摩根摩根菌对碳青霉烯类药物耐药机制的研究

A study on the resistance mechanism of Morganella morganii to carbapenems

目的分析从外科重症监护病房(SICU)分离的对碳青霉烯类药物耐药的3株摩根摩根菌(M1、M2和M3)的耐药机制和传播方式。方法采用琼脂稀释法进行药物敏感性试验;肠杆菌科基因间重复性一致序列(ERIC)-聚合酶链反应(PCR)用于分析菌株的分子流行病学特征;特异性PCR扩增检测细菌编码β-内酰胺酶的基因;接合试验和提取质粒进行酶切分析耐药基因可传递性和耐药质粒的同源性;耐药基因两边序列测序分析耐药基因的遗传背景;提取外膜蛋白电泳分析菌株外膜蛋白的改变。结果 3株分离菌和对应的接合子均扩增出介导碳青霉烯类耐药的肺炎克雷伯菌碳青霉烯酶(KPC)-2型基因,其中M1和M2为相同克隆株;质粒接合试验成功传递了对碳青霉烯类药物的耐药性,KPC-2基因被携带在酶切后片段完全不同的3个质粒上,耐药基因的遗传背景却相同;3株菌均缺失了相对分子质量约为38 000的外膜蛋白而出现36 000的外膜蛋白。结论 3株菌来自2种克隆株,均携带KPC-2基因。该基因由3个不同的质粒携带,同一可移动序列可能介导了KPC-2基因在3株细菌的不同质粒上转移。产KPC-2型碳青霉烯酶及外膜蛋白缺失可能共同导致了所分离的摩根摩根菌对碳青霉烯类抗菌药物耐药。

Objective To investigate resistance mechanisms to carbapenems and transmission modes of 3 Morganella morganii isolates（M1,M2 and M3） isolated from patients in surgical intensive care unit（SICU）.Methods The susceptibility of antimicrobial agents was detected by agar dilution method.The molecular epidemiological characteristics of the isolates were analyzed by enterobacterial repetitive intergenic consensus（ERIC）-polymerase chain reaction（PCR）.The encoding genes of beta-lactamases were analyzed by specific PCR amplification.The transitivity and homology of plasmids were detected by plasmid conjugation and restriction endonuclease analysis.Genetic environments around blaKPC were analyzed by sequencing.The changes of outer membrane permeability were analyzed by the electrophoresis of outer membrane protein.Results The blaKPC-2 was detected by PCR in all 3 isolates and their transconjugants.M1 and M2 were the same clonal type.Carbapenem-resistance was successfully transfered by conjugation experiments.The blaKPC-2 was located on dissimilar plasmids with quite different restriction profiles.The genetic environments around blaKPC-2 were the same.The 3 original Morganella morganii isolates showed a loss of 38 000 outer membrane proteins,and additional 36 000 outer membrane proteins appeared compared with the Morganella morganii isolates susceptible to carbapenems.Conclusions The blaKPC-2 is detected in the 3 Morganella morganii isolates belonging to 2 clonal types.This gene encoded by dissimilar plasmids is likely located on the same mobile genetic elements by which blaKPC-2 is possibly transferred between different plasmids.The KPC-2-producing and lack of outer membrane proteins together cause these 3 Morganella morganii isolates resistant to carbapenems possibly.