Resistance-nodulation-division Efflux Pump Research Articles

Identification of determinants that allow maintenance of high-level fluoroquinolone resistance in Acinetobacter baumannii.

Acinetobacter baumannii is associated with multidrug-resistant infections in healthcare settings, with fluoroquinolones such as ciprofloxacin being currently ineffective. Clinical isolates largely harbor mutations in the GyrA and TopoIV fluoroquinolone targets, as well as mutations that increase expression of drug resistance-nodulation-division (RND) efflux pumps. Factors critical for maintaining fitness levels of pump overproducers are uncharacterized despite their prevalence in clinical isolates. We, here, identify proteins that contribute to the fitness of fluoroquinolone-resistant (FQR) strains overexpressing three known RND systems using high-density insertion mutagenesis. Overexpression of the AdeFGH efflux pump caused hypersensitization to defects in outer membrane homeostatic regulation, including lesions that reduced lipooligosaccharide (LOS) biosynthesis and blocked production of the major A. baumannii porin. In contrast, AdeAB pump hyperexpression, in the absence of elevated adeC expression (the outer membrane component of the pump), was relatively tolerant to loss of these functions, consistent with the outer membrane protein being the primary disruptive component. Surprisingly, overexpression of proton-transporting efflux pumps had little impact on cytosolic pH, consistent with a compensatory response to pump activity. The most striking transcriptional changes were associated with AdeFGH pump overexpression, including the activation of the phenylacetate (PAA) degradation regulon. Disruption of the PAA pathway resulted in cytosolic acidification and defective expression of genes involved in protection from oxidative stress. These results indicate that RND efflux pump overproduction is compensated by maintenance of outer membrane integrity in A. baumannii to facilitate fitness of FQR isolates.IMPORTANCEAcinetobacter baumannii is a pathogen that often causes multidrug-resistant infections in healthcare settings, presenting a threat to the efficacy of known therapeutic interventions. Fluoroquinolones such as ciprofloxacin are currently ineffective against a majority of clinical A. baumannii isolates, many of which express pumps that remove this antibiotic class from within the bacterium. Three of these pumps can be found in most clinical isolates, with one of the three often hyperproduced at all times. In this study, we identify proteins that are necessary for the fitness of pump hyperproducers. The identified proteins are necessary to stabilize the outer membrane and allow the cytoplasm to tolerate the accumulation of ions as a consequence of excess pump activity. These results point to strategies for developing therapies that combine known antibiotics with drugs that target proteins important for survival of strains hyper-expressing efflux pumps.

Deciphering the molecular and functional basis of TMexCD1: the plasmid-encoded efflux pump of resistance-nodulation-division superfamily.

Horizontal gene transfer has been demonstrated to be an important driver for the emergency of multidrug-resistant pathogens. Recently, a transferable gene cluster tmexCD1-toprJ1 of the resistance-nodulation-division (RND) superfamily was identified in the plasmids of animal-derived Klebsiella pneumoniae strains, with a higher efflux capacity for various drugs than the Escherichia coli AcrAB-TolC homolog system. In this study, we focused on the differences in the inner membrane pump of these two systems and identified some key residues that contribute to the robust efflux activity of the TMexCD1 system. With the aid of homologous modeling and molecular docking, eight residues from the proximal binding pocket (PBP) and nine from the distal binding pocket (DBP) were selected and subjected to site-directed mutagenesis. Several of them, such as S134, I139, D181, and A290, were shown to be important for substrate binding in the DBP region, and all residues in PBP and DBP showed certain substrate preferences. Apart from the conservative switch loop (L613-623TMexD1) previously identified in the E. coli AcrB (EcAcrB), a relatively unconservative loop (L665-675TMexD1) at the bottom of PBP was proposed as a critical element for the robust activity of TMexD1, due to variations at sites E669, G670, N673, and S674 compared to EcAcrAB, and the significantly altered efflux activity due to their mutations. The conservation and flexibility of these key factors can contribute to the evolution of the RND efflux pumps and thus serve as potential targets for developing inhibitors to block the widespread of the TMexCD1 system.

Repurposing Mitomycin C in Combination with Pentamidine or Gentamicin to Treat Infections with Multi-Drug-Resistant (MDR) Pseudomonas aeruginosa.

