Imipenem and expression of multidrug efflux pump in Enterobacter aerogenes

Imipenem is often used to treat intensive care unit patients infected by Enterobacter aerogenes, but it is leading to an increasing number of antibiotic resistant strains. Clinical isolates and imipenem resistant variants presented a high level of resistance to beta-lactam antibiotic group and to chemically unrelated drugs. We report here that imipenem selects strains which contain active efflux pumps ejecting various unrelated antibiotics including quinolones, tetracycline, and chloramphenicol. An increase of AcrA, an efflux pump component, was observed in the imipenem resistant variants. The overexpression of marA, involved in the genetic control of membrane permeability via porin and efflux pump expression, indicated the activation of the resistance genetic cascade in imipenem resistant variants.     

外排泵介导鲍曼不动杆菌耐药性的研究进展

目的鲍曼不动杆菌的多重耐药性问题日趋严重,该菌外膜上外排泵过表达是导致其耐药性的重要机制。详尽地研究多药外排泵的机制以及寻找阻断其功能的外排泵抑制剂,将为多耐药鲍曼不动杆菌的治疗开辟新的路径。本文就近年来鲍曼不动杆菌外排泵的研究现状进行综述,着重描述多药外排泵RND家族的耐药谱特征及其表达调控机制,同时,还阐述了MFS和MATE家族外排泵的研究进展。     

外排机制在多重耐药肺炎链球菌耐药中的实验研究

目的探讨我院2005年1月至2007年12月临床分离的多重耐药肺炎链球菌（MDR-SP）的耐药情况及外排机制在部分药物中作用,为我院耐药性监测和临床用药提供依据。方法采用常规方法进行细菌的分离及鉴定;运用VITEK-2全自动细菌分析仪进行细菌的鉴定及药敏分析;M-H琼脂稀释法测定含或不含利血平的青霉素、四环素、红霉素、复方新诺明、氯霉素和加替沙星对MDR-SP的MIC值。结果MDR-SP耐药性较为严重,临床共分离出的178株肺炎链球菌中,MDR-SP为61株;除开青霉素外,外排作用参与了四环素、红霉素、复方新诺明、氯霉素和加替沙星的耐药。结论我院MDR-SP产生率较高（34．3％）,耐药性较为严重;外排作用在耐药中的作用不能忽视。     

结核分枝杆菌外排泵研究进展

结核病是由结核分枝杆菌(Mycobacterium tuberculosis,Mtb)引起的一种传染病。随着多药耐药和广泛耐药结核分枝杆菌的出现,结核病的治疗变得更为艰难。近年来研究发现,结核分枝杆菌存在外排泵是其耐药的原因之一,现已发现结核分枝杆菌的主要易化子超家族(major facilitator superfamily,MFS)、三磷酸腺苷(adenosine-triphosphate,ATP)结合盒超家族(ATP-Binding Cassette,ABC)、耐受小节分裂区家族(resistance-nodulation-division,RND)和小耐多药性家族(small multidrug resistance,SMR)外排泵。但是人们对结核分枝杆菌外排泵介导的耐药现象认识不足,仍缺乏从新药发现角度研发外排泵抑制剂的研究。本文拟对结核分枝杆菌的ABC、MFS、RND和SMR外排泵的结构和功能,以及结核分枝杆菌外排泵抑制剂的研究进展进行综述。     

鲍曼不动杆菌中外排泵介导耐药机制的研究进展

外排泵的过表达是目前导致鲍曼不动杆菌多重耐药的最重要机制之一,详细了解这一复杂机制有助于尽快找到有效的防治策略。目前,鲍曼不动杆菌中已被报道的外排泵家族包括耐药结节细胞分化(resistance-nodulation-cell division,RND)家族、主要协同转运蛋白超家族(major facilitator superfamily,MFS)、多药及毒性化合物外排(multidrug and toxic compound extrusion,MATE)家族、小多重耐药(small multidrug resistance,SMR)家族。它们之中既有通过染色体介导的外排泵,也有通过质粒等遗传元件介导的外排泵。外排底物可呈现多样性,也可呈现专一性。本文就上述外排泵的种类、功能和调控机制进行综述。     

