Family Efflux Pump Research Articles

Identification of novel efflux pump inhibitors for Neisseria gonorrhoeae via multiple ligand-based pharmacophores, e-pharmacophore, molecular docking, density functional theory, and molecular dynamics approaches

Neisseria gonorrhoeae have progressively developed resistance to almost all antibiotics, and it has become imperative to develop novel approaches to combat its multi-drug resistance. Overexpression of the MtrCDE, an RND family efflux pump, is one of the primary causes of antibiotic resistance in the gonococcus and is considered an important target for combating anti-microbial resistance. PaβN, D13–9001, and other EPIs are identified to target the RND efflux pumps, but due to their cytotoxicity, they have failed in clinical trials. Herein, an extensive pharmacophore-based approach was performed to identify novel EPI inhibitors with improved pharmacology/safety profiles. An integrated computational framework comprising pharmacophore generation, virtual screening using HTVS, SP and XP Glide methodology, MM-GBSA analysis, Induced fit docking, QPLD, DFT, ADMET properties calculation, Molecular Dynamics, and MM-PBSA analysis was performed. The comprehensive study leads to the identification of five non-toxic bioactive compounds, namely - ZINC000008764610, ZINC000030879142, ZINC000030879358, ZINC000253414904, and ZINC000225394671, as potential EPIs for RND efflux pump of Neisseria gonorrhoeae. The five compounds were selected based on the pharmacophore mapping, higher dock score than the known EPIs, binding stability, molecular interactions with the critical residues of MtrD protein, higher ADMET properties, non-toxicity, and free energy estimations. In summary, the analysis led to the identification of five top hits from the natural compound subset of the ZINC database that has a higher binding affinity to the MtrD and adequate physiochemical/pharmacokinetic profile that can be used for the generation of novel EPIs against Neisseria gonorrhoeae.

Mathematical Modelling Highlights the Potential for Genetic Manipulation as an Adjuvant to Counter Efflux-Mediated MDR in Salmonella

Bacteria have developed resistance to antibiotics by various mechanisms, notable amongst these is the use of permeation barriers and the expulsion of antibiotics via efflux pumps. The resistance-nodulation-division (RND) family of efflux pumps is found in Gram-negative bacteria and a major contributor to multidrug resistance (MDR). In particular, Salmonella encodes five RND efflux pump systems: AcrAB, AcrAD, AcrEF, MdsAB and MdtAB which have different substrate ranges including many antibiotics. We produce a spatial partial differential equation (PDE) model governing the diffusion and efflux of antibiotic in Salmonella, via these RND efflux pumps. Using parameter fitting techniques on experimental data, we are able to establish the behaviour of multiple wild-type and efflux mutant Salmonella strains, which enables us to produce efflux profiles for each individual efflux pump system. By combining the model with a gene regulatory network (GRN) model of efflux regulation, we simulate how the bacteria respond to their environment. Finally, performing a parameter sensitivity analysis, we look into various different targets to inhibit the efflux pumps. The model provides an in silico framework with which to test these potential adjuvants to counter MDR.

A Model for Allosteric Communication in Drug Transport by the AcrAB-TolC Tripartite Efflux Pump.

RND family efflux pumps are complex macromolecular machines involved in multidrug resistance by extruding antibiotics from the cell. While structural studies and molecular dynamics simulations have provided insights into the architecture and conformational states of the pumps, the path followed by conformational changes from the inner membrane protein (IMP) to the periplasmic membrane fusion protein (MFP) and to the outer membrane protein (OMP) in tripartite efflux assemblies is not fully understood. Here, we investigated AcrAB-TolC efflux pump’s allostery by comparing resting and transport states using difference distance matrices supplemented with evolutionary couplings data and buried surface area measurements. Our analysis indicated that substrate binding by the IMP triggers quaternary level conformational changes in the MFP, which induce OMP to switch from the closed state to the open state, accompanied by a considerable increase in the interface area between the MFP subunits and between the OMPs and MFPs. This suggests that the pump’s transport-ready state is at a more favourable energy level than the resting state, but raises the puzzle of how the pump does not become stably trapped in a transport-intermediate state. We propose a model for pump allostery that includes a downhill energetic transition process from a proposed ‘activated’ transport state back to the resting pump.

