AcrAB-tolC外排泵及marA-soxS-rob调控系统对福氏志贺菌环丙沙星耐药性的影响  

Association of AcrAB-tolC efflux pump and marA-soxS-rob regulon with ciprofloxacin-resistance in Shigella flexneri isolates

作　　者：程玉谦[1] 杨贤[2] 祁伟[2] 

机构地区：[1]天津医科大学第二医院感染免疫科,300211 [2]天津医科大学第二医院感染性疾病研究所,300211

出　　处：《中华临床感染病杂志》2015年第1期47-52,共6页Chinese Journal of Clinical Infectious Diseases

基　　金：天津医科大学科学基金（2010KY47）

摘　　要：目的 探讨AcrAB-tolC外排泵及mar A-soxS-rob调控系统对福氏志贺菌环丙沙星耐药性的影响.方法 连续收集45株2009至2013年分离自天津地区多家三级甲等医院肠道门诊患者粪便样本的福氏志贺菌.采用改良Kirby-Bauer纸片扩散法对菌株进行药物敏感性试验.随机选择10株环丙沙星耐药株和10株环丙沙星敏感株,分析其靶位酶基因（gyrA和parC）,质粒介导的喹诺酮耐药基因（PMQR）,外排泵基因（acrA和acrB）及调控基因（marA、soxS和rob）的携带情况,并进行序列分析.通过泵抑制剂羰基氰氯苯腙（CCCP）抑制试验检验外排泵基因及调控基因对菌株最低抑菌浓度（MIC）的影响,同时应用实时荧光定量PCR（RT-PCR）方法检测外排泵基因及调控基因的表达水平,并采用t检验比较这些基因在环丙沙星耐药株和敏感株中表达水平的差异.结果 10株环丙沙星耐药株均存在gyrA和parC突变,环丙沙星耐药株CR2同时携带qnrS1,环丙沙星耐药株CR5同时携带aac（6＆#39;）-Ib-cr基因.10株环丙沙星敏感株均未检测到外排泵基因及调控基因突变;而10株环丙沙星耐药株中,除CR10外,其余菌株均检出soxRS突变.加入CCCP后,喹诺酮类药物对10株环丙沙星耐药株的MIC均下降至原值的1/4 ～ 1/8,而对10株环丙沙星敏感株的MIC无变化或仅下降至原值的1/2.与环丙沙星敏感株相比,环丙沙星耐药株acrA、acrB、marA和soxS基因的表达水平明显升高（P ＜0.05或P＜0.01）.结论 外排泵机制可能在福氏志贺菌对环丙沙星耐药中发挥重要作用,mar A-soxS-rob调控系统发挥着调节泵基因表达的作用,且soxRS基因序列的改变可能是影响此调控系统的重要原因.Objective To investigate the association of AcrAB-tolC efflux pump and marA-soxS-rob regulon with ciprofloxacin-resistance in Shigella flexneri isolates.Methods Forty five strains of Shigella flexneri were isolated in stool samples collected from outpatient diarrhea clinics in Tianjin from 2009 to 2013.The antimicrobial susceptibility of bacteria was analyzed by Kirby-Bauer disk diffusion method.Ten ciprofloxacin-resistant Shigella flexneri isolates and 10 ciprofloxacin-sensitive isolates were randomly selected.The gyrA and parC genes,plasmid mediated quinolone resistant （PMQR） determinants,efflux pump genes （acrA,acrB） and regulation genes （marA,soxS,rob） were screened and sequenced.Minimum inhibition concentrations （MICs） of the strains were determined before and after efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone （CCCP） was added.The expression of acrA,acrB,marA,soxS and rob genes in ciprofloxacin-resistant and ciprofloxacin-sensitive strains was detected by realtime fluorescence quantitative RCR （RT-PCR）,and the differences of expression were evaluated using t test.Results Both gyrA and parC mutations were detected in all ciprofloxacin-resistant strains; qnrS1 was positive in ciprofloxacin-resistant strain CR2 and aac（6＆#39;）-Ib-cr was positive in ciprofloxacin-resistant strain CR5.Efflux pump genes and regulation genes were not detected in ciprofloxacin-sensitive strains,while soxRS mutation was detected in all ciprofloxacin-resistant strains except CR10.MICs of quinolones in ciprofloxacin-resistant strains decreased to one-fourth or one-eighth when CCCP added,while not changed or only decreased to one-half in ciprofloxacin-sensitive strains.Expressions of acrA,acrB,marA and soxS were significantly higher in ciprofloxacin-resistant strains than those in ciprofloxacin-sensitive strains （P 〈 0.05 or P 〈 0.01）.Conclusion Efflux pump may involve in the high-level ciprofloxacin resistance in Shigella flexneri through activating transcription of efflux pump ge

关 键 词：志贺菌属 抗药性 多药 环丙沙星 外排泵 marA-soxS-rob调控系统 

分 类 号：R446.5[医药卫生—诊断学]