Active efflux,a common mechanism for biocide and antibiotic resistance

Energy-driven drug efflux systems are increasingly recognized as mechanisms of antibiotic resistance. Chromosomally located or acquired by bacteria, they can either be activated by environmental signals or by a mutation in a regulatory gene. Two major categories exist: those systems energized by proton motive force and those dependent on ATP. The pumps may have limited or broad substrates, the so-called multiple drug resistance pumps, which themselves form a number of related families. The multiple antibiotic resistance (mar) locus and mar regulon in Escherichia coli and other members of the Enterobacteriaceae is a paradigm for a generalized response locus leading to increased expression of efflux pumps. One such pump, the AcrAB pump extrudes biocides such as triclosan, chlorhexidine and quaternary ammonium compounds as well as multiple antibiotics. In Pseudomonas aeruginosa, a number of multidrug efflux pumps export a broad range of substrates. Since bacteria expressing these pumps thwart the efficacy of both kinds of therapeutic agents which control infectious diseases--biocides which prevent transmission of infectious disease agents and antibiotics which treat and cure infectious diseases--they are of particular concern. The prudent use of antibiotics and biocides will guard against the selection and propagation of drug-resistant mutants and preserve the efficacy of these valuable anti-infective agents.     

Inner membrane efflux components are responsible for beta-lactam specificity of multidrug efflux pumps in Pseudomonas aeruginosa.

A major feature of the MexAB-OprM multidrug efflux pump which distinguishes it from the MexCD-OprJ and MexEF-OprN multidrug efflux systems in Pseudomonas aeruginosa is its ability to export a wide variety of beta-lactam antibiotics. Given the periplasmic location of their targets it is feasible that beta-lactams exit the cell via the outer membrane OprM without interaction with MexA and MexB, though the latter appear to be necessary for OprM function. To test this, chimeric MexAB-OprJ and MexCD-OprM efflux pumps were reconstituted in delta mexCD delta oprM and delta mexAB delta oprJ strains, respectively, and the influence of the exchange of outer membrane components on substrate (i.e., beta-lactam) specificity was assessed. Both chimeric pumps were active in antibiotic efflux, as evidenced by their contributions to resistance to a variety of antimicrobial agents, although there was no change in resistance profiles relative to the native pumps, indicating that OprM is not the determining factor for the beta-lactam specificity of MexAB-OprM. Thus, one or both of inner membrane-associated proteins MexA and MexB are responsible for drug recognition, including recognition of beta-lactams.     

Bacterial multidrug efflux pumps: Mechanisms,physiology and pharmacological exploitations

Multidrug resistance (MDR) refers to the capability of bacterial pathogens to withstand lethal doses of structurally diverse drugs which are capable of eradicating non-resistant strains. MDR has been identified as a major threat to the public health of human being by the World Health Organization (WHO). Among the four general mechanisms that cause antibiotic resistance including target alteration, drug inactivation, decreased permeability and increased efflux, drug extrusion by the multidrug efflux pumps serves as an important mechanism of MDR. Efflux pumps not only can expel a broad range of antibiotics owing to their poly-substrate specificity, but also drive the acquisition of additional resistance mechanisms by lowering intracellular antibiotic concentration and promoting mutation accumulation. Over-expression of multidrug efflux pumps have been increasingly found to be associated with clinically relevant drug resistance. On the other hand, accumulating evidence has suggested that efflux pumps also have physiological functions in bacteria and their expression is subject tight regulation in response to various of environmental and physiological signals. A comprehensive understanding of the mechanisms of drug extrusion, and regulation and physiological functions of efflux pumps is essential for the development of anti-resistance interventions. In this review, we summarize the development of these research areas in the recent decades and present the pharmacological exploitation of efflux pump inhibitors as a promising anti-drug resistance intervention.     

Preventing drug access to targets: cell surface permeability barriers and active efflux in bacteria.

