【摘要】 目的鲍曼不动杆菌是医院内感染常见的细菌。文中旨在分析其分子流行特征,检测外排泵和调节蛋白基因的携带率,并研究替加环素对外排泵和调节蛋白基因表达的影响。方法选取2017年5月至2019年3月江苏大学附属医院住院患者183株鲍曼不动杆菌。分为耐药组(一类及以上抗菌药物耐药的菌株,139株),敏感组(药敏试验中的药物均为非耐药的菌株,44株)。应用重复序列PCR作菌株的同源性分析,并以脉冲场凝胶电泳(PFGE)为同源性分析的金标准对部分菌株进行验证和比较。PCR检测耐药相关基因。依据同源性分析、外排泵携带率检测及药敏试验结果,选取6株外排泵基因全携带但耐药表型不同的临床菌株作为实验株,包括敏感株(SAB)、多药耐药株(MDRAB)、广泛耐药株(XDRAB),进行替加环素体外诱导,诱导后的菌株为诱导组,以不含替加环素LB培养的相同菌株为对照组。检测AdeB、TetB等外排泵基因及AdeS、BaeS等调节蛋白的基因表达变化。结果同源性分析显示,183株临床分离株包含有45个克隆群。耐药组AdeB、TetB基因(97.84%、98.56%)检出率均高于敏感组(63.64%、20.45%),CraA、AbeM等基因亦高于敏感组(P<0.05)。替加环素诱导前MDRAB、XDRAB中AdeB(4.17±0.94、6.86±3.23)、TetB(13.06±0.38、13.96±0.63)表达较SAB(0.00±3.70、0.00±0.70)升高(P<0.05),MDRAB菌株中MacB、BaeS表达较SAB菌株亦升高(P<0.05)。MDRAB、XDRAB菌株中AbaQ表达较SAB菌株降低(P<0.05)。诱导组AdeB、TetB在各菌株的表达较对照组明显升高(P<0.01);除SAB1菌株外,AbaQ、AbeM在其余菌株中的表达较对照组明显升高(P<0.05);AdeS在SAB1、SAB2、MDRAB1、XDRAB2菌株中的表达较对照组升高(P<0.05);负调节蛋白SoxR在SAB1、XDRAB1菌株中表达较对照组降低(P<0.05)。结论 AdeB、TetB、AbeS、AdeS、BaeS在临床鲍曼不动杆菌多药耐药过程中发挥着重要的作用,其中AdeB和TetB在菌株对替加环素耐药过程中的作用尤为明显,可能是替加环素耐药的优势外排泵基因。更多还原

【Abstract】 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 in?vestigate the effects of tigecycline on the efflux pump and expression of regulatory protein genes.MethodsA 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 sen?sitivity test were all non-resistant strains,44 strains). Repeated sequence PCR was used for homology analysis of the strains,and pulsefield 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 tige?cycline 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.ResultsHomology analysis showed that there were 45 clonal groups in the detected clini?cal isolates,with no obvious outbreak of epidemic clonal groups. Efflux pumps and regulatory proteins were widely distributed in the clini?cal 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 signifi?cantly increase the expression levels of efflux pumps and regulatory proteins.Conclusion A. baumanniiis widely distributed in the clinic,and efflux pumps and regulatory proteins might play an important role in drug resistance process. The unreasonable use of tigecy?cline could enhance the tolerance of A. baumannii by up-regulating the expression of some bacterial efflux pumps.更多还原