Multidrug Efflux System Research Articles

Structural characterization of the EmrAB-TolC efflux complex from E. coli

Gram-negative bacteria export a large variety of antimicrobial compounds by forming two-membrane spanning tripartite multidrug efflux systems composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. Here we present the co-expression, purification and first electron microscopy insights of the Escherichia coli EmrAB–TolC tripartite Major Facilitator Superfamily (MSF) efflux system as a whole complex stabilized by Amphipol polymer. The structure reveals a 33 nm long complex delineated by the Amphipol belt at both extremities. Comparison of projection structures of EmrAB-TolC and AcrAB-TolC indicates that the outer membrane protein TolC linked to the periplasmic adaptor EmrA protein form an extended periplasmic canal. The overall length of EmrAB-TolC complex is similar to that of AcrAB-TolC with a probable tip-to-tip interaction between EmrA and TolC unveiling how the adaptor protein connects TolC and EmrB embedded in the inner membrane.

Antibiotic export by MexB multidrug efflux transporter is allosterically controlled by a MexA-OprM chaperone-like complex

The tripartite multidrug efflux system MexAB-OprM is a major actor in Pseudomonas aeruginosa antibiotic resistance by exporting a large variety of antimicrobial compounds. Crystal structures of MexB and of its Escherichia coli homolog AcrB had revealed asymmetric trimers depicting a directional drug pathway by a conformational interconversion (from Loose and Tight binding pockets to Open gate (LTO) for drug exit). It remains unclear how MexB acquires its LTO form. Here by performing functional and cryo-EM structural investigations of MexB at various stages of the assembly process, we unveil that MexB inserted in lipid membrane is not set for active transport because it displays an inactive LTC form with a Closed exit gate. In the tripartite complex, OprM and MexA form a corset-like platform that converts MexB into the active form. Our findings shed new light on the resistance nodulation cell division (RND) cognate partners which act as allosteric factors eliciting the functional drug extrusion.

The impaired quorum sensing response of Pseudomonas aeruginosa MexAB-OprM efflux pump overexpressing mutants is not due to non-physiological efflux of 3-oxo-C12-HSL.

Multidrug (MDR) efflux pumps are ancient and conserved molecular machineries with relevant roles in different aspects of the bacterial physiology, besides antibiotic resistance. In the case of the environmental opportunistic pathogen Pseudomonas aeruginosa, it has been shown that overexpression of different efflux pumps is linked to the impairment of the quorum sensing (QS) response. Nevertheless, the causes of such impairment are different for each analysed efflux pump. Herein, we performed an in-depth analysis of the QS-mediated response of a P. aeruginosa antibiotic resistant mutant that overexpresses MexAB-OprM. Although previous work claimed that this efflux pump extrudes the QS signal 3-oxo-C12-HSL, we show otherwise. Our results evidence that the observed attenuation in the QS response when overexpressing this pump is related to an impaired production of alkyl quinolone QS signals, likely prompted by the reduced availability of one of their precursors, the octanoate. Together with previous studies, this indicates that, although the consequences of overexpressing efflux pumps are similar (impaired QS response), the underlying mechanisms are different. This 'apparent redundancy' of MDR efflux systems can be understood as a P. aeruginosa strategy to keep the robustness of the QS regulatory network and modulate its output in response to different signals.

Evaluation of Antioxidant, Antimicrobial and Tyrosinase Inhibitory Activities of Extracts from Tricholosporum goniospermum, an Edible Wild Mushroom.

