Molecular characteristics of Acinetobacter baumannii and the changes of efflux pump expression under tigecycline pressure / 医学研究生学报

Objective　Acinetobacter baumannii (A. baumannii) is a commonly infective bacterium in the hospital. This study aims to analyze its molecular epidemiological characteristics, detect the carrying rate of efflux pump and regulatory protein genes, and investigate the effects of tigecycline on the efflux pump and expression of regulatory protein genes.　Methods　A total of 183 A. baumannii strains were collected from inpatients of the affiliated hospital of Jiangsu University from May 2017 to March 2019. They were divided into an antimicrobial-resistant group (one or more antimicrobial-resistant strains, 139 strains) and a sensitive group (the drugs in the drug sensitivity test were all non-resistant strains, 44 strains). Repeated sequence PCR was used for homology analysis of the strains, and pulse-field gel electrophoresis (PFGE) was used as the gold standard for homology analysis to verify and compare some strains. PCR was used to detect the occurrence of drug resistance-related genes. Based on homology analysis, efflux pump carrying rate detection and antibiotics sensitivity test results, 6 clinical strains carrying all efflux pump genes but different resistance phenotypes were selected as experimental strains, including sensitive strains (SAB), the multidrug resistance strain (MDRAB) and the extensively drug-resistant strain (XDRAB). All strains were induced in vitro with the minimum inhibitory concentration (MIC) of tigecycline. The induced strains were categorized as induction group, and the same strains cultured in LB agar without tigecycline was used as a control group. MIC was used to analyze the tigecycline susceptibility, and RT-qPCR was used to detect the gene expression of efflux pumps, such as TetB, AbaQ and regulatory proteins (AdeS and BaeS), in drug-resistant strains.　Results　Homology analysis showed that there were 45 clonal groups in the detected clinical isolates, with no obvious outbreak of epidemic clonal groups. Efflux pumps and regulatory proteins were widely distributed in the clinical isolates, and the expression of AdeB, TetB, AbeS, AdeS in MDRAB and XDRAB is significantly higher than that insensitive group SAB. Continuous in vitro induction with tigecycline could increase the antimicrobial resistance of some clinical strains and even significantly increase the expression levels of efflux pumps and regulatory proteins.　Conclusion　A. baumannii is widely distributed in the clinic, and efflux pumps and regulatory proteins might play an important role in drug resistance process. The unreasonable use of tigecycline could enhance the tolerance of A. baumannii by up-regulating the expression of some bacterial efflux pumps.

Is the efflux pump inhibitor Verapamil a potential booster for isoniazid against Mycobacterium tuberculosis?

The membrane-based efflux pump systems are recognized to have an important role in pathogenicity and drug resistance in Mycobacterium tuberculosis by the extrusion of toxic substrates and drugs from the inner bacillus. This study aimed to investigate the in vitro interaction of Verapamil (VP), an efflux pump inhibitor, with the classical first-line anti-tuberculosis drug isoniazid (INH) in resistant and susceptible M. tuberculosis clinical isolates. Seven multidrug-resistant (MDR), three INH monoresistant and four susceptible M. tuberculosis clinical isolates were tested for the INH and VP combination by modified Resazurin Microtiter Assay Plate (REMA). Fractional Inhibitory Concentration (FIC) and Modulation Factor (MF) were determined. The INH plus VP combination showed no significant change in the Minimum inhibitory concentration (MIC) values of INH (FIC≥ 0.5; MF=1 or 2).The use of VP in tuberculosis therapy should be managed carefully, considering the resistance caused by specific mutation in katG and inhA genes, in which the use of these EPIs may have no success. The use of EPIs as an adjunctive drug in the anti-tuberculosis therapy should be further investigated on a larger number of M. tuberculosis clinical isolates with different resistant profile.