The aims of this study were (i) to determine if the combination of mitomycin C with pentamidine or existing antibiotics resulted in enhanced efficacy versus infections with MDR P. aeruginosa in vivo; and (ii) to determine if the doses of mitomycin C and pentamidine in combination can be reduced to levels that are non-toxic in humans but still retain antibacterial activity. Resistant clinical isolates of P. aeruginosa, a mutant strain over-expressing the MexAB-OprM resistance nodulation division (RND) efflux pump and a strain with three RND pumps deleted, were used. MIC assays indicated that all strains were sensitive to mitomycin C, but deletion of three RND pumps resulted in hypersensitivity and over-expression of MexAB-OprM caused some resistance. These results imply that mitomycin C is a substrate of the RND efflux pumps. Mitomycin C monotherapy successfully treated infected Galleria mellonella larvae, albeit at doses too high for human administration. Checkerboard and time-kill assays showed that the combination of mitomycin C with pentamidine, or the antibiotic gentamicin, resulted in synergistic inhibition of most P. aeruginosa strains in vitro. In vivo, administration of a combination therapy of mitomycin C with pentamidine, or gentamicin, to G. mellonella larvae infected with P. aeruginosa resulted in enhanced efficacy compared with monotherapies for the majority of MDR clinical isolates. Notably, the therapeutic benefit conferred by the combination therapy occurred with doses of mitomycin C close to those used in human medicine. Thus, repurposing mitomycin C in combination therapies to target MDR P. aeruginosa infections merits further investigation.

Characterization of pyridylpiperazine-based efflux pump inhibitors for Acinetobacter baumannii.

In Acinetobacter baumannii, multidrug efflux pumps belonging to the resistance-nodulation-division (RND) superfamily result in decreased antibiotic susceptibility. Improving the activity of current antibiotics via efflux pump inhibitors (EPIs) represents an attractive alternative approach to control this bacterium. Pyridylpiperazines (PyrPips) are a new class of EPIs that can effectively inhibit the Escherichia coli RND efflux pump AcrAB-TolC and boost the activity of several antibiotics. Here we have evaluated and characterized whether the PyrPip chemical family is also able to boost antibiotic activity through inhibition of the RND efflux pumps in A. baumannii. Comparative structural modelling and docking, structure-activity relationship studies alongside molecular genetic approaches were deployed to improve, characterize and validate PyrPips' target. We showed that two enhanced PyrPip EPIs are capable of rescuing the activity of different classes of antibiotics in A. baumannii. By expressing A. baumannii main efflux pumps (AdeB, AdeG and AdeJ) individually in E. coli recombinant strains, we could gain further insights about the EPIs' capacity to act upon each pump. Finally, we showed that PyrPip EPIs are mostly acting through AdeJ inhibition via interactions with two key charged residues, namely E959 and E963. Our work demonstrates that PyrPip EPIs are capable of inhibiting RND efflux pumps of A. baumannii, and thus may present a promising chemical scaffold for further development.

Combination Therapy with Ciprofloxacin and Pentamidine against Multidrug-Resistant Pseudomonas aeruginosa: Assessment of In Vitro and In Vivo Efficacy and the Role of Resistance-Nodulation-Division (RND) Efflux Pumps.

The aim of this work was to (i) evaluate the efficacy of a combination treatment of pentamidine with ciprofloxacin against Galleria mellonella larvae infected with an MDR strain of P. aeruginosa and (ii) determine if pentamidine acts as an efflux-pump inhibitor. Resistant clinical isolates, mutant strains overexpressing one of three RND efflux pumps (MexAB-OprM, MexCD-OprJ, and MexEF-OprN), and a strain with the same three pumps deleted were used. MIC assays confirmed that the clinical isolates and the mutants overexpressing efflux pumps were resistant to ciprofloxacin and pentamidine. The deletion of the three efflux pumps induced sensitivity to both compounds. Exposure to pentamidine and ciprofloxacin in combination resulted in the synergistic inhibition of all resistant strains in vitro, but no synergy was observed versus the efflux-pump deletion strain. The treatment of infected G. mellonella larvae with the combination of pentamidine and ciprofloxacin resulted in enhanced efficacy compared with the monotherapies and significantly reduced the number of proliferating bacteria. Our measurement of efflux activity from cells revealed that pentamidine had a specific inhibitory effect on the MexCD-OprJ and MexEF-OprN efflux pumps. However, the efflux activity and membrane permeability assays revealed that pentamidine also disrupted the membrane of all cells. In conclusion, pentamidine does possess some efflux-pump inhibitory activity, in addition to a more general disruptive effect on membrane integrity that accounts for its ability to potentiate ciprofloxacin activity. Notably, the enhanced efficacy of combination therapy with pentamidine and ciprofloxacin versus MDR P. aeruginosa strains in vivo merits further investigation into its potential to treat infections via this pathogen in patients.