Comparative analysis of carbapenemases, RND family efflux pumps and biofilm formation potential among Acinetobacter baumannii strains with different carbapenem susceptibility

AimThis study has conducted a comparative analysis of common carbapenemases harboring, the expression of resistance-nodulation-cell division (RND) family efflux pumps, and biofilm formation potential associated with carbapenem resistance among Acinetobacter baumannii (A. baumannii) strains with different carbapenem susceptibility. Methods: A total of 90 isolates of A. baumannii from two tertiary hospitals of China were identified and grouped as carbapenem susceptible A. baumannii (CSAB) strains and carbapenem non-susceptible A. baumannii (CnSAB) strains based on the susceptibility to imipenem. Harboring of carbapenemase genes, relative expression of RND family efflux pumps and biofilm formation potential were compared between the two groups. Result: Among these strains, 12 (13.3 %) strains were divided into the CSAB group, and 78 (86.7 %) strains into the CnSAB group. Compared with CSAB strains, CnSAB strains increased distribution of blaOXA−23 (p < 0.001) and ISAba1/blaOXA−51−like (p = 0.034) carbapenemase genes, and a 6.1-fold relative expression of adeB (p = 0.002), while CSAB strains led to biofilm formation by 1.3-fold than CnSAB strains (p = 0.021).ConclusionsClinically, harboring more blaOXA−23−like and ISAba1/blaOXA−51−like complex genes and overproduction of adeABC are relevant with carbapenem resistance, while carbapenem susceptible strains might survive the stress of antibiotic through their ability of higher biofilm formation.

Insight into the AcrAB-TolC Complex Assembly Process Learned from Competition Studies.

The RND family efflux pump AcrAB-TolC in E. coli and its homologs in other Gram-negative bacteria are major players in conferring multidrug resistance to the cells. While the structure of the pump complex has been elucidated with ever-increasing resolution through crystallography and Cryo-EM efforts, the dynamic assembly process remains poorly understood. Here, we tested the effect of overexpressing functionally defective pump components in wild type E. coli cells to probe the pump assembly process. Incorporation of a defective component is expected to reduce the efflux efficiency of the complex, leading to the so called “dominant negative” effect. Being one of the most intensively studied bacterial multidrug efflux pumps, many AcrA and AcrB mutations have been reported that disrupt efflux through different mechanisms. We examined five groups of AcrB and AcrA mutants, defective in different aspects of assembly and substrate efflux. We found that none of them demonstrated the expected dominant negative effect, even when expressed at concentrations many folds higher than their genomic counterpart. The assembly of the AcrAB-TolC complex appears to have a proof-read mechanism that effectively eliminated the formation of futile pump complex.

Efflux Pump and its Inhibitors: Novel Targets to Combat Drug Resistance

The rapid emergence of resistant bacteria is occurring worldwide, endangering the efficacy of antibiotics, which have transformed medicine and saved millions of lives. Antibiotic resistance has become a major clinical problem today. In addition, multidrug resistance also develops various structurally and functionally unrelated toxic compounds. To overcome this problem, a new target efflux pump was recognized that, if inhibited, reduces the level of resistance or potentiates or produces a synergistic effect in combination with antibiotics. Keeping this in view, the present review article aims to describe the families of efflux pumps and the various natural components to be employed as good efflux pump inhibitors.

Identification and Transferability of Tetracycline Resistance in Streptococcus thermophilus during Milk Fermentation, Storage, and Gastrointestinal Transit