Bacteria, being unicellular, are constantly exposed to toxic compounds in their environment. Gram-negative bacteria and mycobacteria are unusually successful in surviving in the presence of toxic compounds because they combine two mechanisms of resistance. They produce effective permeability barriers, comprising the outer membrane and the mycolate-containing cell wall, on the cell surface. Further, they actively pump out drug molecules that trickle through the barrier, often utilizing multidrug efflux pumps. In Gram-negative bacteria, multidrug pumps of exceptionally wide specificity frequently interact with outer membrane channels and accessory proteins, forming multisubunit complexes that extrude drug molecules directly into the medium, bypassing the outer membrane barrier.     

Synthesis and evaluation of inhibitors of bacterial drug efflux pumps of the major facilitator superfamily

Inhibitors of drug efflux pumps have great potential as pharmacological agents that restore the drug susceptibility of multidrug resistant bacterial pathogens. Most attention has been focused on the discovery of small molecules that inhibit the resistance nodulation division (RND) family drug efflux pumps in Gram-negative bacteria. The prototypical inhibitor of RND-family efflux pumps in Gram-negative bacteria is MC-207,110 (Phe-Arg-β-naphthylamide), a C-capped dipeptide. Here, we report that C-capped dipeptides inhibit two chloramphenicol-specific efflux pumps in Streptomyces coelicolor, a Gram-positive bacterium that is a relative of the human pathogen Mycobacterium tuberculosis. Diversity-oriented synthesis of a library of structurally related C-capped dipeptides via an Ugi four component reaction and screening of the resulting compounds resulted in the discovery of a compound that is threefold more potent as a suppressor of chloramphenicol resistance in S. coelicolor than MC-207,110. Since chloramphenicol resistance in S. coelicolor is mediated by major facilitator superfamily drug efflux pumps, our findings provide the first evidence that C-capped dipeptides can inhibit drug efflux pumps outside of the RND superfamily.     

Bacterial efflux systems and efflux pumps inhibitors

It is now well established that bacterial resistance to antibiotics has become a serious problem of public health that concerns almost all antibacterial agents and that manifests in all fields of their application. Among the three main mechanisms involved in bacterial resistance (target modification, antibiotic inactivation or default of its accumulation within the cell), efflux pumps, responsible for the extrusion of the antibiotic outside the cell, have recently received a particular attention. Actually, these systems, classified into five families, can confer resistance to a specific class of antibiotics or to a large number of drugs, thus conferring a multi-drug resistance (MDR) phenotype to bacteria. To face this issue, it is urgent to find new molecules active against resistant bacteria. Among the strategies employed, the search for inhibitors of resistance mechanisms seems to be attractive because such molecules could restore antibiotic activity. In the case of efflux systems, efflux pump inhibitors (EPIs) are expected to block the pumps and such EPIs, if active against MDR pumps, would be of great interest. This review will focus on the families of bacterial efflux systems conferring drug resistance, and on the EPIs that have been identified to restore antibiotic activity.     

Broad-specificity efflux pumps and their role in multidrug resistance of Gram-negative bacteria

Antibiotic resistance mechanisms reported in Gram-negative bacteria are causing a worldwide health problem. The continuous dissemination of 'multidrug-resistant' (MDR) bacteria drastically reduces the efficacy of our antibiotic 'arsenal' and consequently increases the frequency of therapeutic failure. In MDR bacteria, the overexpression of efflux pumps that expel structurally unrelated drugs contributes to the reduced susceptibility by decreasing the intracellular concentration of antibiotics. During the last decade, several clinical data have indicated an increasing involvement of efflux pumps in the emergence and dissemination of resistant Gram-negative bacteria. It is necessary to clearly define the molecular, functional and genetic bases of the efflux pump in order to understand the translocation of antibiotic molecules through the efflux transporter. The recent investigation on the efflux pump AcrB at its structural and physiological levels, including the identification of drug affinity sites and kinetic parameters for various antibiotics, may pave the way towards the rational development of an improved new generation of antibacterial agents as well as efflux inhibitors in order to efficiently combat efflux-based resistance mechanisms.     

