Resistance-nodulation-cell Division Pumps Research Articles

Cryo-EM Structures of AcrD Illuminate a Mechanism for Capturing Aminoglycosides from Its Central Cavity.

The Escherichia coli acriflavine resistance protein D (AcrD) is an efflux pump that belongs to the resistance-nodulation-cell division (RND) superfamily. Its primary function is to provide resistance to aminoglycoside-based drugs by actively extruding these noxious compounds out of E. coli cells. AcrD can also mediate resistance to a limited range of other amphiphilic agents, including bile acids, novobiocin, and fusidic acids. As there is no structural information available for any aminoglycoside-specific RND pump, here we describe cryo-electron microscopy (cryo-EM) structures of AcrD in the absence and presence of bound gentamicin. These structures provide new information about the RND superfamily of efflux pumps, specifically, that three negatively charged residues central to the aminoglycoside-binding site are located within the ceiling of the central cavity of the AcrD trimer. Thus, it is likely that AcrD is capable of picking up aminoglycosides via this central cavity. Through the combination of cryo-EM structural determination, mutagenesis analysis, and molecular simulation, we show that charged residues are critically important for this pump to shuttle drugs directly from the central cavity to the funnel of the AcrD trimer for extrusion. IMPORTANCE Here, we report cryo-EM structures of the AcrD aminoglycoside efflux pump in the absence and presence of bound gentamicin, posing the possibility that this pump is capable of capturing aminoglycosides from the central cavity of the AcrD trimer. The results indicate that AcrD utilizes charged residues to bind and export drugs, mediating resistance to these antibiotics.

Structural Basis of Peptide-Based Antimicrobial Inhibition of a Resistance-Nodulation-Cell Division Multidrug Efflux Pump.

ABSTRACTBacterial efflux pumps in the resistance-nodulation-cell division (RND) family of Gram-negative bacteria contribute significantly to the development of antimicrobial resistance by many pathogens. In this study, we selected the MtrD transporter protein of Neisseria gonorrhoeae as it is the sole RND pump possessed by this strictly human pathogen and can export multiple antimicrobials, including antibiotics, bile salts, detergents, dyes, and antimicrobial peptides. Using knowledge from our previously published structures of MtrD in the presence or absence of bound antibiotics as a model and the known ability of MtrCDE to export cationic antimicrobial peptides, we hypothesized that cationic peptides could be accommodated within MtrD binding sites. Furthermore, we thought that MtrD-bound peptides lacking antibacterial action could sensitize bacteria to an antibiotic normally exported by the MtrCDE efflux pump or other similar RND-type pumps possessed by different Gram-negative bacteria. We now report the identification of a novel nonantimicrobial cyclic cationic antimicrobial peptide, which we termed CASP (cationic antibiotic-sensitizing peptide). By single-particle cryo-electron microscopy, we found that CASP binds within the periplasmic cleft region of MtrD using overlapping and distinct amino acid contact sites that interact with another cyclic peptide (colistin) or a linear human cationic antimicrobial peptide derived from human LL-37. While CASP could not sensitize Neisseria gonorrhoeae to an antibiotic (novobiocin) that is a substrate for RND pumps, it could do so against multiple Gram-negative, rod-shaped bacteria. We propose that CASP (or future derivatives) could serve as an adjuvant for the antibiotic treatment of certain Gram-negative infections previously thwarted by RND transporters.IMPORTANCE RND efflux pumps can export numerous antimicrobials that enter Gram-negative bacteria, and their action can reduce the efficacy of antibiotics and provide decreased susceptibility to various host antimicrobials. Here, we identified a cationic antibiotic-sensitizing peptide (CASP) that binds within the periplasmic cleft of an RND transporter protein (MtrD) produced by Neisseria gonorrhoeae. Surprisingly, CASP was able to render rod-shaped Gram-negative bacteria, but not gonococci, susceptible to an antibiotic that is a substrate for the gonococcal MtrCDE efflux pump. CASP (or its future derivatives) could be used as an adjuvant to treat infections for which RND efflux contributes to multidrug resistance.

Evaluation of Delafloxacin against a Burkholderia pseudomallei Efflux Mutant Panel.

