Expression of the Fluoroquinolones Efflux Pump Genes acrA and mdfA in Urinary Escherichia coli Isolates.
Abdelhamid Sarah M,Abozahra Rania R
Polish journal of microbiology
Escherichia coli is one of the most frequent causes of urinary tract infections. Efflux system overexpression is reported to contribute to E. coli resistance to several antibiotics. Our aim in this study was to investigate the relation between antibiotic resistance and the expression of the efflux pump genes acrA and mdfA in E. coli by real-time reverse transcription-PCR. We tested the in vitro susceptibilities to 12 antibiotics in 28 clinical isolates of E. coli obtained from urine samples. We also determined the minimum inhibitory concentrations of levofloxacin to these samples. We then revealed significant correlations between the overexpression of both mdfA and acrA and MICs of levofloxacin. In conclusion, we demonstrated that the increased expression of efflux pump genes such as mdfA and acrA can lead to levofloxacin resistance in E. coli. These findings contribute to further understanding of the molecular mechanisms of efflux pump systems and how they contribute to antibiotic resistance.
In situ structure and assembly of the multidrug efflux pump AcrAB-TolC.
Shi Xiaodong,Chen Muyuan,Yu Zhili,Bell James M,Wang Hans,Forrester Isaac,Villarreal Heather,Jakana Joanita,Du Dijun,Luisi Ben F,Ludtke Steven J,Wang Zhao
Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug efflux pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug efflux pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled efflux pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with efflux pump inhibitors.
Expression of multidrug efflux pump genes acrAB-tolC, mdfA, and norE in Escherichia coli clinical isolates as a function of fluoroquinolone and multidrug resistance.
Swick Michelle C,Morgan-Linnell Sonia K,Carlson Kimberly M,Zechiedrich Lynn
Antimicrobial agents and chemotherapy
In a single quantitative study, we measured acrA, acrB, tolC, mdfA, and norE expression in Escherichia coli clinical isolates by using real-time PCR. acrA and acrB overexpression strongly correlated with fluoroquinolone and multidrug resistance; tolC, mdfA, and norE expression did not. The order of abundance of efflux pump transcripts in all fluoroquinolone-susceptible isolates was tolC (highest), then acrA and acrB, and then mdfA and norE. Our findings suggest acrAB overexpression is an indicator of multidrug resistance.
Ineffectiveness of topoisomerase mutations in mediating clinically significant fluoroquinolone resistance in Escherichia coli in the absence of the AcrAB efflux pump.
Oethinger M,Kern W V,Jellen-Ritter A S,McMurry L M,Levy S B
Antimicrobial agents and chemotherapy
Fluoroquinolone-resistant mutants, selected from a wild-type Escherichia coli K-12 strain and its Mar mutant by exposure to increasing levels of ofloxacin on solid medium, were analyzed by Northern (RNA) blot analysis, sequencing, and radiolabelled ciprofloxacin accumulation studies. Mutations in the target gene gyrA (DNA gyrase), the regulatory gene marR, and additional, as yet unidentified genes (genes that probably affect efflux mediated by the multidrug efflux pump AcrAB) all contributed to fluoroquinolone resistance. Inactivation of the acrAB locus made all strains, including those with target gene mutations, hypersusceptible to fluoroquinolones and certain other unrelated drugs. These studies indicate that, in the absence of the AcrAB pump, gyrase mutations fail to produce clinically relevant levels of fluoroquinolone resistance.
AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli multiple-antibiotic-resistance (Mar) mutants.
Okusu H,Ma D,Nikaido H
Journal of bacteriology
Multiple-antibiotic-resistance (Mar) mutants of Escherichia coli are resistant to a wide variety of antibiotics, and increased active efflux is known to be responsible for the resistance to some drugs. The identity of the efflux system, however, has remained unknown. By constructing an isogenic set of E. coli K-12 strains, we showed that the marR1 mutation was incapable of increasing the resistance level in the absence of the AcrAB efflux system. This experiment identified the AcrAB system as the major pump responsible for making the Mar mutants resistant to many agents, including tetracycline, chloramphenicol, ampicillin, nalidixic acid, and rifampin.
Contribution of β-lactamase and efflux pump overproduction to tazobactam-piperacillin resistance in clinical isolates of Escherichia coli.
Suzuki Yuuki,Sato Toyotaka,Fukushima Yukari,Nakajima Chie,Suzuki Yasuhiko,Takahashi Satoshi,Yokota Shin-Ichi
International journal of antimicrobial agents
INTRODUCTION:Tazobactam-piperacillin (TZP) is a mixture of a broad-spectrum penicillin and an irreversible β-lactamase inhibitor. TZP is effective against Gram-negative bacteria that produce extended-spectrum β-lactamases, and it is used as a first-line or second-line drug to treat serious infections. METHODS:This study identified three TZP-resistant and two TZP-intermediate strains among 514 clinical isolates of Escherichia coli. RESULTS:These five isolates possessed one or more β-lactamase genes, bla, bla, bla, and/or bla. The expression levels of β-lactamase genes and acrAB genes in the strains were examined by using real-time reverse transcription PCR. The total enzymatic piperacillin-degrading activity in cells was determined. Two TZP-resistance mechanisms were identified: hyperproduction of TEM-1 in the two resistant strains; and simultaneous high production of β-lactamase and efflux pump AcrAB in the two TZP-intermediate isolates. The latter are an international high-risk clone O25b:H4-ST131-H30R. CONCLUSION:TZP resistance is still rare in clinical isolates of E. coli. However, resistance can develop on high production and/or combinations of known antimicrobial resistance mechanisms in different ways.
Mutations that increase expression of the EmrAB-TolC efflux pump confer increased resistance to nitroxoline in Escherichia coli.
