AcrR突变介导鼠伤寒沙门菌耐药的调控  

Drug Resistance of Salmonella enterica Serovar Typhimurium Regulated by Mutated AcrR

作　　者：李小军 李胜利 樊帅奇 邓翔杰 孙亚伟[2] LI Xiao-jun;LI Sheng-li;FAN Shuai-qi;DENG Xiang-jie;SUN Ya-wei(Animal Health Supervision Institution of Jiyuan City,Henan Province,Jiyuan 454650,China;College of Animal Science and Veterinary Medicine,Henan Institute of Science and Technology,Xinxiang 453003,China)

机构地区：[1]河南省济源市动物卫生监督所,河南济源454650 [2]河南科技学院动物科技学院,河南新乡453003

出　　处：《中国兽医杂志》2022年第10期44-52,共9页Chinese Journal of Veterinary Medicine

基　　金：河南省高等学校重点科研项目(22A230008)。

摘　　要：为了阐明临床鼠伤寒沙门菌(CST1、CST2、CST3和CST4)多重耐药机制,本试验通过2倍微量稀释法测定不同抗菌药物在多重耐药泵抑制剂[苯丙氨酸-精氨酸-β-萘胺(PAβN)和羰基氰化物间氯苯腙(CCCP)]、多重耐药泵AcrB和多重耐药泵调控蛋白存在及缺失条件下对鼠伤寒沙门菌的最小抑菌浓度(MIC);用实时荧光定量PCR(Real-time RT-PCR)检测临床菌和鼠伤寒沙门菌CVCC541(ST)的多重耐药泵基因表达水平;随后用PCR扩增临床菌喹诺酮耐药决定区和多重耐药泵调控蛋白编码基因并进行测序分析;依据所测序列,选取发生突变的耐药泵相关蛋白,构建该蛋白重组质粒并在临床突变菌中过表达并检测其对MICs的影响。结果显示:PAβN可导致盐酸四环素、多西环素、加替沙星和恩诺沙星对所测细菌的MICs下降3.00~26.67倍;临床菌acrA表达水平相对ST菌增加3~9倍。CST2、CST3和CST4中,多重耐药泵编码基因acrF、mdfA和emrB表达水平相对ST菌增加5~15倍。AcrB失活后,大部分药物对基因失活菌的MICs下降3~80倍,但多重耐药泵调控蛋白RamA、MarA和SoxS分别失活后,所测药物对基因缺失菌的MICs均未发生显著改变;测序结果表明,CST2和CST3染色体内AcrAB的负调控蛋白AcrR携带Ser216→Ala突变,CST4的acrR由于1个碱基缺失导致其转录提前终止;当ST的acrR在临床突变菌中过表达后,盐酸四环素、头孢噻呋钠、多西环素、恩诺沙星和加替沙星对细菌的MICs下降3.00~21.40倍。本试验首次在临床鼠伤寒沙门菌中检测到AcrR突变,该突变参与细菌耐药调控。In order to investigate the multidrug resistant(MDR)mechanism of Salmonella enterica Serovar Typhimurium(S.Typhimurium)isolates(CST1,CST2,CST3 and CST4),the minimum inhibition concentrations(MICs)of the strains in the presence and absence of efflux pump inhibitors(PAβN and CCCP),MDR efflux pump AcrB and the regulators of MDR efflux pumps were determined by two-fold microdilution method.The expression levels of the genes encoding MDR efflux pumps in these isolates compared to that in S.Typhimurium CVCC541(ST)were tested by real-time RT-PCR.Furthermore,the nucleotide sequences in quinolone resistance determining regions and the genes encoding the regulators of MDR efflux pumps were sequenced and analyzed.Based on the tested sequences,the recombined plasmid containing the gene encoding regulator was constructed and over-expressed in clinical isolate carrying the mutated regulator and its effect on MICs was detected.The results showed that the MICs of tetracycline hydrochloride,doxycycline,gatifloxacin and enrofloxacin against the test strains in the presence of PAβN decreased by 3.00-to 26.67-folds compared to those in ST;the expression level of acrA in clinical isolates increased 3-to 9-folds compared to that in ST;the expression levels of acrF,mdfA and emrB encoding MDR efflux pump in CST2,CST3 and CST4 also increased by 5-to 15-folds compared to those in ST.Deletion of AcrB decreased the MICs of mutants to most antibacterial agents by 3-to 80-folds.However,the susceptibility of the mutants did not exhibit significant change after RamA,MarA and SoxS were individually inactivated in these isolates.Sequence analysis showed that AcrR,a negative regulator of AcrAB from CST2 and CST3,carried a mutation(Ser216→Ala).A frameshift mutation due to a 1-bp deletion was detected in the acrR from CST4,which led to the early termination of acrR reading frame.Complementation of the mutated acrR gene with the wild-type gene caused 3.00-to 21.40-folds reduction in the MICs of part of antibacterial agents(tetracycline hydrochlor

关 键 词：鼠伤寒沙门菌 细菌耐药 ACRB AcrR 

分 类 号：S855.1[农业科学—临床兽医学]