The existence of antibiotic-resistant bacteria in food products, particularly those carrying acquired resistance genes, has increased concerns about the transmission of these genes from beneficial microbes to human pathogens. In this study, we evaluated the antibiotic resistance-susceptibility patterns of 16 antibiotics in eight S. thermophilus strains, whose genome sequence is available, using phenotypic and genomic approaches. The minimal inhibitory concentration values collected revealed intermediate resistance to aminoglycosides, whereas susceptibility was detected for different classes of β-lactams, quinolones, glycopeptide, macrolides, and sulfonamides in all strains. A high tetracycline resistance level has been detected in strain M17PTZA496, whose genome analysis indicated the presence of the tet(S) gene and the multidrug and toxic compound extrusion (MATE) family efflux pump. Moreover, an in-depth genomic analysis revealed genomic islands and an integrative and mobilizable element (IME) in the proximity of the gene tet(S). However, despite the presence of a prophage, genomic islands, and IME, no horizontal gene transfer was detected to Lactobacillus delbrueckii subsp. lactis DSM 20355 and Lactobacillusrhamnosus GG during 24 h of skim milk fermentation, 2 weeks of refrigerated storage, and 4 h of simulated gastrointestinal transit.

RND family efflux pumps: from antibioresistance to chemotherapy resistance

Resistance to chemotherapy can be studied comparatively to the study of resistance in microorganisms. For over 40 years, understanding mechanisms that confer MDR has been a major goal of cancer biologists. Most of the studies toward MDR in cancer cells were about ABC transporters. Unfortunately, inhibition of these transporters often resulted in over toxicity due to the important role of these ABC transporters in healthy cells. The discovery of other targets for MDR of resistant cancer cells is of significant interest. Among the protein superfamily identified as being responsible for multidrug resistance are RND. Its members are widespread in bacterial organisms, but also in Archaea and Eukaryotes. Among the common features of multidrug resistance in RND is the ability of these transmembrane proteins to efflux a broad spectrum of substrates and drugs using the proton motive force. Ptch1, member of the RND family, is overexpressed in many aggressive and metastatic cancers. Like other members of the RND family such as NPC1, it is able to transport cholesterol. It was later shown to transport chemotherapeutic drugs, and its inhibition in resistant cancer cell lines resulted in increasing chemotherapeutic treatment efficacy. However, the drug efflux mechanism of Ptch1 is still unknown. In this review, we will discuss the possibility of a drug efflux mechanism common to the different proteins from the RND family.

&lt;p&gt;Efflux pumps and biofilm formation by both methicillin-resistant and methicillin-sensitive &lt;em&gt;Staphylococcus aureus&lt;/em&gt; strains&lt;/p&gt;

Staphylococcus aureus is the primary cause of nosocomial infections. It produces potent toxins and causes superficial lesions, systemic diseases, and several toxaemic syndromes. In this study, we determined the role of efflux pump in conferring resistance to fluoroquinolones in the biofilm of methicillin resistant Staphylococcus aureus (MRSA) and methicillin sensitive S. aureus (MSSA) strains. We selected five strains of S. aureus comprising three methicillin sensitive strains, including ATCC25923, and two strains of methicillin resistant S . aureus . Thioridazine, chlorpromazine, carbonyl cyanide 3-chloro-phenylhydrazone and reserpine showed activity against MSSA strains. Whereas, thioridazine and chlorpromazine were active against MRSAstrains too. Thioridazine and chlorpromazine showed a significant decrease in biofilm formation of MSSA strain 2493, and MRSA strain UC1079 compared to the control strain ATCC25923. Thioridazine showed a similar effect on MSSA strain N297214 and MRSA strain N307002. Four to eight-fold increase was found in the expression of efflux pump genes with ethidium bromide, ciprofloxacin, and moxifloxacin in these strains. However, a decrease in the expression of norB , norC , abcA and mepA was observed in MSSA, and MRSA strains with thioridazine, chlorpromazine and naringenin. This study, thus demonstrate that the major facilitator superfamily, multidrug and toxin compound extrusion, and ATP binding cassette family of efflux pumps play an essential role in developing antibiotic resistance and biofilm formation. It is the first report on existence of efflux pump genes in biofilm beside antibiotic resistance for fluoroquinolones in MRSA and MSSA strains, which need further characterization