Tricholosporum goniospermum (Bres.) Guzmán ex T.J. Baroni is an excellent edible mushroom whose compounds and biological properties are still unknown. In this study, n-hexane, ethyl acetate and methanol extracts from fruiting bodies and liquid-cultured mycelia were compared for the analysis of phenolic compounds, the evaluation of scavenger (DPPH, ABTS) and reducing (CUPRAC, FRAP) activities, and the enzyme inhibition of α-amylase, acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and tyrosinase. Additionally, T. goniospermum extracts were evaluated for antibacterial and antimycotic activities against Gram+ and Gram− bacteria, and clinical yeast and fungal dermatophytes. Finally, based on the extract content in phenolic compounds, in silico studies, including the docking approach, were conducted to predict the putative targets (namely tyrosinase, lanosterol-14-α-demethylase, the multidrug efflux system transporters of E. coli (mdtK) and P. aeruginosa (pmpM), and S. aureus β-lactamase (ORF259)) underlying the observed bio-pharmacological and microbiological effects. The methanolic extract from mycelia was the richest in gallic acid, whereas the ethyl acetate extract from fruiting bodies was the sole extract to show levels of catechin. Specifically, docking runs demonstrated an affinity of catechin towards all docked proteins, in the micromolar range. These in silico data are consistent, at least in part, with the highest activity of ethyl acetate extract as an antimicrobial and anti-tyrosinase (554.30 mg KAE/g for fruiting bodies and 412.81 mg KAE/g for mycelia) agent. The ethyl acetate extracts were also noted as being the most active (2.97 mmol ACAE/g for fruiting bodies and 2.25 mmol ACAE/g for mycelia) on α-amylase. BChE inhibitory activities varied from 2.61 to 26.78 mg GALAE/g, while the tested extracts were not active on AChE. In conclusion, all mushroom extracts tested in this study had potent antimicrobial activities. Particularly, among the tested extracts, the ethyl acetate extract showed the highest efficacy as both an antimicrobial and anti-tyrosinase agent. This could be related, albeit partially, to its content of catechin. In this regard, the bioinformatics analyses showed interactions of catechin with tyrosinase and specific microbial proteins involved in the resistance to chemotherapeutic drugs, thus suggesting innovative pharmacological applications of T. goniospermum extracts.

Antimicrobial activity of polypyrrole nanoparticles and aqueous extract of Moringa oleifera against Staphylococcus spp. carriers of multi-drug efflux system genes isolated from dairy farms.

Our objectives were to identify genes of the multi-drug efflux system and to evaluate the antimicrobial activities of polypyrrole nanoparticles (PPy-NPs) and aqueous extract of Moringa oleifera against Staphylococcus spp. isolated from dairy farms in Northeast Brazil. Initially, 162 Staphylococcus spp. isolates were subjected to in vitro antimicrobial sensitivity tests. Of these, 35 presented antimicrobial multi-drug resistance phenotypes. These 35 isolates were then referred for the detection of norA, norB, norC, msrA, mgrA, tet-38, and lmrS genes, all of which feature in multi-drug efflux systems. In the isolates carrying the genes, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of PPy-NPs and Moringa oleifera aqueous extract were determined. In the molecular analysis of the 35 isolates norA, norC, tet-38, and msrA genes were detected and for the other genes norB, lmrS and mgrA there was no amplification. Antimicrobial activity was verified of PPy-NPs and aqueous extract of Moringa oleifera in Staphylococcus spp. carrying multi-drug efflux system genes. We concluded that there are multi-drug efflux system genes present in the Staphylococcus spp. from the agricultural environment in Northeast Brazil, and that aqueous extract of Moringa oleifera and PPy-NPs show bactericidal activity against these isolates.

Cryo-EM Structures of a Gonococcal Multidrug Efflux Pump Illuminate a Mechanism of Drug Recognition and Resistance.

Neisseria gonorrhoeae is an obligate human pathogen and causative agent of the sexually transmitted infection (STI) gonorrhea. The most predominant and clinically important multidrug efflux system in N. gonorrhoeae is the multiple transferrable resistance (Mtr) pump, which mediates resistance to a number of different classes of structurally diverse antimicrobial agents, including clinically used antibiotics (e.g., β-lactams and macrolides), dyes, detergents and host-derived antimicrobials (e.g., cationic antimicrobial peptides and bile salts). Recently, it has been found that gonococci bearing mosaic-like sequences within the mtrD gene can result in amino acid changes that increase the MtrD multidrug efflux pump activity, probably by influencing antimicrobial recognition and/or extrusion to elevate the level of antibiotic resistance. Here, we report drug-bound solution structures of the MtrD multidrug efflux pump carrying a mosaic-like sequence using single-particle cryo-electron microscopy, with the antibiotics bound deeply inside the periplasmic domain of the pump. Through this structural approach coupled with genetic studies, we identify critical amino acids that are important for drug resistance and propose a mechanism for proton translocation.IMPORTANCENeisseria gonorrhoeae has become a highly antimicrobial-resistant Gram-negative pathogen. Multidrug efflux is a major mechanism that N. gonorrhoeae uses to counteract the action of multiple classes of antibiotics. It appears that gonococci bearing mosaic-like sequences within the gene mtrD, encoding the most predominant and clinically important transporter of any gonococcal multidrug efflux pump, significantly elevate drug resistance and enhance transport function. Here, we report cryo-electron microscopy (EM) structures of N. gonorrhoeae MtrD carrying a mosaic-like sequence that allow us to understand the mechanism of drug recognition. Our work will ultimately inform structure-guided drug design for inhibiting these critical multidrug efflux pumps.