Puértolas-Balint Fabiola,Warsi Omar,Linkevicius Marius,Tang Po-Cheng,Andersson Dan I
The Journal of antimicrobial chemotherapy
OBJECTIVES:To determine the mechanism of resistance to the antibiotic nitroxoline in Escherichia coli. METHODS:Spontaneous nitroxoline-resistant mutants were selected at different concentrations of nitroxoline. WGS and strain reconstruction were used to define the genetic basis for the resistance. The mechanistic basis of resistance was determined by quantitative PCR (qPCR) and by overexpression of target genes. Fitness costs of the resistance mutations and cross-resistance to other antibiotics were also determined. RESULTS:Mutations in the transcriptional repressor emrR conferred low-level resistance to nitroxoline [nitroxoline MIC (MICNOX)=16 mg/L] by increasing the expression of the emrA and emrB genes of the EmrAB-TolC efflux pump. These resistant mutants showed no fitness reduction and displayed cross-resistance to nalidixic acid. Second-step mutants with higher-level resistance (MICNOX=32-64 mg/L) had mutations in the emrR gene, together with either a 50 kb amplification, a mutation in the gene marA, or an IS upstream of the lon gene. The latter mutations resulted in higher-level nitroxoline resistance due to increased expression of the tolC gene, which was confirmed by overexpressing tolC from an inducible plasmid in a low-level resistance mutant. Furthermore, the emrR mutations conferred a small increase in resistance to nitrofurantoin only when combined with an nfsAB double-knockout mutation. However, nitrofurantoin-resistant nfsAB mutants showed no cross-resistance to nitroxoline. CONCLUSIONS:Mutations in different genes causing increased expression of the EmrAB-TolC pump lead to an increased resistance to nitroxoline. The structurally similar antibiotics nitroxoline and nitrofurantoin appear to have different modes of action and resistance mechanisms.
Correlation of overexpression of efflux pump genes with antibiotic resistance in Escherichia coli Strains clinically isolated from urinary tract infection patients.
Yasufuku Tomihiko,Shigemura Katsumi,Shirakawa Toshiro,Matsumoto Minori,Nakano Yuzo,Tanaka Kazushi,Arakawa Soichi,Kinoshita Shouhiro,Kawabata Masato,Fujisawa Masato
Journal of clinical microbiology
Escherichia coli is one of the most common pathogens in urinary tract infections (UTIs), and antibiotic resistance in E. coli is becoming a serious problem in treating UTI. Efflux system overexpression is reported to contribute to E. coli resistance to several antibiotics. This study investigated the correlation of antibiotic susceptibilities with the overexpression of the efflux pump genes such as marA, yhiU, yhiV, and mdfA and with risk factors for antibiotic resistance in E. coli isolated from UTI patients. We examined the expression level of efflux pump genes using quantitative real-time reverse transcription-PCR (qRT-PCR). We also tested the in vitro susceptibilities to 12 kinds of antibiotics in 64 clinical strains of E. coli isolated from UTI patients. By multivariate analyses we revealed significant relationships between the overexpression of (i) marA and MICs of cefepime (FEP) and nalidixic acid (NAL), (ii) yhiV and MICs of minocycline (MIN), and (iii) mdfA and MICs of sitafloxacin (STX). In our investigation of the efflux pump genes, risk factors such as gender and the previous use of fluoroquinolones correlated with the overexpression of marA, and indwelling catheter use correlated with the overexpression of mdfA. In conclusion, we demonstrated that the increased expression of efflux pump genes such as marA and mdfA can lead to fluoroquinolone resistance in E. coli. These results contribute to our knowledge of the efflux system and raise the possibility of developing new agents, such as efflux pump inhibitors (EPIs), to antibiotic-resistant E. coli.
Contribution of the AcrAB-TolC efflux pump to high-level fluoroquinolone resistance in Escherichia coli isolated from dogs and humans.
Sato Toyotaka,Yokota Shin-ichi,Okubo Torahiko,Ishihara Kanako,Ueno Hiroshi,Muramatsu Yasukazu,Fujii Nobuhiro,Tamura Yutaka
The Journal of veterinary medical science
Fluoroquinolone resistance is mainly caused by mutations in quinolone resistance-determining regions of DNA gyrase and topoisomerase IV in Escherichia coli. The AcrAB-TolC efflux pump contributes to resistance against fluoroquinolone and other antimicrobials. In this study, we investigated a high-level mechanism of fluoroquinolone resistance in E. coli that was isolated from human clinical samples and canine fecal samples. E. coli strains with high levels of fluoroquinolone resistance have been found to be frequently resistant to cephalosporins. Strains with high-level fluoroquinolone resistance exhibited lower intracellular enrofloxacin (ENR) concentrations, higher expression of AcrA, and a greater reduction in the fluoroquinolone minimum inhibitory concentration for treatment with an efflux pump inhibitor. The frequency of strains with enhanced ENR resistance selection and the survival rate of E. coli in the presence of ENR in vitro were correlated well with AcrA protein expression levels in the parental strains. These results suggest that AcrAB-TolC efflux pump over-expression is related to high-level fluoroquinolone resistance and the selection of strains with enhanced fluoroquinolone resistance.
Reviving Antibiotics: Efflux Pump Inhibitors That Interact with AcrA, a Membrane Fusion Protein of the AcrAB-TolC Multidrug Efflux Pump.
Abdali Narges,Parks Jerry M,Haynes Keith M,Chaney Julie L,Green Adam T,Wolloscheck David,Walker John K,Rybenkov Valentin V,Baudry Jerome,Smith Jeremy C,Zgurskaya Helen I
ACS infectious diseases
Antibiotic resistance is a major threat to human welfare. Inhibitors of multidrug efflux pumps (EPIs) are promising alternative therapeutics that could revive activities of antibiotics and reduce bacterial virulence. Identification of new druggable sites for inhibition is critical for the development of effective EPIs, especially in light of constantly emerging resistance. Here, we describe EPIs that interact with periplasmic membrane fusion proteins, critical components of efflux pumps that are responsible for the activation of the transporter and the recruitment of the outer-membrane channel. The discovered EPIs bind to AcrA, a component of the prototypical AcrAB-TolC pump, change its structure in vivo, inhibit efflux of fluorescent probes, and potentiate the activities of antibiotics in Escherichia coli and other Gram-negative bacteria. Our findings expand the chemical and mechanistic diversity of EPIs, suggest the mechanism for regulation of the efflux pump assembly and activity, and provide a promising path for reviving the activities of antibiotics in resistant bacteria.
Identification of Binding Sites for Efflux Pump Inhibitors of the AcrAB-TolC Component AcrA.
Darzynkiewicz Zbigniew M,Green Adam T,Abdali Narges,Hazel Anthony,Fulton Ronnie L,Kimball Joseph,Gryczynski Zygmunt,Gumbart James C,Parks Jerry M,Smith Jeremy C,Zgurskaya Helen I
The overexpression of multidrug efflux pumps is an important mechanism of clinical resistance in Gram-negative bacteria. Recently, four small molecules were discovered that inhibit efflux in Escherichia coli and interact with the AcrAB-TolC efflux pump component AcrA. However, the binding site(s) for these molecules was not determined. Here, we combine ensemble docking and molecular dynamics simulations with tryptophan fluorescence spectroscopy, site-directed mutagenesis, and antibiotic susceptibility assays to probe binding sites and effects of binding of these molecules. We conclude that clorobiocin and SLU-258 likely bind at a site located between the lipoyl and β-barrel domains of AcrA.