鲍曼不动杆菌耐药程度与其主动外排泵蛋白的相关性研究

目的:探讨鲍曼不动杆菌耐药程度与其主动外排泵蛋白的相关性。方法:首先用纸片扩散法检测64株临床鲍曼不动杆菌对8种抗菌药物的敏感性;将其分为A组(0～2种抗生素耐药)、B组(对3～5种抗生素耐药)和C组(对6～8种抗生素耐药);检测64株临床鲍曼不动杆菌对罗丹明6G的外排情况,筛选出罗丹明6G外排明显增加的菌株;并用逆转录-聚合酶链反应(RT-PCR)方法检测主动外排泵基因AdeABC的表达水平。结果:64株鲍曼不动杆菌中有4株对0～2种抗生素耐药(A组),对3～5种抗生素耐药的有33株(B组),对6～8种抗生素耐药的有27株(C组);多重耐药组鲍曼不动杆菌罗丹6G外排明显增高,外排程度A组相似文献   

Isolation and transposon mutagenesis of a Pseudomonas putida KT2442 toluene-resistant variant: involvement of an efflux system in solvent resistance

A toluene-resistant variant of Pseudomonas putida KT2442, strain TOL, was isolated after liquid cultivation under xylene followed by toluene for 1 month in each condition. Almost all the populations of the variant strain formed small but readily visible colonies under toluene within 24 h at 30°C. The toluene-resistant strain also showed an increase in resistance to some unrelated antibiotics. Several toluene-sensitive Tn5 mutants have been isolated from the toluene-resistant strain and showed various levels of sensitivity. Most of these mutations did not cause significant changes in antibiotic resistance; however, one of the mutants (TOL-4) was highly susceptible to both organic solvents and various antibiotics, especially β-lactams. Sequencing analysis revealed that the mutation in TOL-4 had been introduced into a gene that may encode a transporter protein of an efflux system. This efflux system is very similar to one of the multidrug efflux systems of Pseudomonas aeruginosa. These observations indicate that a multidrug efflux system plays a major role in the organic solvent resistance of P. putida TOL. However, several other genes may also be involved. Received: December 18, 1997 / Accepted: March 16, 1998     

Bacterial efflux systems and efflux pumps inhibitors

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

Relative contribution of target gene mutation and efflux to varying quinolone resistance in Irish Campylobacter isolates

The contribution of target gene mutations and active efflux to varying levels of quinolone resistance in Irish Campylobacter isolates was studied. The Thr-86-Ile modification of GyrA did not correlate with the level of quinolone resistance. The efflux pump inhibitor Phe-Arg-beta-Naphthylamide (PAbetaN) had no effect on the MICs to ciprofloxacin. In contrast, a PAbetaN sensitive efflux system contributed to the low-level nalixidic acid resistance phenotype. The lack of effect of PAbetaN in high-level resistant nalidixic isolates may be attributable to mutations identified in the CmeB efflux pump of these isolates. PAbetaN may have limited diagnostic value in the assessment of the contribution of efflux pump activity to ciprofloxacin resistance in Campylobacter.     

Directed evolution of a bacterial efflux pump: adaptation of the E. coli TolC exit duct to the Pseudomonas MexAB translocase

Bacterial multidrug efflux pumps operate by periplasmic recruitment and opening of TolC family outer membrane exit ducts by cognate inner membrane translocases. Directed evolution of active hybrid pumps was achieved by challenging a library of mutated, shuffled TolC variants to adapt to the non-cognate Pseudomonas MexAB translocase, and confer resistance to the efflux substrate novobiocin. Amino acid substitutions in MexAB-adapted TolC variants that endowed high resistance were recreated independently, and revealed that MexAB-adaptation was conferred only by substitutions located in the lower alpha-helical barrel of TolC, specifically the periplasmic equatorial domain and entrance coiled coils. These changes converge to the native MexAB partner OprM, and indicate an interface key to the function and diversity of efflux pumps.     