ABSTRACTIn vitro activities of delafloxacin and ciprofloxacin were evaluated against Burkholderia pseudomallei mutants expressing or lacking defined resistance-nodulation-cell division (RND) efflux pumps using CLSI methodology at pHs of 5.8 and 7.2. Delafloxacin MIC values were as much as 8-fold lower at pH 5.8 than those at pH 7.2, while ciprofloxacin MICs increased as much as 8-fold. The data from this study suggest that compared to ciprofloxacin, delafloxacin may have improved efflux avoidance, notably at acidic pH. In contrast to ciprofloxacin, delafloxacin may thus retain its therapeutic potential, even in BpeEF-OprC efflux-pump-expressing B. pseudomallei strains that compromise the use of fluoroquinolones, such as ciprofloxacin.IMPORTANCE Resistance-nodulation-cell division (RND) efflux pumps play a major role in intrinsic and acquired antibiotic resistance in Burkholderia pseudomallei, and these pumps are its only known multidrug resistance determinants. Fluoroquinolones have performed poorly in clinical settings and are currently not recommended for treatment of B. pseudomallei infections. While the reasons for the poor clinical performance of this pathogen remain unclear, efflux may be partially responsible since fluoroquinolones like ciprofloxacin are prone to efflux by RND pumps, notably BpeEF-OprC. In vitro efficacy testing using a panel of efflux-proficient and efflux-deficient strains allows identification of fluoroquinolones that compared to ciprofloxacin are less prone to efflux.

Characterization of substituted piperazines able to reverse MDR in Escherichia coli strains overexpressing resistance-nodulation-cell division (RND) efflux pumps.

MDR in bacteria is threatening to public health. Overexpression of efflux pumps is an important cause of MDR. The co-administration of antimicrobial drugs and efflux pump inhibitors (EPIs) is a promising approach to address the problem of MDR. To identify new putative EPIs and to characterize their mechanisms of action. The effects of four selected piperazine derivatives on resistance-nodulation-cell division (RND) pumps was evaluated in Escherichia coli strains overexpressing or not expressing RND pumps by assays aimed at evaluating antibiotic potentiation, membrane functionality, ethidium bromide accumulation and AcrB expression. The cytotoxicity of selected piperazines towards primary cultures of human dermal fibroblasts was also investigated. Four molecules enhanced levofloxacin activity against strains overexpressing RND efflux pumps (AcrAB-TolC and AcrEF-TolC), but not against RND pump-deficient strains. They had little effects on membrane potential. Molecule 4 decreased, whereas the other three increased, membrane permeability compared with untreated control cells. The four molecules showed differences in the specificity of interaction with RND efflux pumps, by inactivating the transport of one or more antibiotics, and in the levels of ethidium bromide accumulation and of acrB expression inhibition. Piperazine derivatives are good candidates as inhibitors of RND efflux pumps. They decreased the activity of RND pumps by mixed mechanisms of action. Small structural differences among the molecules can be critical in defining their behaviour.

Role of RND Efflux Pumps in Drug Resistance of Cystic Fibrosis Pathogens.

Drug resistance represents a great concern among people with cystic fibrosis (CF), due to the recurrent and prolonged antibiotic therapy they should often undergo. Among Multi Drug Resistance (MDR) determinants, Resistance-Nodulation-cell Division (RND) efflux pumps have been reported as the main contributors, due to their ability to extrude a wide variety of molecules out of the bacterial cell. In this review, we summarize the principal RND efflux pump families described in CF pathogens, focusing on the main Gram-negative bacterial species (Pseudomonas aeruginosa, Burkholderia cenocepacia, Achromobacter xylosoxidans, Stenotrophomonas maltophilia) for which a predominant role of RND pumps has been associated to MDR phenotypes.

Coordinate overexpression of two RND efflux systems, ParXY and TtgABC, is responsible for multidrug resistance in Pseudomonas putida.

Resistance Nodulation cell Division (RND) efflux pumps are known to contribute to the tolerance of Pseudomonas putida to aromatic hydrocarbons, but their role in antibiotic resistance has not been fully elucidated. In this study, two types of single-step multidrug-resistant (MDR) mutants were selected in vitro from reference strain KT2440. Mutants of the first type were more resistant to fluoroquinolones and β-lactams except imipenem, and overproduced the efflux system TtgABC as a result of mutations occurring in regulator TtgR. In addition to TtgABC, mutants of the second type such as HPG-5 were found to upregulate a novel RND pump, dubbed ParXY/TtgC, which accommodates cefepim, fluoroquinolones and aminoglycosides. As demonstrated by gene deletion experiments, TtgABC and ParXY/TtgC are both under the positive control of a two-component system, PpeRS. Whole-genome sequence analyses revealed that mutant HPG-5 harbours a mutation inactivating the gene (sucD) of succinyl-CoA synthetase, an enzyme of the tricarboxylic cycle. Disruption of sucD in strain KT2440 reproduced the resistance phenotype of HPG-5, and activated the glyoxylate shunt. Finally, identification of two MDR clinical strains of P. putida that jointly overexpress TtgABC and ParXY/TtgC, of which one is a sucD mutant, highlights the role of these efflux systems as determinants of antibiotic resistance.