Effectiveness of Efflux Pump Inhibitors as Biofilm Disruptors and Resistance Breakers in Gram-Negative (ESKAPEE) Bacteria.
Reza Akif,Sutton J Mark,Rahman Khondaker Miraz
Antibiotics (Basel, Switzerland)
Antibiotic resistance represents a significant threat to the modern healthcare provision. The ESKAPEE pathogens ( and ), in particular, have proven to be especially challenging to treat, due to their intrinsic and acquired ability to rapidly develop resistance mechanisms in response to environmental threats. The development of biofilm has been characterised as an essential contributing factor towards antimicrobial-resistance and tolerance. Several studies have implicated the involvement of efflux pumps in antibiotic resistance, both directly, via drug extrusion and indirectly, through the formation of biofilm. As a result, the underlying mechanism of these pumps has attracted considerable interest due to the potential of targeting these protein structures and developing novel adjunct therapies. Subsequent investigations have revealed the ability of efflux pump-inhibitors (EPIs) to block drug-extrusion and disrupt biofilm formation, thereby, potentiating antibiotics and reversing resistance of pathogen towards them. This review will discuss the potential of EPIs as a possible solution to antimicrobial resistance, examining different challenges to the design of these compounds, with an emphasis on Gram-negative ESKAPEE pathogens.
Efflux pump-mediated antibiotics resistance: insights from computational structural biology.
Fischer Nadine,Raunest Martin,Schmidt Thomas H,Koch Dennis C,Kandt Christian
Interdisciplinary sciences, computational life sciences
The continuous rise of bacterial resistance against formerly effective pharmaceuticals is a major challenge for biomedical research. Since the first computational studies published seven years ago the simulation-based investigation of antibiotics resistance mediated by multidrug efflux pumps of the resistance nodulation division (RND) protein super family has grown into a vivid field of research. Here we review the employment of molecular dynamics computer simulations to investigate RND efflux pumps focusing on our group's recent contributions to this field studying questions of energy conversion and substrate transport in the inner membrane antiporter AcrB in Escherichia coli as well as access regulation and gating mechanism in the outer membrane efflux ducts TolC and OprM in E. coli and Pseudomonas aeruginosa.
The pseudo-atomic structure of an RND-type tripartite multidrug efflux pump.
Du Dijun,Voss Jarrod,Wang Zhao,Chiu Wah,Luisi Ben F
Microorganisms encode several classes of transmembrane molecular pumps that can expel a wide range of chemically distinct toxic substances. These machines contribute to the capacity of the organisms to withstand harsh environments, and they help to confer resistance against clinical antimicrobial agents. In Gram-negative bacteria, some of the pumps comprise tripartite assemblies that actively transport drugs and other harmful compounds across the cell envelope. We describe recent structural and functional data that have provided insights into the architecture and transport mechanism of the AcrA-AcrB-TolC pump of Escherichia coli. This multidrug efflux pump is powered by proton electrochemical gradients through the activity of AcrB, a member of the resistance/nodulation/cell division (RND) transporter family. Crystallographic data reveal how the small protein AcrZ binds to AcrB in a concave surface of the transmembrane domain, and we discuss how this interaction may affect the efflux activities of the transporter.
Efflux pump inhibitor CCCP to rescue colistin susceptibility in mcr-1 plasmid-mediated colistin-resistant strains and Gram-negative bacteria.
Baron Sophie A,Rolain Jean-Marc
The Journal of antimicrobial chemotherapy
Objectives:Efflux in bacteria is a ubiquitous mechanism associated with resistance to antimicrobials agents. Efflux pump inhibitors (EPIs) have been developed to inhibit efflux mechanisms and could be a good alternative to reverse colistin resistance, but only CCCP has shown good activity. The aim of our study was to identify CCCP activity in a collection of 93 Gram-negative bacteria with known and unknown colistin resistance mechanisms including isolates with mcr-1 plasmid-mediated colistin resistance. Methods:Colistin MIC was evaluated with and without CCCP and the fold decrease of colistin MIC was calculated for each strain. In order to evaluate the effect of this combination, a time-kill study was performed on five strains carrying different colistin resistance mechanisms. Results:Overall, CCCP was able to reverse colistin resistance for all strains tested. The effect of CCCP was significantly greater on intrinsically colistin-resistant bacteria (i.e. Proteus spp., Serratia marcescens, Morganella morganii and Providencia spp.) than on other Enterobacteriaceae (P < 0.0001). The same was true for bacteria with a heteroresistance mechanism compared to bacteria with other colistin resistance mechanisms (P < 0.0001). A time-kill study showed the combination was bacteriostatic on strains tested. Conclusions:These results suggest an efflux mechanism, especially on intrinsically resistant bacteria and Enterobacter spp., but further analysis is needed to identify the molecular support of this mechanism. EPIs could be an alternative for restoring colistin activity in Gram-negative bacteria. Further work is necessary to identify new EPIs that could be used in humans.
Biased partitioning of the multidrug efflux pump AcrAB-TolC underlies long-lived phenotypic heterogeneity.
Bergmiller Tobias,Andersson Anna M C,Tomasek Kathrin,Balleza Enrique,Kiviet Daniel J,Hauschild Robert,Tkačik Gašper,Guet Călin C
Science (New York, N.Y.)
The molecular mechanisms underlying phenotypic variation in isogenic bacterial populations remain poorly understood. We report that AcrAB-TolC, the main multidrug efflux pump of exhibits a strong partitioning bias for old cell poles by a segregation mechanism that is mediated by ternary AcrAB-TolC complex formation. Mother cells inheriting old poles are phenotypically distinct and display increased drug efflux activity relative to daughters. Consequently, we find systematic and long-lived growth differences between mother and daughter cells in the presence of subinhibitory drug concentrations. A simple model for biased partitioning predicts a population structure of long-lived and highly heterogeneous phenotypes. This straightforward mechanism of generating sustained growth rate differences at subinhibitory antibiotic concentrations has implications for understanding the emergence of multidrug resistance in bacteria.
An allosteric transport mechanism for the AcrAB-TolC multidrug efflux pump.
Wang Zhao,Fan Guizhen,Hryc Corey F,Blaza James N,Serysheva Irina I,Schmid Michael F,Chiu Wah,Luisi Ben F,Du Dijun
Bacterial efflux pumps confer multidrug resistance by transporting diverse antibiotics from the cell. In Gram-negative bacteria, some of these pumps form multi-protein assemblies that span the cell envelope. Here, we report the near-atomic resolution cryoEM structures of the AcrAB-TolC multidrug efflux pump in resting and drug transport states, revealing a quaternary structural switch that allosterically couples and synchronizes initial ligand binding with channel opening. Within the transport-activated state, the channel remains open even though the pump cycles through three distinct conformations. Collectively, our data provide a dynamic mechanism for the assembly and operation of the AcrAB-TolC pump.