Drug resistance and physiological roles of RND multidrug efflux pumps in Salmonella enterica, Escherichia coli and Pseudomonas aeruginosa.

Drug efflux pumps transport antimicrobial agents out of bacteria, thereby reducing the intracellular antimicrobial concentration, which is associated with intrinsic and acquired bacterial resistance to these antimicrobials. As genome analysis has advanced, many drug efflux pump genes have been detected in the genomes of bacterial species. In addition to drug resistance, these pumps are involved in various essential physiological functions, such as bacterial adaptation to hostile environments, toxin and metabolite efflux, biofilm formation and quorum sensing. In Gram-negative bacteria, efflux pumps in the resistance–nodulation–division (RND) superfamily play a clinically important role. In this review, we focus on Gram-negative bacteria, including Salmonella enterica , Escherichia coli and Pseudomonas aeruginosa , and discuss the role of RND efflux pumps in drug resistance and physiological functions.

LexR Positively Regulates the LexABC Efflux Pump Involved in Self-Resistance to the Antimicrobial Di-N-Oxide Phenazine in Lysobacter antibioticus.

Myxin, a di-N-oxide phenazine isolated from the soil bacterium Lysobacter antibioticus, exhibits potent activity against various microorganisms and has the potential to be developed as an agrochemical. Antibiotic-producing microorganisms have developed self-resistance mechanisms to protect themselves from autotoxicity. Antibiotic efflux is vital for such protection. Recently, we identified a resistance-nodulation-division (RND) efflux pump, LexABC, involved in self-resistance against myxin in L. antibioticus. Expression of its genes, lexABC, was induced by myxin and was positively regulated by the LysR family transcriptional regulator LexR. The molecular mechanisms, however, have not been clear. Here, LexR was found to bind to the lexABC promoter region to directly regulate expression. Moreover, myxin enhanced this binding. Molecular docking and surface plasmon resonance analysis showed that myxin bound LexR with valine and lysine residues at positions 146 (V146) and 195 (K195), respectively. Furthermore, mutation of K195 in vivo led to downregulation of the gene lexA. These results indicated that LexR sensed and bound with myxin, thereby directly activating the expression of the LexABC efflux pump and increasing L. antibioticus resistance against myxin. IMPORTANCE Antibiotic-producing bacteria exhibit various sophisticated mechanisms for self-protection against their own secondary metabolites. RND efflux pumps that eliminate antibiotics from cells are ubiquitous in Gram-negative bacteria. Myxin is a heterocyclic N-oxide phenazine with potent antimicrobial and antitumor activities produced by the soil bacterium L. antibioticus. The RND pump LexABC contributes to the self-resistance of L. antibioticus against myxin. Herein, we report a mechanism involving the LysR family regulator LexR that binds to myxin and directly activates the LexABC pump. Further study on self-resistance mechanisms could help the investigation of strategies to deal with increasing bacterial antibiotic resistance and enable the discovery of novel natural products with resistance genes as selective markers.

RND pumps across the genus Acinetobacter: AdeIJK is the universal efflux pump.