Dos distúrbios psiquiátricos à antibioticoterapia: reposicionamento da clorpromazina como agente antibacteriano / From psychiatric disorders to antibiotic therapy: repositioning of chlorpromazine as an antibacterial agent

RESUMO O alarmante aumento na taxa de resistência aos antibióticos põe em check à eficácia da terapia antibacteriana futura. Em contrapartida, as indústrias farmacêuticas negligenciam os investimentos em pesquisa e desenvolvimento de novos fármacos antimicrobianos em virtude de questões financeiras, legais e farmacológicas. Assim sendo, o reposicionamento de agentes disponíveis clinicamente torna-se uma promissora ferramenta para tentar driblar o desinteresse das indústrias. O fármaco antipsicótico clorpromazina (CPZ) destaca-se por possuir uma ampla faixa de atividade antibacteriana, a qual cobre desde patógenos Gram-positivos e Gram-negativos, até as mico-bactérias. A atividade antibacteriana é independente do perfil de susceptibilidade do microrganismo, sendo ela mantida mesmo em cepas altamente resistentes aos antibióticos. Alguns estudos mostram que mesmo nas concentrações clinicamente disponíveis no plasma (entre 0,1-0,5 (g/mL), a CPZ é capaz de matar Staphylococcus aureus e Mycobacterium tuberculosis dentro dos macrófagos. Em adição, estudos clínicos têm revelado os benefícios do uso da CPZ na terapia de suporte para pacientes com infecções em curso. Em conclusão, a CPZ pode eventualmente ser direcionada ao arsenal terapêutico antimicrobiano, especialmente no manejo das infecções causadas por microrganismos intracelulares com fenótipo multirresistente.

SUMMARY The substantial increase in the antibiotic resistance brings on an alarm to the future of the antibiotic therapy. However, the pharmaceutical industry has been neglecting its investments in new drug research and development, mainly because of the pharmacologic, financial and legal factors. Therefore, the drug repositioning of clinic available agents become a promising tool to bypass the lack of interest of the pharmaceutical industry. A drug used to treat psychoses, the Chlorpromazine (CPZ), stands out as a large spectrum antibiotic, which covers Gram-positive and Gram -negative bacteria, and also mycobacteria. Its antibacterial activity is not related to microorganism susceptibility profile, and it could be maintained even on strains highly resistant to the conventional antibiotics. Studies point out that even on serum concentrations clinically available, the CPZ can eliminate Staphylococcus aureus and Mycobacterium tuberculosis inside of macrophages. In addition, clinical trials have revealed its benefits on support therapy for patients suffering from active infections. As a result, the CPZ could be used as an optional antibiotic therapy, especially in case of infections due to intracellular microorganisms with multidrug resistance phenotype.

Efflux pump inhibitors for bacterial pathogens: From bench to bedside

With the advent of antibiotics, bacterial infections were supposed to be a thing of past. However, this instead led to the selection and evolution of bacteria with mechanisms to counter the action of antibiotics. Antibiotic efflux is one of the major mechanisms, whereby bacteria pump out the antibiotics from their cellular interior to the external environment using special transporter proteins called efflux pumps. Inhibiting these pumps seems to be an attractive strategy at a time when novel antibiotic supplies are dwindling. Molecules capable of inhibiting these pumps, known as efflux pump inhibitors (EPIs), have been viewed as potential therapeutic agents that can rejuvenate the activity of antibiotics that are no longer effective against bacterial pathogens. EPIs follow some general mechanisms of efflux inhibition and are derived from various natural as well as synthetic sources. This review focuses on EPIs and identifies the challenges that have kept these futuristic therapeutics away from the commercial realm so far.

RND efflux pump systems in Acinetobacter, with special emphasis on their role in quorum sensing

Acinetobacter is an important opportunistic, multidrug resistant pathogen causing majority of nosocomial infections worldwide. The multidrug resistance is attributed by a plethora of efflux pumps and the overexpression of the same mediates export of antimicrobial agents. Quorum sensing (QS) is the cell-to-cell communication system in which bacteria produces specific signaling molecules which are transported out to the surrounding environment to communicate with other bacterial cells. It has been noticed that multidrug efflux pumps like resistance-nodulation-cell division (RND) efflux pumps play an important role in QS by exporting these signaling molecules. This review discusses various RND efflux pumps and the current understanding of the interrelationship of RND efflux pumps and QS in Acinetobacter spp. Studies demonstrate that RND efflux pumps could be considered as potential targets to block QS thereby reducing pathogenesis and antibiotic resistance. The known RND efflux pump-mediated quorum quenching strategies for Acinetobacter and other bacterial strains are discussed in detail. Finally, the prospective quorum quenching strategies targeting the transcriptional regulators of RND efflux pumps to inhibit multidrug efflux pumps are addressed.