Regulation of the AcrAB-TolC efflux pump in Enterobacteriaceae.
Weston Natasha,Sharma Prateek,Ricci Vito,Piddock Laura J V
Research in microbiology
Bacterial multidrug efflux systems are a major mechanism of antimicrobial resistance and are fundamental to the physiology of Gram-negative bacteria. The resistance-nodulation-division (RND) family of efflux pumps is the most clinically significant, as it is associated with multidrug resistance. Expression of efflux systems is subject to multiple levels of regulation, involving local and global transcriptional regulation as well as post-transcriptional and post-translational regulation. The best-characterised RND system is AcrAB-TolC, which is present in Enterobacteriaceae. This review describes the current knowledge and new data about the regulation of the acrAB and tolC genes in Escherichia coli and Salmonella enterica.
Importance of Real-Time Assays To Distinguish Multidrug Efflux Pump-Inhibiting and Outer Membrane-Destabilizing Activities in Escherichia coli.
Misra Rajeev,Morrison Keith D,Cho Hyun Jae,Khuu Thanh
Journal of bacteriology
UNLABELLED:The constitutively expressed AcrAB multidrug efflux system of Escherichia coli shows a high degree of homology with the normally silent AcrEF system. Exposure of a strain with acrAB deleted to antibiotic selection pressure frequently leads to the insertion sequence-mediated activation of the homologous AcrEF system. In this study, we used strains constitutively expressing either AcrAB or AcrEF from their normal chromosomal locations to resolve a controversy about whether phenylalanylarginine β-naphthylamide (PAβN) inhibits the activities of AcrAB and AcrEF and/or acts synergistically with antibiotics by destabilizing the outer membrane permeability barrier. Real-time efflux assays allowed a clear distinction between the efflux pump-inhibiting activity of PAβN and the outer membrane-destabilizing action of polymyxin B nonapeptide (PMXBN). When added in equal amounts, PAβN, but not PMXBN, strongly inhibited the efflux activities of both AcrAB and AcrEF pumps. In contrast, when outer membrane destabilization was assessed by the nitrocefin hydrolysis assay, PMXBN exerted a much greater damaging effect than PAβN. Strong action of PAβN in inhibiting efflux activity compared to its weak action in destabilizing the outer membrane permeability barrier suggests that PAβN acts mainly by inhibiting efflux pumps. We concluded that at low concentrations, PAβN acts specifically as an inhibitor of both AcrAB and AcrEF efflux pumps; however, at high concentrations, PAβN in the efflux-proficient background not only inhibits efflux pump activity but also destabilizes the membrane. The effects of PAβN on membrane integrity are compounded in cells unable to extrude PAβN. IMPORTANCE:The increase in multidrug-resistant bacterial pathogens at an alarming rate has accelerated the need for implementation of better antimicrobial stewardship, discovery of new antibiotics, and deeper understanding of the mechanism of drug resistance. The work carried out in this study highlights the importance of employing real-time fluorescence-based assays in differentiating multidrug efflux-inhibitory and outer membrane-destabilizing activities of antibacterial compounds.
Molecular mechanism of MBX2319 inhibition of Escherichia coli AcrB multidrug efflux pump and comparison with other inhibitors.
Vargiu Attilio V,Ruggerone Paolo,Opperman Timothy J,Nguyen Son T,Nikaido Hiroshi
Antimicrobial agents and chemotherapy
Efflux pumps of the resistance nodulation division (RND) superfamily, such as AcrB, make a major contribution to multidrug resistance in Gram-negative bacteria. The development of inhibitors of the RND pumps would improve the efficacy of current and next-generation antibiotics. To date, however, only one inhibitor has been cocrystallized with AcrB. Thus, in silico structure-based analysis is essential for elucidating the interaction between other inhibitors and the efflux pumps. In this work, we used computer docking and molecular dynamics simulations to study the interaction between AcrB and the compound MBX2319, a novel pyranopyridine efflux pump inhibitor with potent activity against RND efflux pumps of Enterobacteriaceae species, as well as other known inhibitors (D13-9001, 1-[1-naphthylmethyl]-piperazine, and phenylalanylarginine-β-naphthylamide) and the binding of doxorubicin to the efflux-defective F610A variant of AcrB. We also analyzed the binding of a substrate, minocycline, for comparison. Our results show that MBX2319 binds very tightly to the lower part of the distal pocket in the B protomer of AcrB, strongly interacting with the phenylalanines lining the hydrophobic trap, where the hydrophobic portion of D13-9001 was found to bind by X-ray crystallography. Additionally, MBX2319 binds to AcrB in a manner that is similar to the way in which doxorubicin binds to the F610A variant of AcrB. In contrast, 1-(1-naphthylmethyl)-piperazine and phenylalanylarginine-β-naphthylamide appear to bind to somewhat different areas of the distal pocket in the B protomer of AcrB than does MBX2319. However, all inhibitors (except D13-9001) appear to distort the structure of the distal pocket, impairing the proper binding of substrates.
Random mutagenesis of the multidrug transporter AcrB from Escherichia coli for identification of putative target residues of efflux pump inhibitors.
Schuster Sabine,Kohler Samay,Buck Annika,Dambacher Christine,König Armin,Bohnert Jürgen A,Kern Winfried V
Antimicrobial agents and chemotherapy
Efflux is an important mechanism of bacterial multidrug resistance (MDR), and the inhibition of MDR pumps by efflux pump inhibitors (EPIs) could be a promising strategy to overcome MDR. 1-(1-Naphthylmethyl)-piperazine (NMP) and phenylalanine-arginine-β-naphthylamide (PAβN) are model EPIs with activity in various Gram-negative bacteria expressing AcrB, the major efflux pump of Escherichia coli, or similar homologous pumps of the resistance-nodulation-cell division class. The aim of the present study was to generate E. coli AcrB mutants resistant to the inhibitory action of the two model EPIs and to identify putative EPI target residues in order to better understand mechanisms of pump inhibition. Using an in vitro random mutagenesis approach focusing on the periplasmic domain of AcrB, we identified the double mutation G141D N282Y, which substantially compromised the synergistic activity of NMP with linezolid, was associated with similar intracellular linezolid concentrations in the presence and absence of NMP, and did not impair the intrinsic MICs of various pump substrates and dye accumulation. We propose that these mutations near the outer face of the distal substrate binding pocket reduce NMP trapping. Other residues found to be relevant for efflux inhibition by NMP were G288 and A279, but mutations at these sites also changed the susceptibility to several pump substrates. Unlike with NMP, we were unable to generate AcrB periplasmic domain mutants with resistance or partial resistance to the EPI activity of PAβN, which is consistent with the modes of action of PAβN differing from those of NMP.