Inhibitors of antibiotic efflux pump in resistant Enterobacter aerogenes strains

Enterobacter aerogenes, a nosocomial pathogen, is frequently exhibiting multidrug resistance mechanisms associated with a change in membrane permeability. In clinical isolates, active efflux plays a prominent role in antibiotic resistance. We report here the effect of three unrelated compounds that are able to restore a noticeable antibiotic susceptibility to resistant strains. The targeting of various parameters which contribute to the efficacy of the efflux mechanism, such as energy, flux selectivity, or functional assembly of the membrane complex, increases the intracellular chloramphenicol concentration in resistant isolates.     

Multidrug resistance reversal agent from Jatropha elliptica

As part of an ongoing project to identify plant natural products as resistance-modifying agents, bioassay-guided fractionation of an extract of Jatropha elliptica (Pohl) Muell Arg. led to the isolation of a penta-substituted pyridine, namely 2,6-dimethyl-4-phenyl-pyridine-3,5-dicarboxylic acid diethyl ester (8). The structure was established by spectroscopic methods. This known compound was assayed for in vitro antibacterial and resistance-modifying activities against strains of Staphylococcus aureus possessing the MsrA and NorA resistance efflux mechanisms. Antibiotic efflux studies indicated that (8) acts as an inhibitor of the NorA efflux pump and restores the level of intracellular drug concentration.     

The protein synthesis inhibitors mycalamides A and E have limited susceptibility toward the drug efflux network

The mycalamides belong to a family of protein synthesis inhibitors noted for antifungal, antitumour, antiviral, immunosuppressive, and nematocidal activities. Here we report a systematic analysis of the role of drug efflux pumps in mycalamide resistance and the first isolation of mycalamide E. In human cell lines, neither P-glycoprotein overexpression nor the use of efflux pump inhibitors significantly modulated mycalamide A toxicity in the systems tested. In Saccharomyces cerevisiae, it appears that mycalamide A is subject to efflux by the principle mediator of xenobiotic efflux, Pdr5p along with the major facilitator superfamily pump Tpo1p. Mycalamide E showed a similar efflux profile. These results suggest that future drugs based on the mycalamides are likely to be valuable in situations where efflux pump-based resistance leads to failure of other chemotherapeutic approaches, although efflux may be a mediator of resistance in antifungal applications.     

沙门氏菌抗生素抗性机理研究进展

沙门氏菌的多重耐药性问题已经成为世界范围内的公共卫生和经济问题.目前沙门氏菌抗生素抗性机理的研究主要集中以下方面:(1)基因突变与抗生素抗性;(2)外排泵与抗生素抗性:(3)耐药基因编码的钝化酶和灭活酶引起的抗生素抗性;(4)可移动的细菌遗传耐药基因元件及其转移与抗生素抗性.本文基于以上几个方面综述了与沙门氏菌抗生素抗性机理研究相关的研究动态和研究进展.     

Role of an acrR mutation in multidrug resistance of in vitro-selected fluoroquinolone-resistant mutants of Salmonella enterica serovar Typhimurium

Quinolone resistance in Salmonella spp. is usually attributed to both active efflux and mutations leading to modification of the target enzymes DNA gyrase and topoisomerase IV. Here, we investigated the presence of mutations in the efflux regulatory genes of fluoroquinolone- and multidrug-resistant mutants of Salmonella enterica serovar Typhimurium (S. Typhimurium) selected in vitro with enrofloxacin that both carried a mutation in the target gene gyrA and overproduced the AcrAB efflux pump. No mutations were detected in the global regulatory loci marRAB and soxRS for the four strains studied. A mutation in acrR, the local repressor of acrAB, was found for two ciprofloxacin-resistant selected-mutants, leading to duplication of amino acids Ile75 and Glu76. Complementation experiments with wild-type acrR showed that the mutation identified in acrR partially contributed to the increase in resistance levels to several unrelated antibiotics. The acrR mutation also contributed to acrAB overexpression as shown by RT-PCR. Thus, this study underlines the role of an acrR mutation, in addition to the mutation in gyrA, in the fluoroquinolone and multidrug resistance phenotype of S. Typhimurium mutants, through overexpression of acrAB.     

