-binding pocket dynamics, rather than preventing antibiotic binding. We also reveal that a drug-binding pocket substitution discovered within a multidrug resistant clinical isolate modifies the plasticity of the transport pathway, which could explain its altered substrate efflux. Our results provide insight into the molecular mechanism of drug export and inhibition of a major multidrug efflux pump and the directive role of its dynamics.

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

OBJECTIVES: The aim of this study was to understand the role of ramA in conferring multidrug resistance (MDR) in Salmonella enterica serovar Typhimurium. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.