Defining the RND-binding residues of AcrA

The resistance-nodulation-division (RND) family of efflux pumps confer clinically relevant antibiotic resistance in Gram-negative bacteria, such as Salmonella enterica. RND pumps, including AcrB, are organized as tri-partite systems, consisting of an inner membrane RND pump, a periplasmic adaptor protein (PAP) and an outer membrane channel. Previously, inactivation of the PAPs AcrA and AcrE in S. enterica has been shown to significantly increase susceptibility to antimicrobials and reduce virulence. Therefore, PAPs are seen as attractive targets for the development of efflux pump inhibitors. However, the role of PAPs in the assembly of tri-partite pumps and the residues involved in PAP-RND pump binding is poorly understood. In this study, point mutations in the predicted RND binding residues of AcrA were generated by site-directed mutagenesis. The point mutants were characterised phenotypically through ethidium bromide efflux assays and antimicrobial susceptibility testing. Furthermore, Western blotting was used to verify that the phenotypic effect of the point mutations was not due to destabilisation of the AcrA protein. Point mutations in certain residues, such as G58, F292, R294 and G363 were found to significantly impair efflux activity and increase susceptibility to various antibiotics and dyes, suggesting an important role for these AcrA residues in RND pump binding. Western blotting confirmed that these point mutants were stable and exhibited similar expression levels to the wild-type. These residues could be important targets for the design and development of PAP inhibitors to restore the activity of existing antibiotics and reduce virulence of Salmonella.

Observations of Compound Penetration in Escherichia coli using Ethidium Bromide as a Model Compound

According to the Centers for Disease Control and Prevention, approximately 2 million people are afflicted with antibiotic-resistant infections annually. One of the contributing factors to a bacterium's resistance to antimicrobials is the presence of multidrug efflux pumps. By removing various toxins from the host cell (i.e. dyes to antibiotics), these pumps ensure the survival of the pathogen. Little is known regarding the kinetics of these pumps. Found in virtually all types of bacteria, there are multiple families of efflux pumps. This research focuses on AcrAB-TolC, an active efflux pump that is commonly found in Gram-negative bacterium such as Escherichia coli (E. coli). The overall objective of this research is the development of a mathematical model to accurately represent compound penetration into E. coli and the contribution of efflux in the process. Using multiple concentrations of the fluorescent compound ethidium bromide (EtBr) and stopped-flow spectrometry, observations of changes in fluorescence intensity over time were made using a genetically altered strain of E. coli with a non-functioning AcrAB-TolC pump. Graphical representations of data revealed four possible distinctive patterns or “stages” of influx of the EtBr compound into the live cells. Data also suggests the Langmuir model of adsorption as being a potential mathematical model for the first diffusion stage. Development of a working model that allows for accurate determination of the kinetic behavior of the influx of EtBr into the cell allows for a basis of comparison in strains of E. coli with fully functioning efflux pumps. This would allow for the quantification of an efficiency value can then be used to help determine which compounds are most effectively removed by the AcrAB-TolC pump.

Co-existence of a novel plasmid-mediated efflux pump with colistin resistance gene mcr in one plasmid confers transferable multidrug resistance in Klebsiella pneumoniae

ABSTRACT Tigecycline is considered one of the last-resort antimicrobials for carbapenem-resistant K. pneumoniae. Plasmid-mediated tigecycline resistance remains largely unclear. Here, by utilizing whole genome sequencing, we report a plasmid-mediated tigecycline resistance mechanism, a 6,489 bp Resistance-nodulation-division family (RND) efflux pump (tmexCD1-toprJ1 pump), that confers transferable tigecycline resistance in K pneumoniae isolated from patients and chickens. In addition, we identified high prevalence of the plasmids co-harbouring both tmexCD1-toprJ1 pump and mcr (tmexCD1-mcr co-harbouring plasmid) from human in our nationwide collection. Even worse, the tmexCD1-toprJ1 and mcr co-harbouring plasmid was also co-existed with bla NDM-harbouring IncX3 plasmid in the same host, resulting in pandrug resistance. Phylogenetic analysis suggested that the plasmid-borne tmexCD1-toprJ1 originated from the chromosome of Aeromonas spp. through Tn5393-mediating translocation. Both plasmid-harbored tmexCD1-toprJ1 gene and mcr-8 likely originated from animal isolates and then spread to human. Our findings highlight a substantial threat of tmexCD1-toprJ1-mcr8 co-harbouring IncFIA/IncFII plasmid to public health due to their mobile resistance to both tigecycline and colistin, emphasizing an urgent need for further global surveillance on this plasmid.