Genome-facilitated discovery of RND efflux pump-mediated resistance to cephalosporins in Vibrio spp. isolated from the mummichog fish gut.

This study examined the role of resistance-nodulation-cell division (RND) efflux pumps in resistance to first-generation and third-generation cephalosporins, and the potential contribution to increased virulence in two Vibrio isolates from the gut microbiota of a forage-feeder fish. Phenotypic MIC testing was performed in the presence and absence of an RND efflux pump inhibitor, phenylalanine-arginine-beta-napthylamide (PAβN). Genomes of the two Vibrio spp. were compared to characterise RND efflux pump gene homologs. The study identified 13 and 12 RND operons, respectively, in Vibrio spp. T21 and T9, with Vibrio sp. T21 containing an additional RND operon compared with other V. parahaemolyticus strains. Both the inner-membrane protein (IMP) and the membrane facilitator protein (MFP) sequences of this operon were homologous to VexD and VexC, respectively, which is an RND operon in Vibrio cholerae. More generally, the other RND proteins in these strains showed homology to RND efflux pumps characterised in Escherichia coli and Vibrio cholerae. Decreased resistance to cefoperazone and cephradine was observed in Vibrio sp. T21, and to cefoperazone and cefsulodin in Vibrio sp. T9 in the presence of PaβN. The RND pumps may also mediate transport of kanamycin. By analysing the genomes of two Vibrio spp. isolated from the mummichog fish gut, RND efflux pump-mediated resistance to first-generation and third-generation cephalosporins was discovered in these strains. This work highlights the need for further research into this unique Vibrio spp. operon and, more generally, RND efflux pumps in Vibrio spp., as Vibrio spp. often cause seafood-borne illness.

Substrate Specificities and Efflux Efficiencies of RND Efflux Pumps of Acinetobacter baumannii.

Antibiotic-resistant Acinetobacter baumannii causes infections that are extremely difficult to treat. A significant role in these resistance profiles is attributed to multidrug efflux pumps, especially those belonging to the resistance-nodulation-cell division (RND) superfamily of transporters. In this study, we analyzed functions and properties of RND efflux pumps in A. baumannii ATCC 17978. This strain is susceptible to antibiotics and does not contain mutations that are commonly selected upon exposure to high concentrations of antibiotics. We constructed derivatives of ATCC 17978 lacking chromosomally encoded RND pumps and complemented these strains by the plasmid-borne genes. We analyzed the substrate selectivities and efficiencies of the individual pumps in the context of native outer membranes and their hyperporinated variants. Our results show that inactivation of AdeIJK provides the strongest potentiation of antibiotic activities, whereas inactivation of AdeFGH triggers the overexpression of AdeAB. The plasmid-borne overproduction complements the hypersusceptible phenotypes of the efflux deletion mutants to the levels of the parental ATCC 17978. Only a few antibiotics strongly benefitted from the overproduction of efflux pumps and antibacterial activities of some of those depended on the synergistic interaction with the low permeability barrier of the outer membrane. Either overproduction or inactivation of efflux pumps change dramatically the lipidome of ATCC 17978. We conclude that efflux pumps of A. baumannii are tightly integrated into physiology of this bacterium and that clinical levels of antibiotic resistance in A. baumannii isolates are unlikely to be reached solely due to the overproduction of RND efflux pumps.IMPORTANCE RND-type efflux pumps are important contributors in development of clinical antibiotic resistance in A. baumannii However, their specific roles and the extent of contribution to antibiotic resistance remain unclear. We analyzed antibacterial activities of antibiotics in strains with different permeability barriers and found that the role of active efflux in antibiotic resistance of A. baumannii is limited to a few select antibiotics. Our results further show that the impact of efflux pump overproduction on antibiotic susceptibility is significantly lower than the previously reported for clinical isolates. Additional mechanisms of resistance, in particular those that improve the permeability barriers of bacterial cells and act synergistically with active efflux pumps are likely involved in antibiotic resistance of clinical A. baumannii isolates.