A noncanonical chaperone interacts with drug efflux pumps during their assembly into bacterial outer membranes

Bacteria have membrane-spanning efflux pumps to secrete toxic compounds ranging from heavy metal ions to organic chemicals, including antibiotic drugs. The overall architecture of these efflux pumps is highly conserved: with an inner membrane energy-transducing subunit coupled via an adaptor protein to an outer membrane conduit subunit that enables toxic compounds to be expelled into the environment. Here, we map the distribution of efflux pumps across bacterial lineages to show these proteins are more widespread than previously recognised. Complex phylogenetics support the concept that gene cassettes encoding the subunits for these pumps are commonly acquired by horizontal gene transfer. Using TolC as a model protein, we demonstrate that assembly of conduit subunits into the outer membrane uses the chaperone TAM to physically organise the membrane-embedded staves of the conduit subunit of the efflux pump. The characteristics of this assembly pathway have impact for the acquisition of efflux pumps across bacterial species and for the development of new antimicrobial compounds that inhibit efflux pump function.

A crosslinking study reveals novel insights into how the chaperone TAM helps Gram-negative bacteria insert the drug efflux pump subunit TolC into their outer membrane. Bioinformatic analyses show that TolC-like proteins can be found in all LPS-containing bacteria, but also in some monodermic Firmicutes.     

Functional role of bacterial multidrug efflux pumps in microbial natural ecosystems

Multidrug efflux pumps have emerged as relevant elements in the intrinsic and acquired antibiotic resistance of bacterial pathogens. In contrast with other antibiotic resistance genes that have been obtained by virulent bacteria through horizontal gene transfer, genes coding for multidrug efflux pumps are present in the chromosomes of all living organisms. In addition, these genes are highly conserved (all members of the same species contain the same efflux pumps) and their expression is tightly regulated. Together, these characteristics suggest that the main function of these systems is not resisting the antibiotics used in therapy and that they should have other roles relevant to the behavior of bacteria in their natural ecosystems. Among the potential roles, it has been demonstrated that efflux pumps are important for processes of detoxification of intracellular metabolites, bacterial virulence in both animal and plant hosts, cell homeostasis and intercellular signal trafficking.     

Multidrug-resistance efflux pumps - not just for resistance

It is well established that multidrug-resistance efflux pumps encoded by bacteria can confer clinically relevant resistance to antibiotics. It is now understood that these efflux pumps also have a physiological role(s). They can confer resistance to natural substances produced by the host, including bile, hormones and host-defence molecules. In addition, some efflux pumps of the resistance nodulation division (RND) family have been shown to have a role in the colonization and the persistence of bacteria in the host. Here, I present the accumulating evidence that multidrug-resistance efflux pumps have roles in bacterial pathogenicity and propose that these pumps therefore have greater clinical relevance than is usually attributed to them.     

Multidrug resistance mechanisms: drug efflux across two membranes

A set of multidrug efflux systems enables Gram-negative bacteria to survive in a hostile environment. This review focuses on the structural features and the mechanism of major efflux pumps of Gram-negative bacteria, which expel from the cells a remarkably broad range of antimicrobial compounds and produce the characteristic intrinsic resistance of these bacteria to antibiotics, detergents, dyes and organic solvents. Each efflux pump consists of three components: the inner membrane transporter, the outer membrane channel and the periplasmic lipoprotein. Similar to the multidrug transporters from eukaryotic cells and Gram-positive bacteria, the inner membrane transporters from Gram-negative bacteria recognize and expel their substrates often from within the phospholipid bilayer. This efflux occurs without drug accumulation in the periplasm, implying that substrates are pumped out across the two membranes directly into the medium. Recent data suggest that the molecular mechanism of the drug extrusion across a two-membrane envelope of Gram-negative bacteria may involve the formation of the membrane adhesion sites between the inner and the outer membranes. The periplasmic components of these pumps are proposed to cause a close membrane apposition as the complexes are assembled for the transport.     