A Novel Plasmid-Mediated Efflux Pump Gene Confers Transferable Resistance to Tigecycline in &lt;i&gt;Klebsiella pneumoniae&lt;/i&gt; from Human and Animals

Background: Tigecycline is considered one of the last-resort antimicrobials for carbapenem-resistant K. pneumoniae. Plasmid-mediated tigecycline resistance remains largely unclear except for the recently reported tet (X3) and tet (X4). Methods: To investigate the unknown plasmid-mediated mechanism, comparative analysis of HiSeq and PacBio sequencing data were performed on 5 isolates of K. pneumoniae with similar genetic background but different resistance profile to tigecycline. Conjugation assay and subcloning were utilized to confirm the function of the novel plasmid-mediated gene in tigecycline resistance. The prevalence of this gene was screened in 257 tigecycline-non-susceptible K. pneumoniae (TNSKP) from a nationwide collection of 10536 Gram-negative bacilli. Plasmid characteristics were further evaluated by growth curve assay, whole genome sequencing and phylogenetic analysis. Finding: A novel plasmid-mediated tigecycline resistance gene, pmexAB-oprY, a 6, 489bp RND family efflux pump was identified from K. pneumoniae in both patients and animals. Conjugation and transformation experiments indicated that pmexAB-oprY increased tigecycline MICs by 8-16-fold in the recipient without fitness cost. The pump gene was mainly present in IncFIA, IncFIB and IncFII type plasmids, the size of which ranging from 110-kb to 200-kb. Importantly, 7 animal and 3 inpatient isolates carried both pmexAB-oprY and mcr ( mcr-1, mcr-8 · 1, mcr-3 · 11 and a new mcr-8 variant mcr-8 · 5 ), conferring resistance to tigecycline and colistin. Among them, pmexAB-oprY was present with mcr-8 · 1 or mcr-8 · 5 in one single transferable plasmid in 4 animal isolates. Alarmingly, the plasmid-mediated pmexAB-oprY and mcr co-existed with blaNDM -harboring IncX3 plasmid in the same host, resulting in pandrug resistance. Phylogenetic analysis suggested that pmexAB-oprY originated from the chromosome of Aeromonas spp. through Tn5393-mediating translocation to the plasmid. Both plasmid-harbored pmexAB-oprY gene and mcr-8 likely originated from animal isolates and then spread to human. The prevalence of pmexAB-oprY in TNSKP were 52·4% in animals and 2·5% in patients, respectively. Interpretation: Our findings reveal a novel plasmid-mediated mechanism in TNSKP, highlighting a substantial threat of the pmexAB-mcr8 co-harboring IncFIA/ IncFII plasmid to public health due to their mobile resistance. We emphasize the urgent need for a ‘one-health’ strategy and suggest that further global surveillance on the distribution and dissemination of this plasmid is in great need to assess its clinical significance impact on public health. Funding Statement: National Natural Science Foundation for Distinguished Young Scholars of China Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: Patients information were not included in this study; thus, ethical approval was waived (Acceptance number of Ethics Review Committee of Peking University People's Hospital: 2016-PHB-135).

Multiomic Fermentation Using Chemically Defined Synthetic Hydrolyzates Revealed Multiple Effects of Lignocellulose-Derived Inhibitors on Cell Physiology and Xylose Utilization in Zymomonas mobilis.