Subfunctionalization influences the expansion of bacterial multidrug antibiotic resistance

BackgroundAntibiotic resistance is a major problem for human health. Multidrug resistance efflux pumps, especially those of the Resistance-Nodulation-Cell Division (RND) family, are major contributors to high-level antibiotic resistance in Gram-negative bacteria. Most bacterial genomes contain several copies of the different classes of multidrug resistance efflux pumps. Gene duplication and gain of function by the duplicate copies of multidrug resistance efflux pump genes plays a key role in the expansion and diversification of drug-resistance mechanisms.ResultsWe used two members of the Burkholderia RND superfamily as models to understand how duplication events affect the antibiotic resistance of these strains. First, we analyzed the conservation and distribution of these two RND systems and their regulators across the Burkholderia genus. Through genetic manipulations, we identified both the exact substrate range of these transporters and their eventual interchangeability. We also performed a directed evolution experiment, combined with next generation sequencing, to evaluate the role of antibiotics in the activation of the expression of these systems. Together, our results indicate that the first step to diversify the functions of these pumps arises from changes in their regulation (subfunctionalization) instead of functional mutations. Further, these pumps could rewire their regulation to respond to antibiotics, thus maintaining high genomic plasticity.ConclusionsStudying the regulatory network that controls the expression of the RND pumps will help understand and eventually control the development and expansion of drug resistance.

Effect of efflux pump inhibition on Pseudomonas aeruginosa transcriptome and virulence

Efflux pumps of the resistance-nodulation-cell-division (RND) family increase antibiotic resistance in many bacterial pathogens, representing candidate targets for the development of antibiotic adjuvants. RND pumps have also been proposed to contribute to bacterial infection, implying that efflux pump inhibitors (EPIs) could also act as anti-virulence drugs. Nevertheless, EPIs are usually investigated only for their properties as antibiotic adjuvants, while their potential anti-virulence activity is seldom taken into account. In this study it is shown that RND efflux pumps contribute to Pseudomonas aeruginosa PAO1 pathogenicity in an insect model of infection, and that the well-characterized EPI Phe-Arg-β-naphthylamide (PAβN) is able to reduce in vivo virulence of the P. aeruginosa PAO1 laboratory strain, as well as of clinical isolates. The production of quorum sensing (QS) molecules and of QS-dependent virulence phenotypes is differentially affected by PAβN, depending on the strain. Transcriptomic and phenotypic analyses showed that the protection exerted by PAβN from P. aeruginosa PAO1 infection in vivo correlates with the down-regulation of key virulence genes (e.g. genes involved in iron and phosphate starvation). Since PAβN impacts P. aeruginosa virulence, anti-virulence properties of EPIs are worthy to be explored, taking into account possible strain-specificity of their activity.

Contribution of the Ade Resistance-Nodulation-Cell Division-Type Efflux Pumps to Fitness and Pathogenesis of Acinetobacter baumannii.

ABSTRACTOverexpression of chromosomal resistance-nodulation-cell division (RND)-type efflux systems with broad substrate specificity contributes to multidrug resistance (MDR) in Acinetobacter baumannii. We have shown that modulation of expression of the structural genes for the efflux systems AdeABC and AdeIJK confers MDR and results in numerous alterations of membrane-associated cellular functions, in particular biofilm formation. However, the contribution of these RND pumps to cell fitness and virulence has not yet been studied. The biological cost of an antibiotic resistance mechanism is a key parameter in determining its stability and dissemination. From an entirely sequenced susceptible clinical isolate, we have generated a set of isogenic derivatives having single point mutations resulting in overexpression of each efflux system or with every pump deleted by allelic replacement. We found that overproduction of the pumps results in a significant decrease in fitness of the bacterial host when measured by competition experiments in vitro. Fitness and virulence were also evaluated in vivo both in systemic and pulmonary infection models in immunocompetent mice. A diminished competitiveness of the AdeABC-overexpressing mutant was observed only after intraperitoneal inoculation, but not after intranasal inoculation, the latter mimicking the most frequent type of human A. baumannii infection. However, in mice infected intranasally, this mutant was more virulent and stimulated an enhanced neutrophil activation in the lungs. Altogether, these data account for the observation that adeABC overexpression is common in MDR A. baumannii frequently found in ventilator-associated pneumonia.