Draft genome sequence of Inquilinus limosus strain MP06, a multidrug-resistant clinical isolate

The bacterium, Inquilinus limosus, with its remarkable antimicrobial multiresistant profile, has increasingly been isolated in cystic fibrosis patients. We report draft genome sequence of a strain MP06, which is of considerable interest in elucidating the associated mechanisms of antibiotic resistance in this bacterium and for an insight about its persistence in airways of these patients.

Enhanced resistance to fluoroquinolones in laboratory-grown mutants & clinical isolates of Shigella due to synergism between efflux pump expression & mutations in quinolone resistance determining region.

Background & objectives: There is a worldwide emergence of fluoroquinolone resistance in Shigella species. To understand the molecular mechanisms associated with fluoroquinolone resistance, naturally occurring fluoroquinolone-resistant strains and laboratory-induced spontaneous mutants of Shigella spp. were used and the relative contributions of acrAB-tolC efflux pumps, gyrase and topoisomerase target gene mutations towards fluoroquinolone resistance were determined. Methods: Eight Shigella flexneri and six S. dysenteriae clinical isolates were studied. Three consecutive mutants resistant to ciprofloxacin for S. flexneri SFM1 (≥0.25 μg/ml), SFM2 (≥4 μg/ml) and SFM3 (≥32 μg/ml) were selected in 15 steps from susceptible isolates by serial exposure to increasing concentrations of nalidixic acid and ciprofloxacin. Similarly, two mutants for S. dysenteriae SDM1 (≥0.25 μg/ml) and SDM2 (≥4 μg/ml) were selected in eight steps. After PCR amplification sequence analyses of gyrase and topoisomerase target genes were performed. Expression of efflux genes acrA, acrB, acrR and tolC was measured using real-time PCR. Results: Mutations were observed in gyrA Ser83→Leu, Asp87→Asn/Gly, Val196→Ala and in parC Phe93→Val, Ser80→Ile, Asp101→Glu and Asp110→Glu. Overall, acrA and acrB overexpression was associated with fluoroquinolone resistance (p<0.05); while tolC and acrR expression levels did not. Interpretation & conclusions: Fluoroquinolone resistance in Shigella spp. is the end product of either a single or a combination of mutations in QRDRs and/ or efflux activity. Novel polymorphisms were observed at Val196→Ala in gyrA in clinical isolates and Phe93→Val, Asp101→Glu, Asp110→Glu and in parC in majority of laboratory-grown mutants.

Mechanisms of tigecycline resistance in carbapenems-resistant Acinetobacter baumannii strains / 中华微生物学和免疫学杂志