Overexpression of the Small RNA PA0805.1 in Pseudomonas aeruginosa Modulates the Expression of a Large Set of Genes and Proteins, Resulting in Altered Motility, Cytotoxicity, and Tobramycin Resistance.

Pseudomonas aeruginosa is a motile species that initiates swarming motility in response to specific environmental cues, i.e., a semisolid surface with amino acids as a nitrogen source (relevant to the human lung). Swarming is an intricately regulated process, but to date posttranscriptional regulation has not been extensively investigated. Small noncoding RNAs (sRNAs) are hypothesized to play posttranscriptional regulatory roles, largely through suppression of translation, and we previously demonstrated 20 sRNA species that were dysregulated under swarming conditions. One of these, sRNA PA0805.1 (which was 5-fold upregulated under swarming conditions), when cloned, transformed into wild-type (WT) PAO1, and overexpressed, led to broad phenotypic changes, including reduced swarming, swimming, and twitching motilities, as well as increased adherence, cytotoxicity, and tobramycin resistance. A ΔPA0805.1 deletion mutant was more susceptible to tobramycin than the WT under swarming conditions. The strain overexpressing PA0805.1 was compared to the empty-vector strain by transcriptome sequencing (RNA-Seq) and proteomics under swarming conditions to determine sRNA targets. Broad transcriptional and proteomic profiles showed 1,121 differentially expressed genes and 258 proteins with significantly different abundance. Importantly, these included 106 transcriptional regulators, two-component regulatory systems, and sigma and anti-sigma factors. Downstream of these regulators were found downregulated type IV pilus genes, many upregulated adherence and virulence factors, and two multidrug efflux systems, mexXY and mexGHI-opmD Therefore, the sRNA PA0805.1 appears to be a global regulator that influences diverse bacterial lifestyles, most likely through a regulatory cascade.IMPORTANCE P. aeruginosa is an opportunistic pathogen of humans. With roughly 10% of its genes encoding transcriptional regulators, and hundreds of small noncoding RNAs (sRNAs) interspersed throughout the genome, P. aeruginosa is able to fine-tune its response to adapt and survive in the host and resist antimicrobial agents. Understanding mechanisms of genetic regulation is therefore crucial to combat pathogenesis. The previously uncharacterized sRNA PA0805.1 was overexpressed in P. aeruginosa strain PAO1, resulting in decreased motility, increased adherence, cytotoxicity, and tobramycin resistance. In contrast, a ΔPA0805.1 deletion mutant had increased susceptibility to tobramycin under swarming conditions. Omic approaches uncovered 1,121 transcriptomic and 258 proteomic changes in the overexpression strain compared with the empty-vector strain, which included 106 regulatory factors. Downstream of these regulators were upregulated adherence factors, multidrug efflux systems, and virulence factors in both transcriptomics and proteomics. This study provides insights into the role of the sRNA PA0805.1 in modulating bacterial adaptations.

Whole genome sequencing and antibiotic diffusion assays, provide new insight on drug resistance in the genus Pedobacter.

ABSTRACTA total of four strains of the ‘environmental superbug’ Pedobacter isolated from sludge produced at Norwegian drinking water treatment plants, were characterized by whole genome sequencing and antibiotic susceptibility assays. As with previous studies on members of this genus, we found that the isolates were multi-drug resistant, and that this resistance included clinically important beta-lactams, aminoglycosides and the fluoroquinolone ciprofloxacin. Using the minION sequencing platform (Oxford Nanopore Technologies) combined with HiSeq PE150 Illumina sequencing data, the four isolates were assembled into genomes of single contigs. Analysis of the genomes revealed potential genetic factors possibly underlying some of the specific resistances observed. Metallo-beta-lactamase activity was detected in one isolate, and the same isolate contained a putative metallo-betalactamase gene resembling pedo-2. Furthermore, several genes related to multidrug efflux systems were found using the resistance database CARD. Additionally, the present study extends our knowledge on the phylogeny of this genus, adding four new genomes to the existing 50.