Evaluation of a series of 2-napthamide derivatives as inhibitors of the drug efflux pump AcrB for the reversal of antimicrobial resistance

Drug efflux pumps confer multidrug resistance to dangerous pathogens which makes these pumps important drug targets. We have synthesised a novel series of compounds based on a 2-naphthamide pharmacore aimed at inhibiting the efflux pumps from Gram-negative bacteria. The archeatypical transporter AcrB from Escherichia coli was used as model efflux pump as AcrB is widely conserved throughout Gram-negative organisms. The compounds were tested for their antibacterial action, ability to potentiate the action of antibiotics and for their ability to inhibit Nile Red efflux by AcrB. None of the compounds were antimicrobial against E. coli wild type cells. Most of the compounds were able to inhibit Nile Red efflux indicating that they are substrates of the AcrB efflux pump. Three compounds were able to synergise with antibiotics and reverse resistance in the resistant phenotype. Compound A3, 4-(isopentyloxy)-2-naphthamide, reduced the MICs of erythromycin and chloramphenicol to the MIC levels of the drug sensitive strain that lacks an efflux pump. A3 had no effect on the MIC of the non-substrate rifampicin indicating that this compound acts specifically through the AcrB efflux pump. A3 also does not act through non-specific mechanisms such as outer membrane or inner membrane permeabilisation and is not cytotoxic against mammalian cell lines. Therefore, we have designed and synthesised a novel chemical compound with great potential to further optimisation as inhibitor of drug efflux pumps.     

Channel-tunnels: outer membrane components of type I secretion systems and multidrug efflux pumps of Gram-negative bacteria

For translocation across the cell envelope of Gram-negative bacteria, substances have to overcome two permeability barriers, the inner and outer membrane. Channel-tunnels are outer membrane proteins, which are central to two distinct export systems: the type I secretion system exporting proteins such as toxins or proteases, and efflux pumps discharging antibiotics, dyes, or heavy metals and thus mediating drug resistance. Protein secretion is driven by an inner membrane ATP-binding cassette (ABC) transporter while drug efflux occurs via an inner membrane proton antiporter. Both inner membrane transporters are associated with a periplasmic accessory protein that recruits an outer membrane channel-tunnel to form a functional export complex. Prototypes of these export systems are the hemolysin secretion system and the AcrAB/TolC drug efflux pump of Escherichia coli, which both employ TolC as an outer membrane component. Its remarkable conduit-like structure, protruding 100 ? into the periplasmic space, reveals how both systems are capable of transporting substrates across both membranes directly from the cytosol into the external environment. Proteins of the channel-tunnel family are widespread within Gram-negative bacteria. Their involvement in drug resistance and in secretion of pathogenic factors makes them an interesting system for further studies. Understanding the mechanism of the different export apparatus could help to develop new drugs, which block the efflux pumps or the secretion system. Electronic Publication     

Use of resazurin to detect mefloquine as an efflux-pump inhibitor in Pseudomonas aeruginosa and Escherichia coli

Multi-drug-resistant bacteria can cause serious infections that are extremely difficult to treat. Bacterial efflux pumps are known to contribute to multi-drug resistance and, thus, constitute a promising target for novel antibacterial agents. Resazurin is widely used to monitor bacterial growth because resazurin is reduced to the fluorescent resorufin by live cells. We have shown by flow cytometric analysis and by accumulation studies with wild type and efflux deficient strains that resazurin is a substrate of efflux pumps in Escherichia coli and Pseudomonas aeruginosa. Our investigations showed that the conversion rate of resazurin to resorufin is affected by efflux pumps. This finding was used to design an assay useful to detect efflux pump activity and to find potential efflux-pump inhibitors in a microtiter plate format. Mefloquine was detected as efflux-pump inhibitor when a panel of selected chemical compounds was tested for assay validation purposes.     

Proton-dependent multidrug efflux systems.