Acinetobacter are generally soil-dwelling organisms that can also cause serious human infections. A. baumannii is one of the most common causative agents of Acinetobacter infections and is often multidrug resistant. However, an additional 25 species within the genus have also been associated with infection. A. baumannii encodes six resistance nodulation division (RND) efflux pumps, the most clinically relevant class of efflux pumps for antibiotic export, but the distribution and types of RND efflux pumps across the genus is currently unknown. Sixty-four species making up the genus Acinetobacter were searched for RND systems within their genomes. We also developed a novel method using conserved RND residues to predict the total number of RND proteins including currently undescribed RND pump proteins. The total number of RND proteins differed both within a species and across the genus. Species associated with infection tended to encode more pumps. AdeIJK/AdeXYZ was found in all searched species of Acinetobacter, and through genomic, structural and phenotypic work we show that these genes are actually homologues of the same system. This interpretation is further supported by structural analysis of the potential drug-binding determinants of the associated RND-transporters, which reveal their close similarity to each other, and distinctiveness from other RND-pumps in Acinetobacter, such as AdeB. Therefore, we conclude that AdeIJK is the fundamental RND system for species in the genus Acinetobacter. AdeIJK can export a broad range of antibiotics and provides crucial functions within the cell, for example lipid modulation of the cell membrane, and therefore it is likely that all Acinetobacter require AdeIJK for survival and homeostasis. In contrast, additional RND systems, such as AdeABC and AdeFGH, were only found in a subset of Acinetobacter that are associated with infection. By understanding the roles and mechanisms of RND efflux systems in Acinetobacter, treatments for infections can avoid efflux-mediated resistance and improve patient outcomes.

Identification of key amino acid residues in OqxB mediated efflux of fluoroquinolones using site-directed mutagenesis

OqxB belongs to the RND (Resistance-Nodulation-Division) efflux pump family, recognized widely as a major contributor towards enhancing antimicrobial resistance. It is known to be predominantly present in all Klebsiella spp. and is attributed for its role in increasing resistance against an array of antibiotics like nitrofurantoin, quinolones, β-lactams and colistin. However, the presence of oqxB encoding this efflux pump is not limited only to Klebsiella spp., but is also found to occur via horizontal gene transfer in other bacterial genera like Escherichia coli, Enterobacter cloacae and Salmonella spp. Recently, we reported the crystal structure of OqxB and its structure-function relationship required for the efflux of fluoroquinolones. Extending these findings further, we characterized the structural architecture of this efflux pump along with identifying some critical amino acids at the substrate binding domain of OqxB. Based on our in silico modelling studies, both hydrophobic residues (F180, L280, L621, F626) and polar residues (R48, E50, E184, R157, R774) were found to be located at this site. The present work reports the importance of these key amino acid residues and the crucial ion–pair interactions at the substrate-binding pocket, thereby establishing their role in OqxB mediated efflux and the resultant resistance development against fluoroquinolones.

Role of the multi-drug efflux systems on the baseline susceptibility to ceftazidime/avibactam and ceftolozane/tazobactam in clinical isolates of non-carbapenemase-producing carbapenem-resistant Pseudomonas aeruginosa.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is one of the pathogens that urgently needs new drugs and new alternatives for its control. The primary strategy to combat this bacterium is combining treatments of beta-lactam with a beta-lactamase inhibitor. The most used combinations against P. aeruginosa are ceftazidime/avibactam (CZA) and ceftolozane/tazobactam (C/T). Although mechanisms leading to CZA and C/T resistance have already been described, among which are the resistance-nodulation-division (RND) efflux pumps, the role that these extrusion systems may play in CZA, and C/T baseline susceptibility of clinical isolates remains unknown. For this purpose, 161 isolates of non-carbapenemase-producing (Non-CP) CRPA were selected, and susceptibility tests to CZA and C/T were performed in the presence and absence of the RND efflux pumps inhibitor, Phenylalanine-arginine β-naphthylamide (PAβN). In the absence of PAβN, C/T showed markedly higher activity against Non-CP-CRPA isolates than observed for CZA. These results were even more evident in isolates classified as extremely-drug resistant (XDR) or with difficult-to-treat resistance (DTR), where CZA decreased its activity up to 55.2% and 20.0%, respectively, whereas C/T did it up to 82.8% (XDR), and 73.3% (DTR). The presence of PAβN showed an increase in both CZA (37.6%) and C/T (44.6%) activity, and 25.5% of Non-CP-CRPA isolates increased their susceptibility to these two combined antibiotics. However, statistical analysis showed that only the C/T susceptibility of Non-CP-CRPA isolates was significantly increased. Although the contribution of RND activity to CZA and C/T baseline susceptibility was generally low (two-fold decrease of minimal inhibitory concentrations [MIC]), a more evident contribution was observed in a non-minor proportion of the Non-CP-CRPA isolates affected by PAβN [CZA: 25.4% (15/59); C/T: 30% (21/70)]. These isolates presented significantly higher MIC values for C/T. Therefore, we conclude that RND efflux pumps are participating in the phenomenon of baseline susceptibility to CZA and, even more, to C/T. However, the genomic diversity of clinical isolates is so great that deeper analyzes are necessary to determine which elements are directly involved in this phenomenon.