Vibrio cholerae OmpR Represses the ToxR Regulon in Response to Membrane Intercalating Agents That Are Prevalent in the Human Gastrointestinal Tract.

Multidrug efflux systems belonging to the resistance-nodulation-division (RND) superfamily are ubiquitous in Gram-negative bacteria. RND efflux systems are often associated with multiple antimicrobial resistance and also contribute to the expression of diverse bacterial phenotypes including virulence, as documented in the intestinal pathogen Vibrio cholerae, the causative agent of the severe diarrheal disease cholera. Transcriptomic studies with RND efflux-negative V. cholerae suggested that RND-mediated efflux was required for homeostasis, as loss of RND efflux resulted in the activation of transcriptional regulators, including multiple environmental sensing systems. In this report, we investigated six RND efflux-responsive regulatory genes for contributions to V. cholerae virulence factor production. Our data showed that the V. cholerae gene VC2714, encoding a homolog of Escherichia coli OmpR, was a virulence repressor. The expression of ompR was elevated in an RND-null mutant, and ompR deletion partially restored virulence factor production in the RND-negative background. Virulence inhibitory activity in the RND-negative background resulted from OmpR repression of the key ToxR regulon virulence activator aphB, and ompR overexpression in wild-type cells also repressed virulence through aphB We further show that ompR expression was not altered by changes in osmolarity but instead was induced by membrane-intercalating agents that are prevalent in the host gastrointestinal tract and which are substrates of the V. cholerae RND efflux systems. Our collective results indicate that V. choleraeompR is an aphB repressor and regulates the expression of the ToxR virulence regulon in response to novel environmental cues.

Complete Genome Sequence and Annotation of Campylobacter jejuni YH003, Isolated from Retail Chicken.

The complete genome sequence of Campylobacter jejuni YH003, isolated from retail chicken, was determined using PacBio and Illumina technologies. The assembled genome is 1,743,985 bp (G+C content of 30.3%). Genome annotation revealed several genes encoding virulence and antibiotic resistance factors, including a type VI secretion system, cytolethal distending toxins, and a multidrug efflux system.

Using Selection by Nonantibiotic Stressors to Sensitize Bacteria to Antibiotics.

Evolutionary adaptation of bacteria to nonantibiotic selective forces, such as osmotic stress, has been previously associated with increased antibiotic resistance, but much less is known about potentially sensitizing effects of nonantibiotic stressors. In this study, we use laboratory evolution to investigate adaptation of Enterococcus faecalis, an opportunistic bacterial pathogen, to a broad collection of environmental agents, ranging from antibiotics and biocides to extreme pH and osmotic stress. We find that nonantibiotic selection frequently leads to increased sensitivity to other conditions, including multiple antibiotics. Using population sequencing and whole-genome sequencing of single isolates from the evolved populations, we identify multiple mutations in genes previously linked with resistance to the selecting conditions, including genes corresponding to known drug targets or multidrug efflux systems previously tied to collateral sensitivity. Finally, we hypothesized based on the measured sensitivity profiles that sequential rounds of antibiotic and nonantibiotic selection may lead to hypersensitive populations by harnessing the orthogonal collateral effects of particular pairs of selective forces. To test this hypothesis, we show experimentally that populations evolved to a sequence of linezolid (an oxazolidinone antibiotic) and sodium benzoate (a common preservative) exhibit increased sensitivity to more stressors than adaptation to either condition alone. The results demonstrate how sequential adaptation to drug and nondrug environments can be used to sensitize bacteria to antibiotics and highlight new potential strategies for exploiting shared constraints governing adaptation to diverse environmental challenges.

The Anti-Campylobacter Activity and Mechanisms of Pinocembrin Action.