Multidrug efflux systems display the ability to transport a variety of structurally unrelated drugs from a cell and consequently are capable of conferring resistance to a diverse range of chemotherapeutic agents. This review examines multidrug efflux systems which use the proton motive force to drive drug transport. These proteins are likely to operate as multidrug/proton antiporters and have been identified in both prokaryotes and eukaryotes. Such proton-dependent multidrug efflux proteins belong to three distinct families or superfamilies of transport proteins: the major facilitator superfamily (MFS), the small multidrug resistance (SMR) family, and the resistance/ nodulation/cell division (RND) family. The MFS consists of symporters, antiporters, and uniporters with either 12 or 14 transmembrane-spanning segments (TMS), and we show that within the MFS, three separate families include various multidrug/proton antiport proteins. The SMR family consists of proteins with four TMS, and the multidrug efflux proteins within this family are the smallest known secondary transporters. The RND family consists of 12-TMS transport proteins and includes a number of multidrug efflux proteins with particularly broad substrate specificity. In gram-negative bacteria, some multidrug efflux systems require two auxiliary constituents, which might enable drug transport to occur across both membranes of the cell envelope. These auxiliary constituents belong to the membrane fusion protein and the outer membrane factor families, respectively. This review examines in detail each of the characterized proton-linked multidrug efflux systems. The molecular basis of the broad substrate specificity of these transporters is discussed. The surprisingly wide distribution of multidrug efflux systems and their multiplicity in single organisms, with Escherichia coli, for instance, possessing at least nine proton-dependent multidrug efflux systems with overlapping specificities, is examined. We also discuss whether the normal physiological role of the multidrug efflux systems is to protect the cell from toxic compounds or whether they fulfil primary functions unrelated to drug resistance and only efflux multiple drugs fortuitously or opportunistically.     

In-silico interaction studies suggest RND efflux pump mediates polymyxin resistance in Acinetobacter baumannii

Bacterial efflux pumps have emerged as antibiotic resistance determinants and confers multi-drug resistance to a broad range of antimicrobials as well as non-antibiotic substances. A study about translocation of antibiotic molecules through the efflux transporter, will contribute in determining substrate specificity. In the present study, we have explored RND family efflux pump extensively found in Acinetobacter baumannii i.e. AdeABC. Besides, another well studied RND efflux pump, AcrAB-TolC together with a non-RND efflux pump, NorM was investigated for comparative analysis. We employed a series of computational techniques ranging from molecular docking to binding free energy estimation and molecular dynamics simulations to determine the binding affinity for different classes of drugs, namely aminoglycosides, polymyxins, β-lactams, tetracyclines, glycylcyclines, quinolones and metronidazole with AdeB, AcrB, and NorM efflux proteins. Our results revealed that class polymyxins has the highest binding affinity with the RND efflux pumps i.e. AcrAB-TolC and AdeABC as well as non-RND efflux pump, NorM. The experimental validation study demonstrated bigger zone of inhibition in presence of efflux pump inhibitor than polymyxin alone thus unveiling its specificity toward efflux pump. The reported experimental data comprising of minimum inhibitory concentration of antibiotics toward these efflux pumps also support our finding based on in silico approach. To recapitulate the outcome, polymyxins shows maximum specificity toward RND as well as non-RND efflux pump and may unlatch the way to rationally develop new potential antibacterial agents as well as efflux pump inhibitors in order to combat resistance.     

Assessment of three Resistance-Nodulation-Cell Division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance

Background  

Burkholderia cenocepacia are opportunistic Gram-negative bacteria that can cause chronic pulmonary infections in patients with cystic fibrosis. These bacteria demonstrate a high-level of intrinsic antibiotic resistance to most clinically useful antibiotics complicating treatment. We previously identified 14 genes encoding putative Resistance-Nodulation-Cell Division (RND) efflux pumps in the genome of B. cenocepacia J2315, but the contribution of these pumps to the intrinsic drug resistance of this bacterium remains unclear.