Objective To investigate the mechanisms of tigecycline nonsusceptibility in carbapen-ems-resistant Acinetobacter baumannii ( CRAB) strains in order to provide a theoretical basis for a reasonable use of antibiotics and the control of nosocomial infection .Methods Susceptibility testing of 120 non-dupli-cate CRAB strains to tigecycline was performed by using the broth microdilution method .Minimal inhibitory concentrations ( MIC) of tigecycline against the A.baumannii strains were determined by using the broth mi-crodilution method before and after exposing the strains to Carbonylcyanide-m-chlorophenylhydrazone (CCCP), which was the efflux pump inhibitor .Polymerase chain reaction (PCR) was used to amply the ef-flux pumps genes including adeB, adeJ, adeG, abeM, adeE, adeRS, tetX and tetX1.The real-time PCR was performed to measure the expression of efflux pumps genes including adeB, adeJ, adeG, abeM and adeE.Results A total of 120 CRAB strains were collected including 13 (10.8%) tigecycline non-suscep-tible A.baumannii (TNAB) strains and 107 (89.2%) tigecycline susceptible A.baumannii (TSAB) strains.The MIC values of tigecline to the 120 CRAB strains were in a range of 0.25 μg/ml to 8 μg/ml. The adeR and adeJ genes were detected in 90.0%and 92.5%of the 120 CRAB strains, respectively.The positive rates of adeB, adeS, adeG and abeM genes among the 120 CRAB strains were all 94.2%.None of the three genes including adeE, tetX and tetX1 were detected .The mean expression levels of adeB and adeJ in TNAB strains were respectively increased by 18.69 folds and 5.46 folds as compared with those in sensi-tive strains.No significant increase in the expression of adeG and abeM genes was observed in TNAB strains . A 4-fold decrease in the MIC was observed in 8 out of 13 TNAB isolates treated with 10 μg/ml of CCCP .The CCCP could partially reverse the resistance pattern of tigecycline .Conclusion The efflux pump sys-tems of adeABC and adeIJK rather than the abeFGH and abeM systems might play an important role in reduc-ing the tigecycline susceptibility in carbapenems-resistant A.baumannii strains.

Identification of natural compounds which inhibit biofilm formation in clinical isolates of Klebsiella pneumoniae.

Klebsiella pneumoniae, an important opportunistic pathogen, exists as a biofilm in persistent infections and in-dwelling medical devices. With the objective of identifying natural compounds inhibiting biofilm formation in K. pneumoniae, 35clinical isolates were screened,out of which 7 strong biofilm producers were identified. Six natural compounds were tested for their inhibitory effects on bacterial growth and biofilm formation by determining the minimum inhibitory concentration and minimum concentration for biofilm inhibition (MBIC) for each compound. The results show that reserpine followed by linoleic acid, were the most potent biofilm inhibitors. Reserpine, an efflux pump inhibitor was effective at biofilm inhibition at a concentration of 0.0156 mg/mL, 64-fold lower concentration than its MIC. Linoleic acid, an essential fatty acid was effective as a biofilm inhibitor at 0.0312 mg/mL, which is 32-fold lower than its MIC. Berberine, another plant derived antimicrobial, chitosan and eugenol had an MBIC value of 0.0635 mg/mL. Curcumin, a natural phenolic compound was effective at biofilm inhibition at a concentration of 0.25 mg/mL, which is 50 fold less than its MIC. Notably, the MIC and MBIC data on these 6 natural compounds was reproducible in all seven high biofilm forming isolates of K. pneumoniae. The present report is a comprehensive comparative analysis of the dose dependent inhibition of various natural compounds on biofilm formation in K. pneumoniae.

Suscetibilidade in vitro e resistência a antifúngicos - estudo comparativo entre os tipos moleculares vni de cryptococcus neoformans e vgi, vgii de cryptococcus gattii pela citometria de fluxo / In vitro susceptibility and resistance to antifungal drugs - a comparative study of the molecular types of cryptococcus neoformans vni, cryptococcus gattii vgi and vgii by flow cytometry