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.

Metformin Reverses tmexCD1-toprJ1- and tet(A)-Mediated High-Level Tigecycline Resistance in K. pneumoniae.

Tigecycline (TIG) is one of the last effective options against multidrug resistance bacteria. Recently, the RND (resistance–nodulation–division) efflux pump gene cluster, tmexCD1-toprJ1, and the tetracycline-efflux pump tet(A) mutation were reported to mediate high level resistance to TIG in clinically important pathogens, weakening the efficacy of TIG. In this study, we report the potent synergistic effect of the antidiabetic drug metformin in combination with TIG against tet(A) mutant and tmexCD1-toprJ1 positive K. pneumoniae. The fractional inhibitory concentration index (FICI) of TIG and metformin were less than 0.05 for all the tested isolates. The time–kill curve assay showed that the combination of TIG and metformin exhibited much better antimicrobial effect than TIG alone. The synergistic effect was also confirmed in vivo using a well-studied Galleria mellonella larvae model. Mechanistic studies demonstrated that metformin disrupted the important component of proton motive force, the electric potential (Δψ) and the function of efflux pump, thereby increasing the intracellular concentration of TIG. This finding revealed that metformin might be a possible adjuvant of TIG for combating with superbugs carrying the tet(A) mutant and tmexCD1-toprJ1 genes.

Reducing bacterial antibiotic resistance by targeting bacterial metabolic pathways and disrupting RND efflux pump activity

Antibiotic resistance is a significant issue for the medical community, worldwide. Many bacteria develop drug resistance by utilizing multidrug resistant or MDR efflux pumps that can export antibiotics from bacterial cells. Antibiotics are expelled from bacteria by efflux pumps a part of the resistance nodulation division (RND) family. Types of RND efflux pumps include the AcrAB-TolC tripartite protein pump. There are an excessive number of antibiotic compounds that have been discovered; however, only a few antibiotics are effective against MDR bacteria. Many bacteria become drug resistant when sharing genes that encode MDR efflux pump expression. MDR efflux pump encoding genes are incorporated into plasmids and then shared among bacteria. As a consequence, advancements in genetic engineering can sufficiently target and edit pathogenic bacterial genomes for perturbing drug resistance mechanisms. In this perspective and review, support will be provided for utilizing genetic modifications as an antimicrobial approach and tool that may effectively combat bacterial MDR. Ayhan et al. found that deleting acrB, acrA, and tolC increased the levels of antibiotic sensitivity in Escherichia coli. Researchers also found that glucose, glutamate, and fructose all induced the absorption of antibiotics by upregulating the gene expression of maeA and maeB that is a part of the MAL-pyruvate pathway. Therefore, the current perspective and review will discuss the potential efficacy of reducing antibiotic resistance by inhibiting genes that encode efflux protein pump expression while simultaneously upregulating metabolic genes for increased antibiotic uptake.

Subinhibitory Concentrations of Clinically-Relevant Antimicrobials Affect Resistance-Nodulation-Division Family Promoter Activity in Acinetobacter baumannii.

Efflux pumps contribute to multidrug resistance in Acinetobacter baumannii due to their ability to expel a wide variety of structurally unrelated compounds. This study aimed to characterize the effect of subinhibitory concentrations of clinically-relevant antibiotics and disinfectants on the promoter activity of members of the Resistance-Nodulation-Division (RND) family in A. baumannii. The promoter regions from three RND efflux pumps (AdeABC, AdeFGH and AdeIJK) and the AdeRS regulatory system from three different A. baumannii strains (ATCC 17961, ATCC 17978, and ATCC 19606) were cloned into a luciferase reporter system (pLPV1Z). Promoter activity was quantitatively assessed in both exponential and stationary phase cultures after exposure to subinhibitory concentrations of four antibiotics from different classes (rifampicin, meropenem, tigecycline and colistin) and two disinfectants (ethanol and chlorhexidine). Subinhibitory concentrations of the compounds tested had variable effects on promoter activity that were highly dependent on the A. baumannii strain, the compound tested and the growth phase. Fold changes in AdeABC promoter activity ranged from 1.97 to 113.7, in AdeFGH from −5.6 to 1.13, in AdeIJK from −2.5 to 2, and in AdeRS from −36.2 to −1.32. Taken together, these results indicate that subinhibitory concentrations of clinically-relevant antibiotics and disinfectants affect the promoter activity of RND family members in A. baumannii in a strain and growth phase dependent manner. These results may have important implications for the treatment of infections caused by A. baumannii.