Utilization of both C5 and C6 sugars to produce biofuels and bioproducts is a key goal for the development of integrated lignocellulosic biorefineries. Previously we found that although engineered Zymomonas mobilis 2032 was able to ferment glucose to ethanol when fermenting highly concentrated hydrolyzates such as 9% glucan-loading AFEX-pretreated corn stover hydrolyzate (9% ACSH), xylose conversion after glucose depletion was greatly impaired. We hypothesized that impaired xylose conversion was caused by lignocellulose-derived inhibitors (LDIs) in hydrolyzates. To investigate the effects of LDIs on the cellular physiology of Z. mobilis during fermentation of hydrolyzates, including impacts on xylose utilization, we generated synthetic hydrolyzates (SynHs) that contained nutrients and LDIs at concentrations found in 9% ACSH. Comparative fermentations of Z. mobilis 2032 using SynH with or without LDIs were performed, and samples were collected for end product, transcriptomic, metabolomic, and proteomic analyses. Several LDI-specific effects were observed at various timepoints during fermentation including upregulation of sulfur assimilation and cysteine biosynthesis, upregulation of RND family efflux pump systems (ZMO0282-0285) and ZMO1429-1432, downregulation of a Type I secretion system (ZMO0252-0255), depletion of reduced glutathione, and intracellular accumulation of mannose-1P and mannose-6P. Furthermore, when grown in SynH containing LDIs, Z. mobilis 2032 only metabolized ∼50% of xylose, compared to ∼80% in SynH without LDIs, recapitulating the poor xylose utilization observed in 9% ACSH. Our metabolomic data suggest that the overall flux of xylose metabolism is reduced in the presence of LDIs. However, the expression of most genes involved in glucose and xylose assimilation was not affected by LDIs, nor did we observe blocks in glucose and xylose metabolic pathways. Accumulations of intracellular xylitol and xylonic acid was observed in both SynH with and without LDIs, which decreased overall xylose-to-ethanol conversion efficiency. Our results suggest that xylose metabolism in Z. mobilis 2032 may not be able to support the cellular demands of LDI mitigation and detoxification during fermentation of highly concentrated lignocellulosic hydrolyzates with elevated levels of LDIs. Together, our findings identify several cellular responses to LDIs and possible causes of impaired xylose conversion that will enable future strain engineering of Z. mobilis.

Alliance of Efflux Pumps with β-Lactamases in Multidrug-Resistant Klebsiella pneumoniae Isolates.

Nosocomial infections caused by Klebsiella pneumoniae are primarily characterized by a high prevalence of extended-spectrum β-lactamases (ESBL's) and a soaring pace of carbapenemase dissemination. Availability of limited antimicrobial agents as a therapeutic option for multidrug-resistant bacteria raises an alarming concern. This study aimed at the molecular characterization of multidrug-resistant K. pneumoniae clinical isolates and studied the role of efflux pumps in β-lactam resistance. Thirty-three isolates confirmed as ESBL-positive K. pneumoniae that harbored resistance genes to major classes of antibiotics. The results showed that CTX-M15 was the preeminent β-lactamase along with carbapenemases in ESBL-positive isolates. However, the efficacy of different antibiotics varied in the presence of lactamase inhibitors and efflux pump inhibitors (EPIs). Those showing increased efficacy of antibiotics with EPI were further explored for the expression of efflux pump genes and expressed a significantly different level of efflux pumps. We found that an isolate had higher expression of kpnF (SMR family) and kdeA (MATE family) pump genes relative to RND family pump genes. No mutations were observed in the genes for porins. Together, the findings suggest that β-lactamases are not the only single factor responsible for providing resistance against the existing β-lactam drugs. Resistance may increase many folds by simultaneous expression of RND family (the most prominent family in Gram-negative bacteria) and other efflux pump family.

Efflux Pumps in Chromobacterium Species Increase Antibiotic Resistance and Promote Survival in a Coculture Competition Model.