Identification of Efflux Pump Genes and Detection of Mutation in Efflux Repressor from Omeprazole Responsive Multidrug-Resistant Isolates Causing Urinary Tract Infections.
Chowdhury Nandan,Suhani Sabrina,Purkaystha Auditi,Begum Musammat Kulsuma,Raihan Topu,Alam Md Jahangir,Islam Kamrul,Azad Abul Kalam
Antimicrobial resistance poses a threat in the treatment of infectious diseases in Bangladesh as well as in the world. Multidrug-resistant (MDR) Enterobacteriaceae, the most common cause of one such infectious disease, urinary tract infection (UTI), has contributed to the escalating problem of selecting empiric antibiotics against UTIs. The aim of this study was to investigate the presence of the efflux pump in MDR isolates from UTI in the North-East region of Bangladesh, to isolate and characterize the AcrAB-TolC efflux pump genes of these locally isolated strains and to do mutation analysis of the efflux pump repressor gene to understand the AcrAB-TolC efflux pump mechanism. In the presence of omeprazole, an efflux pump inhibitor, every MDR isolate showed increased susceptibility to at least 1 of the 7 antibiotics investigated, indicating that efflux pump might be involved in their antibiotic resistance. Omeprazole decreased the minimum inhibitory concentration of every antibiotics being investigated by 2- to 8-fold. DNA and the deduced amino acid sequences of the polymerase chain reaction (PCR) products analyzed by bioinformatics tools revealed that the chromosomal and genes were present in all MDR and antibiotic-susceptible isolates. However, the deduced amino acid sequences of the amplification refractory mutation system (ARMS) PCR product of the gene revealed that the substitution of arginine to cysteine at position 45 of AcrR was observed only in the MDR whose antibiotic susceptibility increased in the presence of omeprazole. Data reported herein support the notion that the increased antibiotic susceptibility of the MDR isolates in the presence of omeprazole might be due to efflux pump(s) inhibition and the AcrAB-TolC efflux pump might be a contributor to antibiotic resistance when the mutation of arginine to cysteine occurs at position 45 of AcrR.
AcrAB-TolC efflux pump system plays a role in carbapenem non-susceptibility in Escherichia coli.
Chetri Shiela,Bhowmik Deepshikha,Paul Deepjyoti,Pandey Piyush,Chanda Debadatta Dhar,Chakravarty Atanu,Bora Debajyoti,Bhattacharjee Amitabha
BACKGROUND:Efflux pump mediated antibiotic resistance is an unnoticed and undetected mechanism in clinical microbiology laboratory. RND efflux systems are known for aminoglycoside and tetracycline resistance whereas their role in carbapenem non-susceptibility is not established. The study was undertaken to investigate the role of efflux pump in providing resistance against carbapenems and their response against concentration gradient carbapenem stress on the transcriptional level of the AcrAB gene in the clinical isolates of Escherichia coli from a tertiary referral hospital of Northeast India. RESULTS:Out of 298 non-susceptible Escherichia coli isolates 98 isolates were found to have efflux pump mediated carbapenem non-susceptibility. Among them thirty-five were non carbapenemase producers and their expressional levels were verified using qRT-PCR under concentration gradient carbapenem stress. In this study, a strong correlation between ertapenem resistance and AcrA overexpression was observed which has not been reported previously. Further, it was observed that imipenem stress increased AcrB expression in Escherichia coli which holds the novelty of this study. Additionally, the transcription of AcrR was insistently increased which is much higher than the transcriptional level of AcrA under concentration gradient carbapenem stress condition. CONCLUSION:The study established that AcrAB pump is a relevant antibiotic resistance determinant in bacterial pathogen, has an important role in developing resistance against carbapenem group of antibiotics.
Structural and mechanistic insights into polymyxin resistance mediated by EptC originating from Escherichia coli.
Zhao Yanqun,Meng Qiang,Lai Yujie,Wang Li,Zhou Dan,Dou Chao,Gu Yijun,Nie Chunlai,Wei Yuquan,Cheng Wei
The FEBS journal
Gram-negative bacteria defend against the toxicity of polymyxins by modifying their outer membrane lipopolysaccharide (LPS). This modification mainly occurs through the addition of cationic molecules such as phosphoethanolamine (PEA). EcEptC is a PEA transferase from Escherichia coli (E. coli). However, unlike its homologs CjEptC (Campylobacter jejuni) and MCR-1, EcEptC is unable to mediate polymyxin resistance when overexpressed in E. coli. Here, we report crystal structures of the C-terminal putative catalytic domain (EcEptCΔN, 205-577 aa) of EcEptC in apo and Zn -bound states at 2.10 and 2.60 Å, respectively. EcEptCΔN is arranged into an α-β-α fold and equipped with the zinc ion in a conserved mode. Coupled with isothermal titration calorimetry (ITC) data, we provide insights into the mechanism by which EcEptC recognizes Zn . Furthermore, structure comparison analysis indicated that disulfide bonds, which play a key role in polymyxin resistance, were absent in EcEptCΔN. Supported by structural and biochemical evidence, we reveal mechanistic implications for disulfide bonds in PEA transferase-mediated polymyxin resistance. Significantly, because the structural effects exhibited by disulfide bonds are absent in EcEptC, it is impossible for this protein to participate in polymyxin resistance in E. coli. DATABASE: Structural data are available in the PDB under the accession numbers 6A82 and 6A83. ENZYME: EC 220.127.116.11.
Plasmid-mediated fluoroquinolone resistance associated with extra-intestinal isolates from hospital samples.