A criptococose é uma micose sistêmica adquirida pela inalação de basidiosporos ou leveduras desidratadas de Cryptococus neoformans e Cryptococus gattii, estas duas espécies podem causar criptococose oportunista e primária respectivamente. C. neoformans está constituído de tipos moleculares VNI-VNIV e C. gattii de VGI-VGIV que apresentam distribuição geográfica diferenciada, como por exemplo, o tipo VNI é cosmopolita e está associado a AIDS e VGI predominando na Austrália e EUA, o tipo VGII predominando no Brasil e America Latina. Este trabalho tem por objetivo realizar estudo comparado dos tipos moleculares VNI de C. neoformans, VGI e VGII de C. gattii analisando diferentes aspectos tais como: 1- Determinar o perfil da suscetibilidade in vitro da concentração inibitória mínima (CIM) de fluconazol (FLZ), itraconazol (ITZ), 5-fluorocitosina (5FC) e anfotericina B (AMB), isoladamente e de forma combinada de AMB com 5FC e AMB com Voriconazol (VRZ); 2- Determinar CIM pela citometria de fluxo (CMF) frente a FLZ, ITZ e AMB; 3- Definir a concentração mínima letal (CML) de AMB e 5FC, isoladamente e em combinação; 4- Avaliar a ação da melanina frente a 5FC e AMB na forma combinada e isolada de 5FC; 5- Induzir a resistência in vitro para FLZ e padronizar os fluorocromos: acetoximetil - calceína (calceina-AM), acetoximetil - 2’, 7’ -bis-(2-carboxietil)-5-(e -6)- carboxifluoresceína (BCECF-AM), rodamina 123 (Rh123) e iodeto de 3, 3’ –dipentiloxacarbocianina (DiOC5) na CMF para verificar a expressão de bombas de efluxo; 6- Comparar a expressão de bombas de efluxo...

Cryptococcosis is a systemic mycosis acquired by inhalation of dried yeasts or basidiospores of Cryptococus neoformans and Cryptococus gattii, these species can cause cryptococcosis opportunistic and primary respectively. C. neoformans is composed of molecular types VNI - VNIV and C. gattii VGI - VGIV they have different geographical distribution, the VNI type is cosmopolitan and is associated with AIDS, VGI type is predominant in Australia and the USA; while VGII type occurs in Brazil and Latin America. This paper aims to conduct a comparative study of the molecular types VNI C. neoformans and VGI, VGII C. gattii analyzing different aspects such as: 1 – The susceptibility profile in vitro of fluconazole ( FLZ ), itraconazole ( ITZ ), 5 - fluorocytosine ( 5FC ) and amphotericin B ( AMB ) alone and in combination with 5FC and AMB with voriconazole ( VRZ ) 2 – The minimum inhibitory concentration ( MIC ) by flow cytometry ( FCM ) comparing MIC of FLZ, ITZ and AMB with CLSI; 3 - The minimum lethal concentration ( MLC ) of AMB and 5FC, alone and in combination; 4 - The action of melanin against 5FC and AMB alone and combined 5FC, 5 – The induced resistance in vitro to FLZ and standardize the fluorochromes: acetoximetil - calceína (calceina-AM), acetoximetil - 2’, 7’ -bis-(2-carboxietil)-5-(e -6)- carboxifluoresceína (BCECF-AM), rodamina 123 (Rh123) e iodeto de 3, 3’ – dipentiloxacarbocianina (DiOC5) in FCM to verify expression of efflux pumps; 6 - The compare the expression of efflux pumps...

Metallo-β-lactamase-production in meropenem-susceptible Pseudomonas aeruginosa isolates: risk for silent spread

The aim of this study was to characterize two metallo-β-lactamases (MBLs)-producing Pseudomonas aeruginosa clinical isolates showing meropenem susceptibility. Antimicrobial susceptibility was assessed by automated testing and Clinical and Laboratory Standards Institute agar dilution method. MBL production was investigated by phenotypic tests. Molecular typing was determined by pulsed field gel electrophoresis (PFGE). MBL-encoding genes, as well as their genetic context, were identified by polymerase chain reaction (PCR) and sequencing. The location of blaIMP-16 was determined by plasmid electrophoresis, Southern blot and hybridization. Transcriptional levels of blaIMP-16, mexB, mexD, mexF, mexY, ampC and oprD were determined by semi-quantitative real time PCR. The P. aeruginosa isolates studied, Pa30 and Pa43, showed imipenem and meropenem susceptibility by automated testing. Agar dilution assays confirmed meropenem susceptibility whereas both isolates showed low level of imipenem resistance. Pa30 and Pa43 were phenotypically detected as MBL producers. PFGE revealed their clonal relatedness. blaIMP-16 was identified in both isolates, carried as a single cassette in a class 1 integron that was embedded in a plasmid of about 60-Kb. Pa30 and Pa43 overexpressed MexAB-OprM, MexCD-OprJ and MexXY-OprM efflux systems and showed basal transcriptional levels of ampC and oprD. MBL-producing P. aeruginosa that are not resistant to meropenem may represent a risk for therapeutic failure and act as silent reservoirs of MBL-encoding genes.

