粘质沙雷菌碳青霉烯类耐药机制研究

The mechanism of carbapenems resistance in Serratia marcescens strains

目的：了解临床分离的粘质沙雷菌碳青霉烯类药物耐药机制及流行特点。方法收集温州医科大学附属第一医院2006-2012年临床分离的147株粘质沙雷菌，用Vitek2 Compact及配套革兰阴性细菌药敏卡检测其药敏情况；筛选出的11株碳青霉烯类耐药的粘质沙雷菌，用琼脂稀释法测定其对10种常见抗菌药物的MIC值；改良Hodge试验进行碳青霉烯酶表型检测；PCR检测碳青霉烯酶、AmpC酶、外排泵及外膜蛋白基因的携带情况；琼脂稀释法测定加入外排泵抑制剂CCCP前后碳青霉烯类药物MIC值的变化；SDS-PAGE分析菌株外膜蛋白有无缺失；对碳青霉烯耐药菌株进行接合转移试验，PCR扩增并测定接合子的MIC，PFGE分析菌株之间的同源性。结果11株粘质沙雷菌对青霉素类、头孢菌素类抗生素和厄他培南全部耐药，其中10株菌同时对亚胺培南和美罗培南耐药，但对氟喹诺酮类和氨基糖苷类药物有较好的敏感性；11株菌中10株携带blaKPC-2，1株携带blaIMP-1，8株菌同时携带blaEBC和blaMOX ，1株同时携带blaEBC和blaDHA ，1株菌同时携带blaEBC、blaMOX和blaDHA基因，其他基因未检出；加入CCCP后有7株菌对亚胺培南的MIC值降低4～64倍，有3株菌对厄他培南的MIC值降低8～256倍，而对美罗培南的MIC值没有变化；11株菌均未检出外膜蛋白缺失；有7株菌的blaKPC-2基因成功转移到受体菌，接合子与受体菌相比MIC值有不同程度提高；PFGE结果显示11株菌中有8株菌属于一个克隆型。结论产KPC-2碳青霉烯酶是本院粘质沙雷菌对碳青霉烯类耐药的主要原因，本研究表明KPC-2在温州地区存在克隆播散并且可以水平传播，故应引起高度重视。

Objective To investigate the mechanism of carbapenems resistance in Serratia marces-cens strains isolated from Wenzhou and their epidemiological characteristics.Methods 147 non-duplicated Serratia marcescens isolates were collected from the First Affiliated Hospital of Wenzhou Medical University during 2006 to 2012.The antimicrobial susceptibility test for all isolates was performed by using Vitek2 Compact to screen carbapenems-resistant Serratia marcescens strains.The minimum inhibitory concentrations （ MICs） of 10 commonly used antibiotics against carbapenems-resistant Serratia marcescens strains were de-termined by agar dilution method.The phenotypes of carbapenemase were analyzed by using the modified Hodge test.PCR analysis was used to detect the genes encoding carbapenemase, AmpC enzyme, efflux pump and outer membrane proteins.The changes of MICs before and after using CCCP efflux pump inhibitor were measured by agar dilution method.Outer membrane proteins were detected by SDS-PAGE.Carbapene-ms resistance genes were transferred from carbapenems-resistant Serratia marcescens strains to recipient strains by conjugation.The transconjugants were amplified by PCR and measured for MICs.Pulsed-field gel electrophoresis （ PFGE） was used to analyze homology among strains.Results 11 isolates resistant to car-bapenems were screened out from 147 Serratia marcescens isolates and all of them were resistant to penicil-lins, cephalosporins and ertapenem.10 out of the 11 isolates were both resistant to imipenem and meropen-em, but remained susceptible to fluoroquinolones and aminoglycoside.Among the 11 isolates, 10 carried blaKPC-2 gene, 1 carried blaIMP-1 gene, 8 harbored both blaEBC and blaMOX genes, 1 harbored both blaEBC and blaDHA genes, and 1 carried blaEBC , blaMOX and blaDHA genes.No additional genes were identified by PCR.The MICs of imipenem to 7 isolates and the MICs of ertapenem to 3 isolates were respectively decreased by 4-64 folds and 8-256 folds after using CCCP.CCCP had no effects on the MICs of meropenem.Loss of outer membrane protein was not detected among the 11 isolates.The blaKPC-2 genes were successfully transferred from 7 isolates into recipient strains.The MICs against the transconjugants were higher than those against the recipient strains in varying degrees.PFGE analysis demonstrated that 8 out of 11 Serratia marcescens strains belonged to one clonotype.Conclusion KPC-2 carbapenemase played an essential role in carbapenems re-sistance in Serratia marcescens strains isolated from Wenzhou.Attention should be paid to the clonal spread of KPC-2 and its horizontal transmission in Wenzhou.