Members of the Chromobacterium genus include opportunistic but often-fatal pathogens and soil saprophytes with highly versatile metabolic capabilities. In previous studies of Chromobacterium subtsugae (formerly C. violaceum) strain CV017, we identified a resistance nodulation division (RND)-family efflux pump (CdeAB-OprM) that confers resistance to several antibiotics, including the bactobolin antibiotic produced by the soil saprophyte Burkholderia thailandensis Here, we show the cdeAB-oprM genes increase C. subtsugae survival in a laboratory competition model with B. thailandensis We also demonstrate that adding sublethal bactobolin concentrations to the coculture increases C. subtsugae survival, but this effect is not through CdeAB-OprM. Instead, the increased survival requires a second, previously unreported pump we call CseAB-OprN. We show that in cells exposed to sublethal bactobolin concentrations, the cseAB-oprN genes are transcriptionally induced, and this corresponds to an increase in bactobolin resistance. Induction of this pump is highly specific and sensitive to bactobolin, while CdeAB-OprM appears to have a broader range of antibiotic recognition. We examine the distribution of cseAB-oprN and cdeAB-oprM gene clusters in members of the Chromobacterium genus and find the cseAB-oprN genes are limited to the nonpathogenic C. subtsugae strains, whereas the cdeAB-oprM genes are more widely distributed among members of the Chromobacterium genus. Our results provide new information on the antibiotic resistance mechanisms of Chromobacterium species and highlight the importance of efflux pumps for saprophytic bacteria existing in multispecies communities.IMPORTANCE Antibiotic efflux pumps are best known for increasing antibiotic resistance of pathogens; however, the role of these pumps in saprophytes is much less well defined. This study describes two predicted efflux pump gene clusters in the Chromobacterium genus, which is comprised of both nonpathogenic saprophytes and species that cause highly fatal human infections. One of the predicted efflux pump clusters is present in every member of the Chromobacterium genus and increases resistance to a broad range of antibiotics. The other gene cluster has more narrow antibiotic specificity and is found only in Chromobacterium subtsugae, a subset of entirely nonpathogenic species. We demonstrate the role of both pumps in increasing antibiotic resistance and demonstrate the importance of efflux-dependent resistance induction for C. subtsugae survival in a dual-species competition model. These results have implications for managing antibiotic-resistant Chromobacterium infections and for understanding the evolution of efflux pumps outside the host.

Short-chain diamines are the physiological substrates of PACE family efflux pumps

Acinetobacter baumannii has rapidly emerged as a major cause of gram-negative hospital infections worldwide. A. baumannii encodes for the transport protein AceI, which confers resistance to chlorhexidine, a widely used antiseptic. AceI is also the prototype for the recently discovered proteobacterial antimicrobial compound efflux (PACE) family of transport proteins that confer resistance to a range of antibiotics and antiseptics in many gram-negative bacteria, including pathogens. The gene encoding AceI is conserved in the core genome of A. baumannii, suggesting that it has an important primordial function. This is incongruous with the sole characterized substrate of AceI, chlorhexidine, an entirely synthetic biocide produced only during the last century. Here we investigated a potential primordial function of AceI and other members of the PACE family in the transport of naturally occurring polyamines. Polyamines are abundant in living cells, where they have physiologically important functions and play multifaceted roles in bacterial infection. Gene expression studies revealed that the aceI gene is induced in A. baumannii by the short-chain diamines cadaverine and putrescine. Membrane transport experiments conducted in whole cells of A. baumannii and Escherichia coli and also in proteoliposomes showed that AceI mediates the efflux of these short-chain diamines when energized by an electrochemical gradient. Assays conducted using 8 additional diverse PACE family proteins identified 3 that also catalyze cadaverine transport. Taken together, these results demonstrate that short-chain diamines are common substrates for the PACE family of transport proteins, adding to their broad significance as a novel family of efflux pumps.

SGI-4 in Monophasic Salmonella Typhimurium ST34 Is a Novel ICE That Enhances Resistance to Copper.

A multi drug resistant Salmonella enterica 4,[5],12:i- of sequence type 34 (monophasic S. Typhimurium ST34) is a current pandemic clone associated with livestock, particularly pigs, and numerous outbreaks in the human population. A large genomic island, termed SGI-4, is present in the monophasic Typhimurium ST34 clade and absent from other S. Typhimurium strains. SGI-4 consists of 87 open reading frames including sil and pco genes previously implicated in resistance to copper (Cu) and silver, and multiple genes predicted to be involved in mobilization and transfer by conjugation. SGI-4 was excised from the chromosome, circularized, and transferred to recipient strains of S. Typhimurium at a frequency influenced by stress induced by mitomycin C, and oxygen tension. The presence of SGI-4 was associated with increased resistance to Cu, particularly but not exclusively under anaerobic conditions. The presence of silCBA genes, predicted to encode an RND family efflux pump that transports Cu from the periplasm to the external milieu, was sufficient to impart the observed enhanced resistance to Cu, above that commonly associated with S. Typhimurium isolates. The presence of these genes resulted in the absence of Cu-dependent induction of pco genes encoding multiple proteins linked to Cu resistance, also present on SGI-4, suggesting that the system effectively limits the Cu availability in the periplasm, but did not affect SodCI-dependent macrophage survival.