Shetty Shruthi S,Deekshit Vijaya Kumar,Jazeela Kadeeja,Vittal Rajeshwari,Rohit Anusha,Chakraborty Anirban,Karunasagar Indrani
The Indian journal of medical research
Background & objectives:Infection from fluoroquinolone-resistant extra-intestinal Escherichia coli is a global concern. In this study, isolation and characterization of fluoroquinolone-resistant extra-intestinal E. coli isolates obtained from hospital samples were undertaken to detect plasmid-mediated quinolone resistance (PMQR) genes. Methods:Forty three isolates of E. coli obtained from patients with extra-intestinal infections were subjected to antibiogram to detect fluoroquinolone resistance. The mechanism of fluoroquinolone resistance was determined by the detection of PMQR genes and mutations in quinolone resistance determining region (QRDR). Results:Of the 43 isolates, 36 were resistant to nalidixic acid (83.72%) and 28 to ciprofloxacin (65.11%). Eight E. coli isolates showed total resistance to both the antimicrobials without any minimum inhibitory concentration. The detection of PMQR genes with qnr primers showed the presence of qnrA in two, qnrB in six and qnrS in 21 isolates. The gene coding for quinolone efflux pump (qepA) was not detected in any of the isolates tested. The presence of some unexpressed PMQR genes in fluoroquinolone sensitive isolates was also observed. Interpretation & conclusions:The detection of silent PMQR genes as observed in the present study presents a risk of the transfer of the silent resistance genes to other microorganisms if present in conjugative plasmids, thus posing a therapeutic challenge to the physicians. Hence, frequent monitoring is to be done for all resistance determinants.
Distribution of quinolone resistance gene () in producing and in Lomé, Togo.
Salah Fortune Djimabi,Soubeiga Serge Théophile,Ouattara Abdoul Karim,Sadji Adodo Yao,Metuor-Dabire Amana,Obiri-Yeboah Dorcas,Banla-Kere Abiba,Karou Simplice,Simpore Jacques
Antimicrobial resistance and infection control
Background: genes are known to confer a low-level resistance to fluoroquinolone in . They are often found on the same resistance plasmids as extended spectrum β-lactamase (ESBL) and constitute the most common antibiotic resistance mechanism. This study aimed to detect the presence of r genes in -producing and . Methods:From May 2013 to July 2015, 91 and 64 strains with phenotypic resistance to quinolone were collected from several specimens and analyzed for the detection of genes and the β-lactamase resistance genes (, , ) using simplex and multiplex PCR. Results:In the present study, 107 (69%; 61 and 46 ) of 155 bacterial strains tested were found harboring at least one gene consisting of 74 (47.74%) , 73 (47.10%) and 4 (2.58%) . Of the 107 strains encoding genes, 102, 96 and 52 carried , and type ESBL respectively. Conclusion:This study identified quinolone resistance () gene in -producing and in Togo. These finding which suggest a possible resistance to quinolone are of high interest for better management of patients and control of antimicrobial resistance in Togo.
Contribution of AcrAB-TolC to multidrug resistance in an Escherichia coli sequence type 131 isolate.
Schuster Sabine,Vavra Martina,Schweigger Tobias M,Rossen John W A,Matsumura Yasufumi,Kern Winfried V
International journal of antimicrobial agents
Drug efflux by resistance-nodulation-cell division (RND)-type transporters, such as AcrAB-TolC of Escherichia coli, is an important resistance mechanism in Gram-negative bacteria; however, its contribution to multidrug resistance (MDR) in clinical isolates is poorly defined. We inactivated acrB of a sequence type 131 E. coli human isolate that showed high-level MDR, but had no mutations within the known efflux-associated local or global regulators. The resistance profile of the acrB deletion mutant revealed significantly increased susceptibility to drugs from seven antibiotic classes, including agents usually inactive against Gram-negative bacteria, notably the new oxazolidinone, tedizolid (512-fold enhanced susceptibility). AcrB deficiency reduced, but did not abolish, the efflux of dyes, which indicates the activity of at least one more efflux transporter. The findings demonstrate the efficacy of AcrAB-TolC-mediated broad-spectrum drug efflux, including agents primarily developed for Gram-positive pathogens, in a clinical isolate representative of a globally-emerging lineage. The results illustrate the need to develop molecules modified to impede their transport by AcrAB-TolC and its homologues and new efflux inhibitors.
Minicells as a Damage Disposal Mechanism in Escherichia coli.
Rang Camilla U,Proenca Audrey,Buetz Christen,Shi Chao,Chao Lin
Many bacteria produce small, spherical minicells that lack chromosomal DNA and therefore are unable to proliferate. Although minicells have been used extensively by researchers as a molecular tool, nothing is known about why bacteria produce them. Here, we show that minicells help cells to rid themselves of damaged proteins induced by antibiotic stress. By comparing the survival and growth rates of wild-type strains with the mutant, which produces excess minicells, we found that the mutant was more resistant to streptomycin. To determine the effects of producing minicells at the single-cell level, we also tracked the growth of lineages by microscopy. We were able to show that the mutant increased the production of minicells in response to a higher level of the antibiotic. When we compared two sister cells, in which one produced minicells and the other did not, the daughters of the former had a shorter doubling time at this higher antibiotic level. Additionally, we found that minicells were more likely produced at the mother's old pole, which is known to accumulate more aggregates. More importantly, by using a fluorescent IbpA chaperone to tag damage aggregates, we found that polar aggregates were contained by and ejected with the minicells produced by the mother bacterium. These results demonstrate for the first time the benefit to bacteria for producing minicells. Bacteria have the ability to produce minicells, or small spherical versions of themselves that lack chromosomal DNA and are unable to replicate. A minicell can constitute as much as 20% of the cell's volume. Although molecular biology and biotechnology have used minicells as laboratory tools for several decades, it is still puzzling that bacteria should produce such costly but potentially nonfunctional structures. Here, we show that bacteria gain a benefit by producing minicells and using them as a mechanism to eliminate damaged or oxidated proteins. The elimination allows the bacteria to tolerate higher levels of stress, such as increasing levels of streptomycin. If this mechanism extends from streptomycin to other antibiotics, minicell production could be an overlooked pathway that bacteria are using to resist antimicrobials.
Antimicrobial susceptibility and mechanisms of fosfomycin resistance in extended-spectrum β-lactamase-producing Escherichia coli strains from urinary tract infections in Wenzhou, China.
Bi Wenzi,Li Bin,Song Jiangning,Hong Youliang,Zhang Xiaoxiao,Liu Haiyang,Lu Hong,Zhou Tieli,Cao Jianming
International journal of antimicrobial agents
Fosfomycin in combination with various antibiotics represents an excellent clinically efficacious regimen for the treatment of urinary tract infections (UTIs) caused by extended-spectrum β-lactamase (ESBL)-producing Escherichia coli. Underlying mechanisms of fosfomycin resistance remain largely uncharacterised. To investigate the antibacterial efficacy of fosfomycin against ESBL-producing E. coli, 356 non-repetitive ESBL-producing E. coli clinical isolates were collected from urine specimens from patients with UTI in Wenzhou, China, from January 2011 to December 2015. Antimicrobial sensitivity testing indicated that 6.7% (24/356) of the ESBL-producing E. coli strains were resistant to fosfomycin. The fosA3 gene encoding a fosfomycin-modifying enzyme was detected in 20 isolates by PCR and sequencing, alone or in combination with other ESBL determinants. Conjugation experiments and Southern blotting demonstrated that 70% (14/20) of the fosA3-positive isolates possessed transferable plasmids (ca. 54.2 kb) co-harbouring the ESBL resistance gene bla and the fosfomycin resistance gene fosA3. Among the four fosfomycin-resistant fosA3-negative E. coli isolates, three contained amino acid substitutions (Ile28Asn and Phe30Leu in MurA and Leu297Phe in GlpT). The results indicate that presence of the fosA3 gene is the primary mechanism of fosfomycin resistance in ESBL-producing E. coli isolates in Wenzhou, China. In addition, a plasmid (ca. 54.2 kb) co-harbouring fosA3 and bla genes is horizontally transferable. Furthermore, a low degree of homology in the fosfomycin-resistant E. coli was confirmed using multilocus sequence typing (MLST), suggesting that there is no obvious phenomenon of clonal dissemination.