Efflux pump-mediated drug resistance in Burkholderia.

Several members of the genus Burkholderia are prominent pathogens. Infections caused by these bacteria are difficult to treat because of significant antibiotic resistance. Virtually all Burkholderia species are also resistant to polymyxin, prohibiting use of drugs like colistin that are available for treatment of infections caused by most other drug resistant Gram-negative bacteria. Despite clinical significance and antibiotic resistance of Burkholderia species, characterization of efflux pumps lags behind other non-enteric Gram-negative pathogens such as Acinetobacter baumannii and Pseudomonas aeruginosa. Although efflux pumps have been described in several Burkholderia species, they have been best studied in Burkholderia cenocepacia and B. pseudomallei. As in other non-enteric Gram-negatives, efflux pumps of the resistance nodulation cell division (RND) family are the clinically most significant efflux systems in these two species. Several efflux pumps were described in B. cenocepacia, which when expressed confer resistance to clinically significant antibiotics, including aminoglycosides, chloramphenicol, fluoroquinolones, and tetracyclines. Three RND pumps have been characterized in B. pseudomallei, two of which confer either intrinsic or acquired resistance to aminoglycosides, macrolides, chloramphenicol, fluoroquinolones, tetracyclines, trimethoprim, and in some instances trimethoprim+sulfamethoxazole. Several strains of the host-adapted B. mallei, a clone of B. pseudomallei, lack AmrAB-OprA, and are therefore aminoglycoside and macrolide susceptible. B. thailandensis is closely related to B. pseudomallei, but non-pathogenic to humans. Its pump repertoire and ensuing drug resistance profile parallels that of B. pseudomallei. An efflux pump in B. vietnamiensis plays a significant role in acquired aminoglycoside resistance. Summarily, efflux pumps are significant players in Burkholderia drug resistance.

Bacterial Multidrug Efflux Transporters

Infections caused by bacteria are a leading cause of death worldwide. Although antibiotics remain a key clinical therapy, their effectiveness has been severely compromised by the development of drug resistance in bacterial pathogens. Multidrug efflux transporters--a common and powerful resistance mechanism--are capable of extruding a number of structurally unrelated antimicrobials from the bacterial cell, including antibiotics and toxic heavy metal ions, facilitating their survival in noxious environments. Transporters of the resistance-nodulation-cell division (RND) superfamily typically assemble as tripartite efflux complexes spanning the inner and outer membranes of the cell envelope. In Escherichia coli, the CusCFBA complex, which mediates resistance to copper(I) and silver(I) ions, is the only known RND transporter specific to heavy metals. Here, we describe the current knowledge of individual pump components of the Cus system, a paradigm for efflux machinery, and speculate on how RND pumps assemble to fight diverse antimicrobials.

Characterization and regulation of the resistance-nodulation-cell division-type multidrug efflux pumps MdtABC and MdtUVW from the fire blight pathogen Erwinia amylovora.

BackgroundThe Gram-negative bacterium Erwinia amylovora is the causal agent of the devastating disease fire blight in rosaceous plants such as apple, pear, quince, raspberry, and cotoneaster. In order to survive and multiply in a host, microbes must be able to circumvent the toxic effects of antimicrobial plant compounds, such as flavonoids and tannins. E. amylovora uses multidrug efflux transporters that recognize and actively export toxic compounds out of the cells. Here, two heterotrimeric resistance-nodulation-cell division (RND)-type multidrug efflux pumps, MdtABC and MdtUVW, from E. amylovora were identified. These RND systems are unusual in that they contain two different RND proteins forming a functional pump.ResultsTo find the substrate specificities of the two efflux systems, we overexpressed the transporters in a hypersensitive mutant lacking the major RND pump AcrB. Both transporters mediated resistance to several flavonoids, fusidic acid and novobiocin. Additionally, MdtABC mediated resistance towards josamycin, bile salts and silver nitrate, and MdtUVW towards clotrimazole. The ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock was reduced. Quantitative RT-PCR analyses revealed that the expression of the transporter genes was induced during infection of apple rootstock. The polyphenolic plant compound tannin, as well as the heavy metal salt tungstate was found to induce the expression of mdtABC. Finally, the expression of the mdtABC genes was shown to be regulated by BaeR, the response regulator of the two-component system BaeSR, a cell envelope stress response system that controls the adaptive responses to changes in the environment.ConclusionsThe expression of MdtABC and MdtUVW is induced during growth of E. amylovora in planta. We identified the plant polyphenol tannin as inducer of mdtABC expression. The reduced ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock suggests that the efflux pumps are involved in resistance to plant antimicrobials, maybe including flavonoids, which were identified as substrates of both pumps. Furthermore, we found that the mdtABC operon belongs to the regulon of the two-component regulator BaeR suggesting a role of this RND transporter in the cell envelope stress response of E. amylovora.