We investigated the anti-Campylobacter activity of pinocembrin and its mechanism of action, as well as Campylobacter responses to pinocembrin treatment at the genetic and phenotypic levels, using C. jejuni NCTC 11168 and a multidrug efflux system repressor mutant (11168ΔcmeR). At its minimal inhibitory concentration, pinocembrin significantly increased cell membrane permeability of Campylobacter. Interestingly, at sub-inhibitory concentrations, pinocembrin did not significantly alter membrane functionality and it increased bacterial fitness. Treatment with pinocembrin evoked decreased expression of ribosomal proteins and down-regulation of several NADH dehydrogenase I chain subunits and proteins involved in iron uptake. This suggests altered protein production and redox cycle and iron metabolism. Interestingly, the chelation of Fe ions during the treatment with pinocembrin increased C. jejuni survival, although there was no increase in the formation of reactive oxygen species. Pre-treatment of C. jejuni with sub-inhibitory concentrations of pinocembrin for 2 h resulted in a 1 log decrease in C. jejuni colony forming units in mice liver at 8 days post-infection, compared to untreated C. jejuni. These findings suggest that pinocembrin modulates the metabolic activity of C. jejuni and that pre-treatment of C. jejuni with pinocembrin influences its virulence potential in mice. This anti-Campylobacter potential of pinocembrin warrants further investigation.

Short communication: Whole-genome sequence analysis of 4 fecal blaCMY-2-producing Escherichia coli isolates from Holstein dairy calves

This study was carried out to determine the antimicrobial resistance (AMR) genes and mobile genetic elements of 4 fecal blaCMY-2-producing Escherichia coli isolated from Holstein dairy calves on the same farm using whole-genome sequencing. Genomic analysis revealed that 3 of the 4 isolates shared similar genetic features, including sequence type (ST), serotype, plasmid characteristics, insertion ST, and virulence genes. In addition to genes encoding for complex multidrug resistance efflux systems, all 4 isolates were carriers of genes conferring resistance to β-lactams (blaCMY-2, blaTEM-1B), tetracyclines (tetA, tetB, tetD), aminoglycosides [aadA1, aph(3")-lb, aph(6)-ld], sulfonamides (sul2), and trimethoprim (dfrA1). We also detected 4 incompatibility plasmid groups: Inc.F, Inc.N, Inc.I, and Inc.Q. A novel ST showing a new purA and mdh allelic combination was found. The 4 isolates were likely enterotoxigenic pathotypes of E. coli, based on serotype and presence of the plasmid Inc.FII(pCoo). This study provides information for comparative genomic analysis of AMR genes and mobile genetic elements. This analysis could give some explanation to the multidrug resistance characteristics of bacteria colonizing the intestinal tract of dairy calves in the first few weeks of life.

AcrB: a mean, keen, drug efflux machine.

Gram-negative bacteria are intrinsically resistant against cytotoxic substances by means of their outer membrane and a network of multidrug efflux systems, acting in synergy. Efflux pumps from various superfamilies with broad substrate preferences sequester and pump drugs across the inner membrane to supply the highly polyspecific and powerful tripartite resistance-nodulation-cell division (RND) efflux pumps with compounds to be extruded across the outer membrane barrier. In Escherichia coli, the tripartite efflux system AcrAB-TolC is the archetype RND multiple drug efflux pump complex. The homotrimeric inner membrane component acriflavine resistance B (AcrB) is the drug specificity and energy transduction center for the drug/proton antiport process. Drugs are bound and expelled via a cycle of mainly three consecutive states in every protomer, constituting a flexible alternating access channel system. This review recapitulates the molecular basis of drug and inhibitor binding, including mechanistic insights into drug efflux by AcrB. It also summarizes 17 years of mutational analysis of the gene acrB, reporting the effect of every substitution on the ability of E. coli to confer resistance toward antibiotics (http://goethe.link/AcrBsubstitutions). We emphasize the functional robustness of AcrB toward single-site substitutions and highlight regions that are more sensitive to perturbation.

Natural Alkaloid Berberine Activity against Pseudomonas aeruginosa MexXY-Mediated Aminoglycoside Resistance: In Silico and in Vitro Studies.

The multidrug efflux system MexXY-OprM, inside the resistance-nodulation-division family, is a major determinant of aminoglycoside resistance in Pseudomonas aeruginosa. In the fight aimed to identify potential efflux pump inhibitors among natural compounds, the alkaloid berberine emerged as a putative inhibitor of MexXY-OprM. In this work, we elucidated its interaction with the extrusor protein MexY and assessed its synergistic activity with aminoglycosides. In particular, we built an in silico model for the MexY protein in its trimeric association using both AcrB (E. coli) and MexB (P. aeruginosa) as 3D templates. This model has been stabilized in the bacterial cytoplasmic membrane using a molecular dynamics approach and used for ensemble docking to obtain the binding site mapping. Then, through dynamic docking, we assessed its binding affinity and its synergism with aminoglycosides focusing on tobramycin, which is widely used in the treatment of pulmonary infections. In vitro assays validated the data obtained: the results showed a 2-fold increase of the inhibitory activity and 2-4 log increase of the killing activity of the association berberine-tobramycin compared to those of tobramycin alone against 13/28 tested P. aeruginosa clinical isolates. From hemolytic assays, we preliminarily assessed berberine's low toxicity.

