非多重耐药及多重耐药鲍曼不动杆菌外排泵及其表达
Relationship between efflux pump and gene expression of non-multidrug resistant and multidrug resistant Acinetobacter baumannii

摘要：目的 探讨多重耐药鲍曼不动杆菌(multidrug resistant Acinetobacter baumannii, MDRAB)及非多重耐药鲍曼不动杆菌(non-multidrug resistant Acinetobacter baumannii, N-MDRAB)的外排泵表型、基因型和外排泵基因表达情况。方法 应用K-B法将120株临床分离的鲍曼不动杆菌，分成MDRAB 96株和N-MDRAB 24株两组；参照Sylvia Valdezate方法检测鲍曼不动杆菌的外排泵表型，即采用微量肉汤稀释法，观察加入泵抑制剂羰基氰氯苯腙(CCCP)前后其MIC值的变化，筛选出加入泵抑制剂后MIC值比原值降低1/4倍或以上的菌株为AB外排泵表型阳性；PCR法扩增外排泵蛋白基因并测序进行序列分析。结果 96株MDRAB对β-内酰胺类、头孢菌素类、碳青霉烯类、氨基糖苷类、喹诺酮类、四环素类、磺胺类、多黏菌素类抗菌药物的耐药率分别为76.04%～92.71%、73.96%～97.92%、40.63%～42.71%、92.71%～98.96%、93.75%～98.96%、98.96%、79.17%、0。24株N-MDRAB对头孢菌素类、β-内酰胺类、碳青霉烯类、氨基糖苷类、喹诺酮类、四环素类、磺胺类、多黏菌素类抗菌药物的耐药率分别为16.52%～20.43%、11.22%～15.65%、0、17.83%～18.26%、20.43%～23.39%、14.78%、19.57%、0。外排泵抑制检测结果显示，96株MDRAB有34株外排泵阳性，外排泵基因检测有33株adeB、32株adeR、33株adeS、33株adeJ、0株adeE和33株abeＭ，检出阳性率分别为97.06%、94.12%、97.06%、97.06%、0、97.06%； 24株N-MDRAB未检测到外排泵阳性菌株，但外排泵基因检测有12株adeB、20株adeR、16株adeS、18株adeJ、0株adeE和16株abeＭ，检出阳性率分别为50%、83.33%、66.67%、75%、0、66.67%。对adeB、adeR、adeS、adeJ、abeM基因进行测序,经比对,所测序列与GenBank中序列同源性为100%。结论 外排泵基因广泛存在于MDRAB中，但在N-MDRAB中亦可以发现主动外排泵基因的存在。
ABSTRACT: Objective To explore the efflux pump phenotype, genotype and gene expressions of multidrug Acinetobacter baumannii (MDRAB) and non-multidrug resistant Acinetobacter baumannii (N-MDRAB). Methods K-B method was used to detect 120 strains of Acinetobacter baumannii including 96 multidrug resistant strains and 24 non-multidrug resistant strains. We detected efflux pump genotype and gene expressions of multidrug and non-multidrug resistant Acinetobacter baumannii with broth microdilution method by the addition of carbonyl cyanide chlorobenzene hydrazone pump inhibitors with Sylvia Valdezate method. Strains whose MIC decreased to 1/4 or less of the original values were positive ones. Characteristics of efflux pump protein gene sequence were studied by PCR amplification and sequencing. Results The Acinetobacter baumannii resistance rate of 96 strains to cephalosporins, beta-lactams, penicillium carbon alkenes, aminoglycosides, quinolones, tetracyclines, tetracyclines, sulfas, and polymyxins was 73.96%-97.92%, 76.04%-92.71%, 40.63%-42.71%, 92.71%-98.96%, 93.75%-98.96%, 98.96%, 79.17%, and 0. The Acinetobacter baumannii resistance rate of 24 strains was 16.52%-20.43%, 11.22%-15.65%, 0, 17.83%-18.26%, 20.43%-23.39%, 14.78%, 19.57%, and 0. There were 34 positive efflux pump phenotypes in 96 multidrug resistant Acinetobacter baumannii strains, including 33 adeB strains, 32 adeR strains, 33 adeS strains, 33 adeJ strains, 0 adeE strains, 33 abeＭstrains, with positive detection rate of 97.06%, 94.12%, 97.06%, 97.06%, 0, and 97.06%, respectively. Positive efflux pump