Overexpression of Resistance-Nodulation-Division Efflux Pump Genes Contributes to Multidrug Resistance in Aeromonas hydrophila Clinical Isolates.

Aeromonas hydrophila is a Gram-negative bacterium that is a critical causative agent of infections in fish and is occasionally responsible for human infections following contact with contaminated water or food. Currently, the extensive use of antibiotics in clinical practice has led to increased number of isolates of multidrug-resistant (MDR) Aeromonas and has posed a serious public health challenge. The efflux pump system is a critical mechanism of antibiotic resistance in most Gram-negative bacteria. However, the role of resistance-nodulation-division (RND)-type efflux pumps in MDR A. hydrophila is not fully understood. We aimed to evaluate the contribution of the RND efflux pump system to MDR A. hydrophila clinical isolates. PCR results indicated a considerable variation in the presence of RND efflux pump genes in clinical isolates compared to that of the environmental reference strain ATCC7966T. Compared to non-MDR clinical isolates, the expression levels of three putative RND efflux pump genes, AHA0021, AHA1320, and AheB, were significantly elevated in MDR strains. The minimal inhibitory concentrations of piperacillin/tazobactam, imipenem, erythromycin, and polymyxin B were significantly reduced by phenylalanine-arginine β-naphthylamide (PAβN), further supporting the contribution of the RND efflux system in MDR A. hydrophila. We provided evidence supporting the contribution of the RND efflux system to multidrug resistance in A. hydrophila clinical isolates. Further studies are warranted to elucidate the detailed mechanisms that confer intrinsic resistance to antimicrobials in A. hydrophila.

Structure and function relationship of OqxB efflux pump from Klebsiella pneumoniae

OqxB is an RND (Resistance-Nodulation-Division) efflux pump that has emerged as a factor contributing to the antibiotic resistance in Klebsiella pneumoniae. OqxB underwent horizontal gene transfer and is now seen in other Gram-negative bacterial pathogens including Escherichia coli, Enterobacter cloacae and Salmonella spp., further disseminating multi-drug resistance. In this study, we describe crystal structure of OqxB with n-dodecyl-β-D-maltoside (DDM) molecules bound in its substrate-binding pocket, at 1.85 Å resolution. We utilize this structure in computational studies to predict the key amino acids contributing to the efflux of fluoroquinolones by OqxB, distinct from analogous residues in related transporters AcrB and MexB. Finally, our complementation assays with mutated OqxB and minimum inhibitory concentration (MIC) experiments with clinical isolates of E. coli provide further evidence that the predicted structural features are indeed involved in ciprofloxacin efflux.

Ever-Adapting RND Efflux Pumps in Gram-Negative Multidrug-Resistant Pathogens: A Race against Time.

The rise in multidrug resistance (MDR) is one of the greatest threats to human health worldwide. MDR in bacterial pathogens is a major challenge in healthcare, as bacterial infections are becoming untreatable by commercially available antibiotics. One of the main causes of MDR is the over-expression of intrinsic and acquired multidrug efflux pumps, belonging to the resistance-nodulation-division (RND) superfamily, which can efflux a wide range of structurally different antibiotics. Besides over-expression, however, recent amino acid substitutions within the pumps themselves—causing an increased drug efflux efficiency—are causing additional worry. In this review, we take a closer look at clinically, environmentally and laboratory-evolved Gram-negative bacterial strains and their decreased drug sensitivity as a result of mutations directly in the RND-type pumps themselves (from Escherichia coli, Salmonella enterica, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Acinetobacter baumannii and Legionella pneumophila). We also focus on the evolution of the efflux pumps by comparing hundreds of efflux pumps to determine where conservation is concentrated and where differences in amino acids can shed light on the broad and even broadening drug recognition. Knowledge of conservation, as well as of novel gain-of-function efflux pump mutations, is essential for the development of novel antibiotics and efflux pump inhibitors.