Gene Targets in Ocular Pathogenic Escherichia coli for Mitigation of Biofilm Formation to Overcome Antibiotic Resistance.

The present work is an attempt to establish the functionality of genes involved in biofilm formation and antibiotic resistance in an ocular strain of Escherichia coli (L-1216/2010) which was isolated and characterized from the Vitreous fluid of a patient with Endophthalmitis. For this purpose, seven separate gene-specific knockout mutants were generated by homologous recombination in ocular E. coli. The genes that were mutated included three transmembrane genes ytfR (ABC transporter ATP-binding protein), mdtO (multidrug efflux system) and tolA (inner membrane protein), ryfA coding for non-coding RNA and three metabolic genes mhpA (3-3-hydroxyphenylpropionate 1,2-dioxygenase), mhpB (2,3-di hydroxyphenylpropionate 1,2-dioxygenase), and bdcR (regulatory gene of bdcA). Mutants were validated by sequencing and Reverse transcription-PCR and monitored for biofilm formation by XTT method and confocal microscopy. The antibiotic susceptibility of the mutants was also ascertained. The results indicated that biofilm formation was inhibited in five mutants (ΔbdcR, ΔmhpA, ΔmhpB, ΔryfA, and ΔtolA) and the thickness of biofilm reduced from 17.2 μm in the wildtype to 1.5 to 4.8 μm in the mutants. Mutants ΔytfR and ΔmdtO retained the potential to form biofilm. Complementation of the mutants with the wild type gene restored biofilm formation potential in all mutants except in ΔmhpB. The 5 mutants which lost their ability to form biofilm (ΔbdcR, ΔmhpA, ΔmhpB, ΔtolA, and ΔryfA) did not exhibit any change in their susceptibility to Ceftazidime, Cefuroxime, Ciprofloxacin, Gentamicin, Cefotaxime, Sulfamethoxazole, Imipenem, Erythromycin, and Streptomycin in the planktonic phase compared to wild type ocular E. coli. But ΔmdtO was the only mutant with altered MIC to Sulfamethoxazole, Imipenem, Erythromycin, and Streptomycin both in the planktonic and biofilm phase. This is the first report demonstrating the involvement of the metabolic genes mhpA and mhpB and bdcR (regulatory gene of bdcA) in biofilm formation in ocular E. coli. In addition we provide evidence that tolA and ryfA are required for biofilm formation while ytfR and mdtO are not required. Mitigation of biofilm formation to overcome antibiotic resistance could be achieved by targeting the genes bdcR, mhpA, mhpB, ryfA, and tolA.

Phenotypical profile and global transcriptomic profile of Hypervirulent Klebsiella pneumoniae due to carbapenemase-encoding plasmid acquisition

BackgroundPlasmids play an vital role in driving the rapid global spread of antimicrobial resistance and adaptation to changing ambient conditions. It has been suggested that the presence of plasmids can pose tremendous impacts on the host physiology. However, little is known regarding the contributions of carbapenemase-encoding plasmid carriage on the physiology and pathogenicity of hypervirulent K. pneumoniae (hvKP).ResultsHere we performed a transcriptomic analysis of hvKP with or without carbapenemase-encoding plasmid p24835-NDM5. The results had shown 683 genes with differential expression (false discovery rate, ≤0.001; > 2-fold change), of which 107 were up-regulated and 576 were down-regulated. Gene groups with functions relating to carbohydrate metabolism and multidrug efflux system were increased in genes with increased expression, and those relating to capsule biosynthesis and virulence factors were increased in the genes with decreased expression. In agreement with these changes, survival rate of TfpNDM-hvKP in the presence of normal human serum decreased, and competitive index (CI values) indicated significant fitness defects in the plasmid-carrying hvKP strain when co-cultured with its plasmid-free isogenic ancestor and the ATCC control. Moreover, the p24835-NDM5-containing hvKP strain retained its high neutrophil-mediated phagocytosis and murine lethality.ConclusionThese data indicate that hvKP responds to carbapenemase-encoding plasmid by altering the expression of genes involved in carbohydrate metabolism, antibiotic resistance, capsule biosynthesis and virulence expression. Apart from antibiotic resistance selective advantages, carbapenemase-encoding plasmid carriage may also lead to virulence change or adaption to specific habitats in hvKP strain.