Deciphering the Role of RND Efflux Transporters in Burkholderia cenocepacia

Burkholderia cenocepacia J2315 is representative of a highly problematic group of cystic fibrosis (CF) pathogens. Eradication of B. cenocepacia is very difficult with the antimicrobial therapy being ineffective due to its high resistance to clinically relevant antimicrobial agents and disinfectants. RND (Resistance-Nodulation-Cell Division) efflux pumps are known to be among the mediators of multidrug resistance in Gram-negative bacteria. Since the significance of the 16 RND efflux systems present in B. cenocepacia (named RND-1 to -16) has been only partially determined, the aim of this work was to analyze mutants of B. cenocepacia strain J2315 impaired in RND-4 and RND-9 efflux systems, and assess their role in the efflux of toxic compounds. The transcriptomes of mutants deleted individually in RND-4 and RND-9 (named D4 and D9), and a double-mutant in both efflux pumps (named D4-D9), were compared to that of the wild-type B. cenocepacia using microarray analysis. Microarray data were confirmed by qRT-PCR, phenotypic experiments, and by Phenotype MicroArray analysis. The data revealed that RND-4 made a significant contribution to the antibiotic resistance of B. cenocepacia, whereas RND-9 was only marginally involved in this process. Moreover, the double mutant D4-D9 showed a phenotype and an expression profile similar to D4. The microarray data showed that motility and chemotaxis-related genes appeared to be up-regulated in both D4 and D4–D9 strains. In contrast, these gene sets were down-regulated or expressed at levels similar to J2315 in the D9 mutant. Biofilm production was enhanced in all mutants. Overall, these results indicate that in B. cenocepacia RND pumps play a wider role than just in drug resistance, influencing additional phenotypic traits important for pathogenesis.

Inactivation of MuxABC-OpmB transporter system in Pseudomonas aeruginosa leads to increased ampicillin and carbenicillin resistance and decreased virulence

Resistance-Nodulation-Cell Division (RND) pumps play important roles in bacterial resistance to antibiotics. Pseudomonas aeruginosa is an important human pathogen which exhibits high level resistance to antibiotics. There are total of 12 RND pumps present in the P. aeruginosa PAOl genome. The recently characterized MuxABC-OpmB system has been shown to play a role in resistance to novobiocin, aztreonam, macrolides, and tetracycline in a multiple knockout mutation. In this study, we examined the expression levels of all the 12 RND pump gene clusters and tested the involvement of MuxABC-OpmB in pathogenicity. The results indicated that in addition to the four known constitutively expressed RND pumps, mexAB-oprM, mexGHI-opmD, mexVW, and mexXY, relatively high levels of expression were observed with mexJK and muxABC-opmB in the conditions tested. Inactivation of muxA in the muxABC-opmB operon resulted in elevated resistance to ampicillin and carbenicillin. The mutant also showed attenuated virulence in both Brassica rapa pekinensis and Drosophila melanogaster infection models. The decreased virulence at least in part was due to decreased twitching motility in the mutant. These results indicate that the RND pump MuxABC-OpmB is associated with ampicillin and carbenicillin susceptibility and also involved in pathogenesis in P. aeruginosa.