The role of chemotaxis and efflux pumps on nitrate reduction in the toxic regions of a ciprofloxacin concentration gradient.

Spatial concentration gradients of antibiotics are prevalent in the natural environment. Yet, the microbial response in these heterogeneous systems remains poorly understood. We used a microfluidic reactor to create an artificial microscopic ecosystem that generates diffusive gradients of solutes across interconnected microenvironments. With this reactor, we showed that chemotaxis toward a soluble electron acceptor (nitrate) allowed Shewanella oneidensis MR-1 to inhabit and sustain metabolic activity in highly toxic regions of the antibiotic ciprofloxacin (>80× minimum inhibitory concentration, MIC). Acquired antibiotic resistance was not observed for cells extracted from the reactor, so we explored the role of transient adaptive resistance by probing multidrug resistance (MDR) efflux pumps, ancient elements that are important for bacterial physiology and virulence. Accordingly, we constructed an efflux pump deficient mutant (∆mexF) and used resistance-nodulation-division (RND) efflux pump inhibitors (EPIs). While batch results showed the importance of RND efflux pumps for microbial survival, microfluidic studies indicated that these pumps were not necessary for survival in antibiotic gradients. Our work contributes to an emerging body of knowledge deciphering the effects of antibiotic spatial heterogeneity on microorganisms and highlights differences of microbial response in these systems versus well-mixed batch conditions.

Resistance-Nodulation-Division Efflux Pump, LexABC, Contributes to Self-Resistance of the Phenazine Di-N-Oxide Natural Product Myxin in Lysobacter antibioticus.

Antibiotic-producing microorganisms have developed several self-resistance mechanisms to protect them from autotoxicity. Transporters belonging to the resistance- nodulation-division (RND) superfamily commonly confer multidrug resistance in Gram-negative bacteria. Phenazines are heterocyclic, nitrogen-containing and redox-active compounds that exhibit diverse activities. We previously identified six phenazines from Lysobacter antibioticus OH13, a soil bacterium emerging as a potential biocontrol agent. Among these phenazines, myxin, a di-N-oxide phenazine, exhibited potent activity against a variety of microorganisms. In this study, we identified a novel RND efflux pump gene cluster, designated lexABC, which is located far away in the genome from the myxin biosynthesis gene cluster. We found a putative LysR-type transcriptional regulator encoding gene lexR, which was adjacent to lexABC. Deletion of lexABC or lexR gene resulted in significant increasing susceptibility of strains to myxin and loss of myxin production. The results demonstrated that LexABC pump conferred resistance against myxin. The myxin produced at lower concentrations in these mutants was derivatized by deoxidation and O-methylation. Furthermore, we found that the abolishment of myxin with deletion of LaPhzB, which is an essential gene in myxin biosynthesis, resulted in significant downregulation of the lexABC. However, exogenous supplementation with myxin to LaPhzB mutant could efficiently induce the expression of lexABC genes. Moreover, lexR mutation also led to decreased expression of lexABC, which indicates that LexR potentially positively modulated the expression of lexABC. Our findings reveal a resistance mechanism against myxin of L. antibioticus, which coordinates regulatory pathways to protect itself from autotoxicity.

Expression of RND efflux pumps mediated antibiotic resistance in Pseudomonas aeruginosa clinical strains

Resistance-Nodulation-Division (RND) efflux pumps are responsible for multidrug resistance in Pseudomonas aeruginosa. The present study aimed to evaluate the overexpression of RND efflux pumps and its role in the antibiotic resistance of P. aeruginosa clinical isolates. A number of 122 isolates were obtained from three military hospitals in Tehran, Iran. In order to determine the antibiotic resistance, the isolates were identified and assessed by the disk diffusion and agar dilution methods. This study investigated the gene expression of four multi-drug efflux pump systems (MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY (-OprA)) and its correlation with antibiotic resistance. The isolates indicated that the highest resistance rate was against ticarcillin (80%), followed by ciprofloxacin (74%) and meropenem (71%). Most of them expressed mexB (69%), mexC (28.7%), mexE (43.4%), and mexY (74.6%), suggesting that mexB and mexY were highly expressed in the studied strains. The overexpression of mexB and mexY was significantly more prevalent in the ICU wards (p = 0.033). Furthermore, there was a significant correlation between the expression of RND-type efflux pumps and the resistance to most anti-pseudomonal antibiotics.