Gene expression analysis of the SdeAB multidrug efflux pump in antibiotic-resistant clinical isolates of Serratia marcescens.

Purpose: Many isolates of Serratia marcescens, a well-known opportunistic pathogen, can be multidrug resistant. Fluoroquinolones are among the most important groups of antibiotics used for treatment of these organisms. However, fluoroquinolone resistance among S. marcescens isolates is fast increasing. Drug extrusion through efflux pumps like SdeAB/ HasF is one of the major mechanisms of resistance to fluoroquinolones. This study was carried out to analyze, through gene expression analysis of sdeB, the relative contribution of this mechanism toward fluoroquinolone resistance in clinical isolates of Serratia. Materials and Methods: Total RNA from 45 clinical isolates of S. marcescens was isolated. Quantitative real-time RT PCR was performed on the extracted RNA to study the gene expression of sdeB and was normalized to the sdeB expression in the standard strain of S. marcescens. Results: Of the 45 isolates analyzed, sdeB expression was found to be elevated in 20 isolates (44%). Of these 20 isolates, eight (40%) were fully resistant to at least one of the fluoroquinolones studied. Conversely, of the 20 isolates that over-expressed sdeB, 12 (60%) were fully sensitive to all fluoroquinolones tested. Conclusions: Drug efflux pumps are an important means of fluoroquinolone resistance among clinically important species ofSerratia. The expression of these pumps can be up-regulated in the presence of antibiotics and have the potential for changing the phenotype from sensitive to resistant, thus contributing to therapeutic failures.

Mechanisms of fluconazole resistance in clinical and experimental induced isolates of Candida glabrata / 中华微生物学和免疫学杂志

Objective To investigate the mechanisms of fluconazole resistance in clinical and experimental induced isolates of C.glabrata.Methods Efflux of rhodamine 6G was performed to evaluate the effects of efflux pumps.The expression levels of transporter genes CDR1,CDR2,SNQ2 and ERG11 were examined by real-time RT-PCR.Meanwhile,sequence of PDR1 was determined by PCR based DNA sequencing.Results Efflux pumps of all fluconazole-resistant isolates had stronger effects than that of susceptible isolates,consistently with significant upregulation of CDR1,but no obvious difference was found in CDR2 or SNQ2.Also,no notable change in the expression level of ERG11 between susceptible and resistant isolates.PDR1 mutations existed in both clinical and experimental induced isolates of C.glabrata,among which P927S,L543P and S947L haven't been reported previously.Conclusion Mutations of PDR1 were induced by fluconazole both in vivo andin vitro,which will result in overexpression of CDR1 and strengthen the effect of efflux pump.

Pseudomonas aeruginosa multirresistente: um problema endêmico no Brasil / Multidrug-resistant Pseudomonas aeruginosa: an endemic problem in Brazil

Relatos mundiais têm documentado a problemática da endemicidade de isolados clínicos de Pseudomonas aeruginosa multirresistente (MDR) aliada a elevados índices de morbidade/mortalidade. No Brasil, surtos de infecção ocasionados por P. aeruginosa têm sido relacionados com uma disseminação clonal da espécie. Atualmente, as opções terapêuticas para o tratamento das infecções causadas por esse microrganismo são limitadas, muitas vezes restringindo-se ao uso de carbapenêmicos (p. ex., imipenem [IPM]). Assim, a resistência ao IPM é uma questão de saúde pública, uma vez que esse antibiótico é empregado como último recurso no tratamento de infecções de origem hospitalar, causadas por bactérias Gram-negativas multirresistentes. No Brasil, os principais mecanismos relacionados com fenótipos multirresistentes de P. aeruginosa são produção de metalobetalactamase (MBL) do tipo SPM-1, presença de metilase 16S rRNA RmtD, perda de porina OprD e superexpressão de bombas de efluxo, o que pode explicar os altos índices de resistência a carbapenêmicos e aminoglicosídeos. A emergência de cepas com essas características é preocupante, tendo em vista a escassez de terapias efetivas no tratamento de infecções por esse patógeno. Finalmente, com base em relatos nacionais, publicados por diferentes grupos de pesquisa, podemos deduzir que a convergência de múltiplos mecanismos de resistência em P. aeruginosa tem sido um evento favorável para a seleção de diferentes clones endêmicos multirresistentes disseminados no Brasil.