Efflux pump inhibitors of clinically relevant multidrug resistant bacteria.

Bacterial infections are an increasingly serious issue worldwide. The inability of existing therapies to treat multidrug-resistant pathogens has been recognized as an important challenge of the 21st century. Efflux pumps are important in both intrinsic and acquired bacterial resistance and identification of small molecule efflux pump inhibitors (EPIs), capable of restoring the effectiveness of available antibiotics, is an active research field. In the last two decades, much effort has been made to identify novel EPIs. However, none of them has so far been approved for therapeutic use. In this article, we explore different structural families of currently known EPIs for multidrug resistance efflux systems in the most extensively studied pathogens (NorA in Staphylococcus aureus, AcrAB-TolC in Escherichia coli, and MexAB-OprM in Pseudomonas aeruginosa). Both synthetic and natural compounds are described, with structure-activity relationship studies and optimization processes presented systematically for each family individually. In vitro activities against selected test strains are presented in a unifying manner for all the EPIs described, together with the most important toxicity, pharmacokinetic and in vivo efficacy data. A critical evaluation of lead-likeness characteristics and the potential for clinical development of the most promising inhibitors of the three efflux systems is described. This overview of EPIs is a good starting point for the identification of novel effective antibacterial drugs.

A Molecular Modeling Approach to Identify Novel Inhibitors of the Major Facilitator Superfamily of Efflux Pump Transporters.

Multidrug efflux systems play a prominent role in medicine, as they are important contributors to bacterial antibiotic resistance. NorA is an efflux pump transporter from the major facilitator superfamily that expels numerous drug compounds across the inner membrane of Staphylococcus aureus (S. aureus). The design of novel inhibitors to combat drug efflux could offer new opportunities to avoid the problem of antibiotic resistance. In this study, we performed molecular modeling studies in an effort to discover novel NorA efflux pump inhibitors. A group of over 673 compounds from the PubChem database with a high (>80%) level of similarity to the chemical structure of capsaicin was used to study the binding affinity of small molecule compounds for the NorA efflux pump. Ten potential lead compounds displayed a good druggability profile, with one in particular (CID 44330438) providing new insight into the molecular mechanism of the inhibition of major facilitator superfamily (MFS) efflux pump transporters. It is our hope that the overall strategy described in this study, and the structural information of the potential novel inhibitors thus identified, will stimulate others to pursue the development of better drugs to tackle multidrug resistance in S. aureus.

Macrolides and Fluoroquinolones Resistance Mechanisms in Campylobacters and their Incidence in Egypt; a Review Article

Campylobacter species are primarily zoonotic pathogens and recognized as a major cause of human illnesses. Poultry, especially, chicken is the main reservoir of Campylobacter species. The uncontrolled use of antibiotics in prophylaxis and treatment of animals caused an increase in antibiotic resistance to macrolides and fluoroquinolones (FQ) which are considered the drugs of choice for treatment of Campylobacter infection. Recently, studies suggested that multidrug efflux systems have the main role in lowering the efficacy of new and old antibiotics. Consequently, efforts are made to find suitable substances to reverse the action of the efflux pumps and prevent antimicrobial resistance. The substances used for evaluation of efflux pumps modulation are either efflux pump inhibitors (EPIs) or efflux pump inducers. The different types of EPIs; phenylalanine arginyl β-naphthylamide, verapamil and phenothiazines, of different mode of actions were used to suppress the activity of different types of efflux pumps. Aspirin, a drug in livestock and poultry, is a nonsteroidal anti-inflammatory, which induces non-heritable resistance of different bacteria to multiple antibiotics. Moreover, it also increases campylobacter resistance to antimicrobials. Alpha-tocopherol represents a new alternative approach against bacterial resistance. It shows modulatory activity on efflux system, showing clinically relevant results. Therefore, it is important to study different resistance mechanisms of campylobacters.