An EmrB multidrug efflux pump in Burkholderia thailandensis with unexpected roles in antibiotic resistance

The antibiotic trimethoprim is frequently used to manage Burkholderia infections, and members of the resistance-nodulation-division (RND) family of efflux pumps have been implicated in multidrug resistance of this species complex. We show here that a member of the distinct Escherichia coli multidrug resistance B (EmrB) family is a primary exporter of trimethoprim in Burkholderia thailandensis, as evidenced by increased trimethoprim sensitivity after inactivation of emrB, the gene that encodes EmrB. We also found that the emrB gene is up-regulated following the addition of gentamicin and that this up-regulation is due to repression of the gene encoding OstR, a member of the multiple antibiotic resistance regulator (MarR) family. The addition of the oxidants H2O2 and CuCl2 to B. thailandensis cultures resulted in OstR-dependent differential emrB expression, as determined by qRT-PCR analysis. Specifically, OstR functions as a rheostat that optimizes emrB expression under oxidizing conditions, and it senses oxidants by a unique mechanism involving two vicinal cysteines and one distant cysteine (Cys3, Cys4, and Cys169) per monomer. Paradoxically, emrB inactivation increased resistance of B. thailandensis to tetracycline, a phenomenon that correlated with up-regulation of an RND efflux pump. These observations highlight the intricate mechanisms by which expression of genes that encode efflux pumps is optimized depending on cellular concentrations of antibiotics and oxidants.

DNA Binding and Sensor Specificity of FarR, a Novel TetR Family Regulator Required for Induction of the Fatty Acid Efflux Pump FarE in Staphylococcus aureus.

Divergent genes in Staphylococcus aureus USA300 encode the efflux pump FarE and TetR family regulator FarR, which confer resistance to antimicrobial unsaturated fatty acids. To study their regulation, we constructed USA300 ΔfarER, which exhibited a 2-fold reduction in MIC of linoleic acid. farE expressed from its native promoter on pLIfarE conferred increased resistance to USA300 but not USA300 ΔfarER Complementation of USA300 ΔfarER with pLIfarR also had no effect, whereas resistance was restored with pLIfarER or through ectopic expression of farE In electrophoretic mobility shift assays, FarR bound to three different oligonucleotide probes that each contained a TAGWTTA motif, occurring as (i) a singular motif overlapping the -10 element of the P farR promoter, (ii) in palindrome PAL1 immediately in the 3' direction of P farR , or (iii) within PAL2 upstream of the predicted P farE promoter. FarR autorepressed its expression through cooperative binding to PAL1 and the adjacent TAGWTTA motif in P farR Consistent with reports that S. aureus does not metabolize fatty acids through acyl coenzyme A (acyl-CoA) intermediates, DNA binding activity of FarR was not affected by linoleoyl-CoA. Conversely, induction of farE required fatty acid kinase FakA, which catalyzes the first metabolic step in the incorporation of unsaturated fatty acids into phospholipid. We conclude that FarR is needed to promote the expression of farE while strongly autorepressing its own expression, and our data are consistent with a model whereby FarR interacts with a FakA-dependent product of exogenous fatty acid metabolism to ensure that efflux only occurs when the metabolic capacity for incorporation of fatty acid into phospholipid is exceeded.IMPORTANCE Here, we describe the DNA binding and sensor specificity of FarR, a novel TetR family regulator (TFR) in Staphylococcus aureus Unlike the majority of TFRs that have been characterized, which function to repress a divergently transcribed gene, we find that FarR is needed to promote expression of the divergently transcribed farE gene, encoding a resistance-nodulation-division (RND) family efflux pump that is induced in response to antimicrobial unsaturated fatty acids. Induction of farE was dependent on the function of the fatty acid kinase FakA, which catalyzes the first metabolic step in the incorporation of exogenous unsaturated fatty acids into phospholipid. This represents a novel example of TFR function.