Expression of resistance-nodulation-cell-division efflux pumps in commonly used Burkholderia pseudomallei strains and clinical isolates from northern Australia

Burkholderia pseudomallei is the aetiological agent of melioidosis. Therapy for this disease is lengthy and limited to only a few antibiotics because of this bacterium's intrinsic antibiotic resistance to many clinically useful antibiotics. These properties of B. pseudomallei may partially be due to expression of efflux pumps of the resistance-nodulation-cell-division (RND) family. The patterns and magnitude of RND efflux pump expression in commonly used strains and clinical isolates of B. pseudomallei from the Royal Darwin Hospital, Darwin, Australia, were assessed in cells grown to late exponential phase using quantitative real-time PCR (qRT-PCR). Expression of the three previously identified RND efflux pumps AmrAB-OprA, BpeAB-OprB and BpeEF-OprC, as well as four other yet uncharacterized pumps, was found to be widespread in the clinical isolates. In 45 of 50 isolates (90%), mRNA was detected for at least one of the seven RND pumps. Of these 45 isolates, 41 (82%) expressed multiple pumps with nine strains expressing all seven pumps tested. While these studies revealed no striking correlation between RND efflux pump expression and clinically significant antibiotic resistance, the data support the notion that RND pumps probably play important roles in this bacterium's physiology, defence against toxic compounds, and perhaps virulence.

PigZ, a TetR/AcrR family repressor, modulates secondary metabolism via the expression of a putative four‐component resistance‐nodulation‐cell‐division efflux pump, ZrpADBC, in Serratia sp. ATCC 39006

The Gram-negative enterobacterium, Serratia sp. ATCC 39006 synthesizes several secondary metabolites, including prodigiosin (Pig) and a carbapenem antibiotic (Car). A complex hierarchical network of regulatory proteins control Pig and Car production. In this study we characterize a TetR family regulator, PigZ, which represses transcription of a divergently transcribed putative resistance-nodulation-cell-division (RND) efflux pump, encoded by zrp (PigZ repressed pump) ADBC, via direct binding to the zrpA-pigZ intergenic region. Unusually, this putative RND pump contains two predicted membrane fusion proteins (MFPs), ZrpA and ZrpD. A mutation in pigZ resulted in multiple phenotypic changes, including exoenzyme production, motility and differential regulation of Pig and Car production. A polar suppressor mutation, within zrpA, restored all tested phenotypes to parental strain levels, indicating that the changes observed are due to the increase in expression of ZrpADBC in the absence of the repressor, PigZ. Genomic deletions of zrpA and zrpD indicate that the MFP ZrpD, but not ZrpA, is essential for activity of the putative pump. Bioinformatic analysis revealed that putative RND efflux pumps encoding two MFP components are not uncommon, particularly among plant-associated, Gram-negative bacteria. In addition, based on phylogenetic analysis, we propose that these pairs of MFPs consist of two distinct subtypes.

Identification and Characterization of TriABC-OpmH, a Triclosan Efflux Pump of Pseudomonas aeruginosa Requiring Two Membrane Fusion Proteins

Pseudomonas aeruginosa achieves high-level (MIC>1 mg/ml) triclosan resistance either by constitutive expression of MexAB-OprM, an efflux pump of the resistance nodulation cell division (RND) family, or expression of MexCD-OprJ, MexEF-OprN, and MexJK-OpmH in regulatory mutants. A triclosan-resistant target enzyme and perhaps other mechanisms probably act synergistically with efflux. To probe this notion, we exposed the susceptible Delta(mexAB-oprM) Delta(mexCD-oprJ) Delta(mexEF-oprN) Delta(mexJK) Delta(mexXY) strain PAO509 to increasing triclosan concentrations and derived a resistant strain, PAO509.5. This mutant overexpressed the PA0156-PA0157-PA0158 pump, which only effluxed triclosan, but not closely related compounds, antibiotics, and divalent cations, and was therefore renamed TriABC. Constitutive expression of the triABC operon was due to a single promoter-up mutation. Deletion of two adjacent genes, pcaR and PA0159, encoding transcriptional regulators had no effect on expression of this operon. TriABC is the only P. aeruginosa RND pump which contains two membrane fusion proteins, TriA and TriB, and both are required for efflux pump function. Probably owing to tight transcriptional coupling of the triABC genes, complementation of individual mutations was only partially achievable. Full complementation was only observed when a complete triABC operon was provided in trans, either in single or multiple copies. TriABC associated with OpmH, but not OprM, for assembly of a functional triclosan efflux pump. TriABC is the fifth RND pump in P. aeruginosa shown to efficiently efflux triclosan, supporting the notion that efflux is the primary mechanism responsible for this bacterium's high intrinsic and acquired triclosan resistance.

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

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 2x the MIC in agar, in the presence and absence of Phe-Arg-beta-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-beta-naphthylamide (20 microg/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.