Population-based inference of aminoglycoside resistance mechanisms in Escherichia coli.
Mancini Stefano,Marchesi Martina,Imkamp Frank,Wagner Karoline,Keller Peter M,Quiblier Chantal,Bodendoerfer Elias,Courvalin Patrice,Böttger Erik C
BACKGROUND:Interpretative reading of antimicrobial susceptibility test (AST) results allows inferring biochemical resistance mechanisms from resistance phenotypes. For aminoglycosides, however, correlations between resistance pathways inferred on the basis of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints and expert rules versus genotypes are generally poor. This study aimed at developing and validating a decision tree based on resistance phenotypes determined by disc diffusion and based on epidemiological cut-offs (ECOFFs) to infer the corresponding resistance mechanisms in Escherichia coli. METHODS:Phenotypic antibiotic susceptibility of thirty wild-type and 458 aminoglycoside-resistant E. coli clinical isolates was determined by disc diffusion and the genomes were sequenced. Based on well-defined cut-offs, we developed a phenotype-based algorithm (Aminoglycoside Resistance Mechanism Inference Algorithm - ARMIA) to infer the biochemical mechanisms responsible for the corresponding aminoglycoside resistance phenotypes. The mechanisms inferred from susceptibility to kanamycin, tobramycin and gentamicin were analysed using ARMIA- or EUCAST-based AST interpretation and validated by whole genome sequencing (WGS) of the host bacteria. FINDINGS:ARMIA-based inference of resistance mechanisms and WGS data were congruent in 441/458 isolates (96·3%). In contrast, there was a poor correlation between resistance mechanisms inferred using EUCAST CBPs/expert rules and WGS data (418/488, 85·6%). Based on the assumption that resistance mechanisms can result in therapeutic failure, EUCAST produced 63 (12·9%) very major errors (vME), compared to only 2 (0·4%) vME with ARMIA. When used for detection and identification of resistance mechanisms, ARMIA resolved >95% vMEs generated by EUCAST-based AST interpretation. INTERPRETATION:This study demonstrates that ECOFF-based analysis of AST data of only four aminoglycosides provides accurate information on the resistance mechanisms in E. coli. Since aminoglycoside resistance mechanisms, despite having in certain cases a minimal effect on the minimal inhibitory concentration, may compromise the bactericidal activity of aminoglycosides, prompt detection of resistance mechanisms is crucial for therapy. Using ARMIA as an interpretative rule set for editing AST results allows for better predictions of in vivo activity of this drug class.
The resistance mechanism of induced by ampicillin in laboratory.
Li Mengchen,Liu Qiaoli,Teng Yanli,Ou Liuyang,Xi Yuanlin,Chen Shuaiyin,Duan Guangcai
Infection and drug resistance
Background:Multi-drug-resistant poses a great threat to human health, especially resistant to ampicillin (AMP), but the mechanism of drug resistance is not very clear. Purpose:To understand the mechanism of resistance of to beta-lactam antibiotics by inducing drug resistance of sensitive bacteria in laboratory. Methods:Clinical sensitive strain was induced into resistance strain by 1/2 minimum inhibitive concentration (MIC) induced trails of AMP. The drug resistance spectrum was measured by modified K-B susceptibility test. Whole-genome sequencing analysis was used to analyze primary sensitive strain, and resequencing was used to analyze induced strains. Protein tertiary structure encoded by the gene containing single nucleotide polymorphism (SNP) was analyzed by bioinformatics. Results:After 315 hrs induced, the MIC value of reached to 256 µg/mL, 64 times higher than that of the sensitive bacteria. During the induction process, the bacterial resistance process is divided into two stages. The rate of drug resistance occurs rapidly before reaching the critical concentration of 32 µg/mL, and then the resistance rate slows down. Sequencing of the genome of resistant strain showed that drug-resistant strain with the MIC values of 32 and 256 µg/mL contained four and eight non-synonymous SNPs, respectively. These non-synonymous SNPs were distributed in the genes of , and Conclusion:These studies will improve our understanding of the molecular mechanism of AMP resistance of , and may provide the basis for prevention and control of multi-drug-resistant bacteria and generation of new antibiotics to treat infection.
Comprehensive proteomic and metabolomic profiling of mcr-1-mediated colistin resistance in Escherichia coli.
Li Hui,Wang Yingyu,Meng Qingshi,Wang Yang,Xia Guoliang,Xia Xi,Shen Jianzhong
International journal of antimicrobial agents
Spread of the mcr-1 gene in human and veterinary medicine has jeopardised the use of polymyxins, last-resort antibiotics against life-threatening multidrug-resistant Gram-negative bacteria. As a lipid-modifying gene, whether mcr-1 causes proteomic and metabolomic changes in bacteria and affects the corresponding metabolic pathway is largely unknown. In this study, label-free quantitative proteomics and untargeted metabolomics were used to profile comprehensive proteome and metabolome characteristics of mcr-1-mediated colistin-resistant and -susceptible Escherichia coli in order to gain further insight into the colistin resistance mechanism. Large sets of differentially expressed proteins (DEPs) and metabolites were identified that contributed to mcr-1-mediated antimicrobial resistance, predominantly in different growth conditions with and without colistin. mcr-1 caused downregulated expression of most proteins in order to adapt to drug pressure. Pathway analysis showed that metabolic processes were significantly affected, mainly related to glycerophospholipid metabolism, thiamine metabolism and lipopolysaccharide (LPS) biosynthesis. The substrate phosphoethanolamine (PEA) for mcr-1 to mediate colistin resistance was accumulated in colistin-resistant E. coli. Notably, mcr-1 not only caused PEA modification of the bacterial cell membrane lipid A but also affected the biosynthesis and transport of lipoprotein in colistin resistance by disturbing the expression of efflux pump proteins involved in the cationic antimicrobial peptide (CAMP) resistance pathway. Overall, disturbed glycerophospholipid metabolism and LPS biosynthesis as well as accumulation of the substrate PEA was closely related with mcr-1-mediated colistin resistance. These findings could provide further valuable information to inhibit colistin resistance by blocking this metabolic process.