Direct observation of substrate induction of resistance mechanism in Pseudomonas aeruginosa using single live cell imaging

The resistance mechanism in three strains of Pseudomonas aeruginosa, Delta ABM (devoid of MexAB-OprM), WT, and nalB-1 (overexpression of MexAB-OprM), was investigated using real-time single live cell imaging and fluorescence spectroscopy. Time courses of fluorescence intensity of these three strains in ethidium bromide (EtBr) showed that accumulation kinetics and extrusion machinery were highly dependent upon pump substrate (EtBr) concentration. At high substrate concentration (100 microM), the accumulation kinetic profiles in the cells at earlier incubation times were similar to those observed in low concentration. As EtBr accumulated in the cells reached a critical concentration, the fluorescence intensity of Delta ABM decreased below the fluorescence intensity of EtBr in buffer solution. This result suggested an inductive mechanism in the development of substrate resistance in P. aeruginosa. Substrates appeared to trigger the degradation of EtBr in Delta ABM. Unlike bulk measurements, single live cell imaging overcame the ensemble measurement of bulk analysis and showed that efflux machinery and resistance mechanism in individual cells were not synchronized.     

Contribution of the multidrug efflux pump LfrA to innate mycobacterial drug resistance

Multidrug resistance (MDR) in bacteria has been associated with efflux pumps that export structurally unrelated compounds and decrease cytoplasmic drug accumulation. To investigate MDR in mycobacteria, we studied the Mycobacterium smegmatis mutant mc(2)11, which is resistant to doxorubicin, tetracycline, rhodamine, ethidium bromide and the hydrophilic fluoroquinolones. A genomic library constructed from this mutant was used to select clones conferring resistance to doxorubicin. Surprisingly, the clone selected encodes the efflux pump LfrA, which has been reported to confer resistance to hydrophilic fluoroquinolones, ethidium bromide, rhodamine, and acriflavine. To define the contribution of LfrA to the innate mycobacterial drug resistance and to the MDR phenotype in mc(2)11, the lfrA gene was disrupted in both the mc(2)11 mutant and the mc(2)155 wild-type parent. LfrA disruption of the wild-type strain decreased resistance to ethidium bromide and acriflavine, and increased accumulation of ethidium bromide. However, disruption of lfrA gene results only in a 2-fold decrease in minimal inhibitory concentrations (MICs) for ciprofloxacin, doxorubicin, rhodamine, and accumulation of [(14)C]ciprofloxacin was unchanged. LfrA disruption of the MDR strain mc(2)11 produced a similar phenotype. Thus, LfrA contributes significantly to the intrinsic MICs of M. smegmatis for ethidium bromide and acriflavine, but not for ciprofloxacin, doxorubicin or rhodamine.     

Efflux pump inhibition by 11H-pyrido[2,1-b]quinazolin-11-one analogues in mycobacteria

Mycobacterium tuberculosis infection causes 1.8 million deaths worldwide, of which half a million has been diagnosed with resistant tuberculosis (TB). Emergence of multi drug resistant and extensive drug resistant strains has made all the existing anti-TB therapy futile. The major involvement of efflux pump in drug resistance has made it a direct approach for therapeutic exploration against resistant M. tuberculosis. This study demarcates the role of 11H-pyrido[2,1-b]quinazolin-11-one (quinazolinone) analogues as efflux pump inhibitor in Mycobacterium smegmatis. Sixteen quinazolinone analogues were synthesized by treating 2-aminopyridine and 2-fluorobenzonitrile with K^tOBu. Analogues were tested, and 3a, 3b, 3c, 3g, 3j, 3l, 3m, and 3p were found to modulate EtBr MIC by >4 whereas 3a, 3g, 3i and 3o showed >4 modulation on norfloxacin MIC. 3l and 3o in addition to their very low toxicity they showed high EtBr and norfloxacin accumulation respectively. Time kill curve showed effective log reduction in colony forming unit in presence of these analogues, thus confirming their role as efflux pump inhibitor. Through docking and alignment studies, we have also shown that the LfrA amino acid residues that the analogues are interacting with are present in Rv2333c and Rv2846c of M. tuberculosis. This study have shown for the first time the possibility of developing the 11H-pyrido[2,1-b]quinazolin-11-one analogues as efflux pump inhibitors for M. smegmatis and hence unbolts the scope to advance this study against resistant M. tuberculosis as well.     

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

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