鲍曼不动杆菌美罗培南体外诱导后对常用抗菌药物敏感性降低及其机制研究

Reduced antibiotics sensitivity of Acinetobaoter baumannii induced by meropenem in vitro and related mechanism

目的研究美罗培南体外诱导后的鲍曼不动杆菌对药物的敏感性及相关机制。方法将临床分离的3株对碳青霉烯类敏感的鲍曼不动杆菌用美罗培南体外诱导后获得突变株（MS1、MS2和MS3），对诱导前后的菌株，采用全自动药敏分析仪测定抗菌药物最低抑菌浓度（MIC）的变化，采用肠杆菌科细菌间重复共有序列聚合酶链反应（ERIC—PCR）分析其同源性，改良Hodge试验和EDTA—Na，双纸片协同法分别检测碳青霉烯酶和金属β-内酰胺酶，PCR法检测碳青霉烯酶基因，对产物进行测序分析，荧光定量PCR检测外排泵基因adeB的表达差异，并用SDS-聚丙烯酰胺凝胶电泳检测外膜蛋白表达变化。采用t检验分析数据。结果美罗培南对鲍曼不动杆菌突变株的MIC上升，除亚胺培南、阿米卡星和多黏菌素对突变株的MIC无变化外，其他大部分抗菌药物对突变株的MIC也上升，且MS2和MS3对美罗培南的敏感性下降可稳定传代。同源性分析显示，亲本株和突变株100％同源。亲本株和突变株碳青霉烯酶和金属酶均阴性，只检测到OXA-51耐药基因。突变株adeB基因表达量为24．26±0．91，亲本株为22．81±0．38，二者差异无统计学意义（t=2．534，P〉0．05）。MS1缺失相对分子质量为54000外膜蛋白，MS2和MS3缺失相对分子质量为47000外膜蛋白。结论美罗培南体外诱导后，鲍曼不动杆菌对美罗培南和常用抗菌药物的敏感性下降，可能与相对分子质量为47000外膜蛋白缺失有关。

Objective To investigate the mechanism related to reduced antibiotic sensitivity of Aciuetobacter baumannii inducted by meropenem in vitro. Methods Three strains of clinically isolated carbapenems-sensitive Acinetobacter baumannii were induced by meropenem in vitro, and the mutant strains （MS1, MS2 and MS3） were obtained. Minimal inhibitory concentrations （MICs） of antimicrobial agents to strains before and after induction were determined by automatic drug sensitivity analyzer. The homology of strains was analyzed by Enterobacterial repetitive intergenic consensus-polymerase chain reaction （ERIC- PCR）. Modified Hodge test and EDTA-Na2-double disk synergy test were used to detect carbapenemase and metallo-β-1actamase （MBL）, respectively. Main carbapenemase genes were detected by PCR and followed by DNA sequencing. Expressions of adeB and outer membrane proteins in strains before and after induction were detected with fluorescence quantitative PCR and SDS-polyacrylamide gel electrophoresis, respectively, t test was used for data analysis. Results The sensitivity of mutant Acinetobacter baumannii strains to meropenem and most antibiotics was reduced, except to imipenem, amikacin and polymyxin; and the reduced sensitivity to meropenem in MS2 and MS3 was of genetic stability. ERIC-PCR showed 100% homology between the mutant strains and parental strains. Both carbapenemase and metallo-β-1actamase were negative in mutant strains and parental strains, and only OXA-51 gene was found. The expressions of adeB gene in mutant strains were 24.26 ± 0. 91, while those in parental strains were 22.81 ± 0.38, and the difference was not significant （ t = 2. 534, P 〉 0. 05 ）. Outer membrane protein with molecular weight 54 000 was missing in MS1, while that with molecular weight 47 000 was missing in MS2 and MS3. Conclusion Reduced antibiotics sensitivity in meropenem-induced Acinetobacter baumannii may be correlated with the deficiency of outer membrane protein with molecular weight 47 000.