Global reports have documented the endemicity of multidrug-resistant (MDR) Pseudomonas aeruginosa associated with high levels of morbidity/mortality. In Brazil, outbreaks of MDR P. aeruginosa have been related to clonal dissemination. Currently, therapeutic options for the treatment of these infections are restricted to carbapenemic antibiotics (i.e., imipenem [IPM]). Thus, carbapenem resistance is a public health issue, since carbapenems are considered the last resort to nosocomial infections caused by MDR Gram-negative bacteria. In Brazil, the main mechanisms associated with MDR P. aeruginosa phenotypes are metallo-betalactamase (MBL) production (SPM-1 enzyme), presence of 16S rRNA methylase RmtD, loss of OprD porin, and overexpression of efflux pumps, which may explain the high level of carbapenem and aminoglycoside resistance. Accordingly, the emergence and dissemination of MDR strains is worrisome. Finally, based on national reports published by different groups of investigators, it is deduced that the convergence of multiple mechanisms of P. aeruginosa resistance has played a major role in the selection of endemic MDR clones widespread in Brazil.

The effect of small interfering RNA against Pseudomonas aeruginosa expressing MexA-MexB-OprM efflux pumps / 中华微生物学和免疫学杂志

Objective To identify the efficacy small interfering RNA against Pseudomonas aeruginosa expressing MexA-MexB-OprM efflux pumps.Methods Four siRNA ( siRNA1,siRNA2,siRNA3 and siRNA4) against mexB gene were designed and prepared by electricity transference in vitro.MICs of antibiotic combined with efflux pump inhibitors against multiple resistant strain PAO1 and PAO3 were determined by E-test method.The mRNA expression levels of efflux pump gene (mexB) were quantified by real time fluorescent quantitative PCR.Results siRNA expression vectors were constructed success by enzyme cut method.48 after PAO1 and multiple drug resistant PAO3 transfected with siRNA4,the sensibilities to antibiotic were enhanced.48 after PAO1 and multiple drug resistant PAO3 transfected with siRNAl,siRNA2 and siRNA3,the sensibilities to antibiotic didn't change obviously.48 after PAO1 and multiple drug resistant PAO3 ttransfected with siRNA4,the expression level of mexB was decreased obviously (P ＜ 0.05 ).48 after PAO1 and multiple drug resistant PAO3 transfected with siRNA1,siRNA2 and siRNA3,the expression level of mexB didn't change obviously.Conclusion siRNA against Pseudomonas aeruginosa expressing MexA-MexB-OprM efflux pumps enhanced the sensibility to antibiotic and inhibited the expression of mexB gene.Our results demonstrate the using RNAi may be potential targets for novel therapies directed against treatment of antibiotic-resistant infections.

Distribution and effects of gene expression of four efflux pumps in Pseudomonas aeruginosa with multi-drug resistance in Hunan province / 中华微生物学和免疫学杂志