Detection of floR gene and active efflux mechanism of Escherichia coli in Ningxia, China.
Wang Yi-Hui,Li Xiao-Na,Chen Cheng,Zhang Jing,Wang Gui-Qin
Florfenicol is an antibiotic, a fluorinated structural analogue of thiamphenicol and chloramphenicol, approved exclusively for use in Asia for aquaculture since the 1980's. Our study examined the prevalence of florfenicol resistance in bovine mastitis Escherichia coli isolates. A total of 245 E. coli isolates were collected from bovine mastitis in Ningxia Province, China between May 2016 to July 2017 and screened for florfenicol resistance gene, floR gene by PCR analysis. About 7.35% (15/245) of the isolates were positive for floR gene. Minimal inhibitory concentration (MIC) results showed that 9 isolates were susceptible strains and 6 isolates were highly resistant to florfenicol. HPLC (high performance liquid chromatography) results showed that the amounts of florfenicol was significantly improved in the presence of carbonyl cyanide 3-chlorophenylhydrazone (CCCP) as an efflux pump inhibitor. This, therefore, indicates that the employment of florfenicol in conjunction with CCCP in future drug formulations should be considered.
Prevalence of the mcr-1 colistin resistance gene in extended-spectrum β-lactamase-producing Escherichia coli from human faecal samples collected in 2012 in rural villages in Shandong Province, China.
Bi Zhenwang,Berglund Björn,Sun Qiang,Nilsson Maud,Chen Baoli,Tärnberg Maria,Ding Lilu,Stålsby Lundborg Cecilia,Bi Zhenqiang,Tomson Göran,Yao Jingjing,Gu Zhanying,Yin Xiao,Kou Zengqiang,Nilsson Lennart E
International journal of antimicrobial agents
Since its initial discovery in China in 2015, the plasmid-mediated colistin resistance gene mcr-1 has been reported in Escherichia coli isolated from clinical samples, animals and meat worldwide. In this study, 706 extended-spectrum β-lactamase (ESBL)-producing E. coli from 411 persons were detected in a collection of faecal samples from 1000 rural residents in three counties in Shandong Province, China. These isolates were screened for mcr-1 and phenotypic colistin resistance. The gene was found in 3.5% of the isolates (from 4.9% of persons) from all three counties. All isolates with phenotypic colistin resistance carried mcr-1. These data indicate that commensal carriage of ESBL-producing E. coli with mcr-1 among persons in rural China was already present in 2012 and that mcr-1 was the most important colistin resistance mechanism. Interventions are necessary to minimise further dissemination of mcr-1, which would limit the future usefulness of colistin as a last-resort antibiotic.
Antimicrobial resistance and the presence of extended-spectrum beta-lactamase genes in Escherichia coli isolated from the environment of horse riding centers.
Wolny-Koładka Katarzyna,Lenart-Boroń Anna
Environmental science and pollution research international
The aim of the study was to determine the antimicrobial resistance profile and the occurrence of extended-spectrum beta-lactamase genes and to analyze the genetic diversity of Escherichia coli strains isolated from the environment of horse riding centers. The study was conducted using E. coli strains isolated from the air, manure, and horse nostril swabs in three horse riding centers differing in the system of horse keeping-stable (OJK Pegaz and KJK Szary) and free-range (SKH Nielepice). Resistance to antibiotics was determined using the disk-diffusion method, and the PCR technique was employed to detect the extended-spectrum β-lactamase (ESBL) genes, while the genetic diversity of strains was assessed by rep-PCR. A total of 200 strains were collected during the 2-year study, with the majority isolated from KJK Szary, while the smallest number was obtained from SKH Nielepice. The strains were mostly resistant to ampicillin, aztreonam, and ticarcillin. The tested strains were most frequently resistant to one or two antibiotics, with a maximum of ten antimicrobials at the same time. Two multidrug-resistant (MDR) strains were detected in OJK Pegaz while in KJK Szary there were two MDR and one extensively drug-resistant (XDR) strain. The ESBL mechanism was most frequently observed in OJK Pegaz (20.31% of strains) followed by KJK Szary (15.53% of strains) and SKH Nielepice (15.15% of strains). Among the ESBL-determining genes, only blaTEM and blaCTXM-9 were detected-blaTEM was mostly found in KJK Szary (53.40% of strains), while the second detected gene-blaCTXM-9-was most frequent in SKH Nielepice (6.06% of strains). The rep-PCR genotyping showed high variation among the analyzed strains, whereas its degree differed between the studied facilities, indicating that the type of horse keeping (stable vs. free-range) affects the genetic diversity of the E. coli strains. Having regard to the fact that the tested strains of E. coli were derived from non-hospitalized horses that were not treated pharmacologically, we can assume that the observed antimicrobial resistance may be of both-natural origin, i.e., not the result of the selection pressure, and acquired, the source of which could be people present in the horse riding facilities, the remaining horses which were not included in the study, and air, as well as water, fodder, and litter of the animals. Therefore, it can be concluded that the studied horses are the source of resistant E. coli and it is reasonable to continue monitoring the changes in antimicrobial resistance in those bacteria.
Covalently Linked Trimers of RND (Resistance-Nodulation-Division) Efflux Transporters to Study Their Mechanism of Action: Escherichia coli AcrB Multidrug Exporter as an Example.
Methods in molecular biology (Clifton, N.J.)
Transporters undergo large conformational changes in their functional cycle. RND (Resistance-Nodulation-Division) family efflux transporters usually exist as homotrimers, and each protomer was proposed to undergo a cycle of conformational changes in succession so that at any given time the trimer would contain three protomers of different conformations, the functionally rotating mechanism of transport. This mechanism implies that the inactivation of one protomer among three will inactivate the entire trimeric ensemble by blocking the functional rotation. We describe a biochemical approach to test this prediction by first producing a giant protein in which the three protomers of Escherichia coli AcrB efflux pump are covalently linked together through linker sequences, and then testing for its function by inactivation of a single protomer unit. Inactivation can be done permanently by mutating a residue involved in proton relay, or in "real time" by using a protein in which one protomer contains two Cys residues on both sides of the large cleft in the periplasmic domain and then by rapidly inactivating this protomer with a methanethiosulfonate cross-linker.