Objective To investigate the effects and distribution of 4 efflux pumps and correlated mutation of regulatory gene in multi-drug resistance Pseudomonas aeruginosa (Pa) in Hunan province. Methods Forty non-duplicated clinical strains of multiple-drug-resistant Pseudomonas aeruginosa were collected in Hunan in 2008, then the phenotype was screened with efflux pumps inhibitor phenylalanine-L-β-naphthylamide and 4 antimicrobial susceptibility test discs. Genes of the efflux pump membrane fusion protein were amplified by PCR, and correlated efflux pump regulatory genes were amplified and sequenced to analyze the role of efflux pump gene expression in multi-drug resistance compared to that of 18 strains with non-multi-drug resistance. Re-suits The positivity rates of MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY-OprM were 45.0% (18/40), 30.0% (12/40), 42.5% (17/40) and 12.5% (5/40) respectively with phenotype screening in multi-drug resistance group. The positivity rates of mexA, mexC, mexE and mexX were 100% (58/58), 22.5% (9/40), 45.0% (18/40) and 22.5% (9/40) with RT-PCR. The overexpressed positivity rates of mexA and mexX were 55.0% (22/40) and 22.5% (9/40) respectively by semi-quantitative analysis with real-time PCR. However, no over-expression of mexA and mexX in non-multi-drug resistance group with real-time PCR. The positive expression rates of mexC and mexE with RT-PCR were 11.1% (2/18), 38.9% (7/18) in non-multi-drug resistance group. The difference of overexpression of mexA and mexX was significant(P<0.001, P=0.045) between two groups. The strain Pa20 with mexA overexpressian displayed gene mutations in mexR(164GTC→GAG) and amino acid substitution(126Val→Glu), the strain Pa34 with overexpression of mexX had 164GCG→GAG,55Ala→GIu. Conclusion Most of Pa with multi-drug resistance contained the resistant mechanism of efflux pumps and most-ly due to the overexpression of MexAB-OprM and MexXY-OprM in Hunan. There are mostly regulatory genes mutation in the strains with overexpression of MexAB-OprM and MexXY-OprM.

Efflux pumps in multidrug resistant Escherichia coli / 中华临床感染病杂志

Objective To explore the prevalence of 11 efflux pumps in isolates of multidrugresistant Escherichia coli(MDR-ECO). Methods Efflux pumps emrB, emrD , emrE, mdfA, sugE, mdtl,tehA, oqxA, qacE△1, qacE and smr-2 were detected by polymerase chain reaction(PCR)in 20 MDR-ECOs isolated from clinical samples. Results Efflux pumps emrB, emrD, emrE, mdfA, sugE, mdtI, qacE △1, tehA and oqxA were detected, and 8 efflux pumps were found in the same strain. Conclusion Multidrug- resistance in Escherichia coli may be related with efflux pumps.

Bombas de expulsión multidrogas en Acinetobacter baumannii y resistencia a antimicrobianos / Multi-drug efflux pumps and antibiotic resistance in Acinetobacter baumannii

Los sistemas multidrogas bacterianos contribuyen al desarrollo del fenotipo de multi-resistencia presentado por cepas de Acinetobacter baumannü, patógeno intrahospi-talario, que durante los últimos años ha incrementado su importancia por la creciente resistencia a carbapenémicos. El fenotipo de multi-resistencia está otorgado por la combinación de varios mecanismos de resistencia entre los cuales se encuentran estos sistemas de bombas de expulsión. El sistema multidroga AdeABC se ha detectado en muchas de estas cepas multi-resistentes de A. baumannü y, se ha relacionado con resistencia a diversos grupos de antimicrobianos, incluidos tigeciclina y meropenem. La inhibición de dichos sistemas multidrogas permitiría aumentar la eficacia de la terapia antimicrobiana. La siguiente revisión se enfoca en las bombas de expulsión multidrogas presentes en A. baumannü, con particular énfasis en el sistema AdeABC.

Bacterial multi-drugs systems contribute to the development of multi-resistance patterns of Acinetobacter baumannii, a nosocomial pathogen of increasing importance due to its emerging resistance to carbapenems. The multi-resistance phenomena is generated by a combination of mechanisms, one of which the efflux pump system. Many of these multiresistant isolates of A. baumannii harbor genes for the AdeABC multi-drug efflux system, related with resistance to various groups of antibacterial agents, including tygecicline and meropenem. Inhibition of these systems would allow to increase the efficacy of this antimicrobial. This review focuses on the multi-drug efflux pump system oí A. baumanni with special emphasis in the AdeABC system.