肺炎克雷伯菌多黏菌素耐药机制研究

Molecular mechanism of colistin resistance in Klebsiella pneumoniae

目的 探讨肺炎克雷伯菌对多黏菌素的耐药机制.方法 对临床分离的3株多黏菌素耐药肺炎克雷伯菌（FK1149、FK1920和FK1934）和3株体外诱导多黏菌素耐药肺炎克雷伯菌（FK660R、FK713R和FK729R）,通过PCR检测多黏菌素耐药相关基因,包括pmrAB、phoPQ、mgrB、crrAB、mcr-1和mcr-2;采用PROVEAN平台预测耐药相关蛋白质的生物功能改变情况;采用实时荧光定量PCR检测pmrH、pmrC、mgrB和phoP基因的相对表达量;SDS-PAGE银染试验检测3株临床分离的多黏菌素耐药菌株中是否存在脂多糖（LPS）缺失现象;同时通过接合转移试验检测是否存在耐药性转移的可移动性元件.结果 本研究检测到多黏菌素耐药株中存在PmrA（G53V）、PmrB（T157P和R256G）、MgrB（F44C）和CrrB（E189K）等多种有义突变,在FK713R和FK729R中分别检测到ISkpn14和IS5-like插入序列;所有耐药菌株中均未检测到mcr基因;与对照菌株相比,所有临床多黏菌素耐药菌株和诱导多黏菌素耐药菌株中pmrH和pmrC基因的表达量均增加,phoP和mgrB的表达水平也发生了变化;临床耐药株FK1149中存在LPS部分缺失.结论 本研究中,pmrAB或mgrB基因突变引起的LPS修饰是肺炎克雷伯菌对多黏菌素耐药的主要机制;首次在临床分离的肺炎克雷伯菌中检测到可能与多黏菌素耐药相关的PmrA（G53V）、MgrB（F44C）和CrrB（E189K）突变.

Objective To investigate the molecular mechanism of colistin resistance in Klebsiella pneumoniae （K. pneumoniae ）. Methods Three clinical isolates of eolistin-resistant K. pneumoniae （FKl149, FK1920 and FK1934） and three colistin-resistant mutants （FK660R, FK713R and FK729R） were investigated. Resistance genes of pmrAB, phoPQ, mgrB, crrAB, mcr-1 and mcr-2 were detected by PCR and then analyzed by sequencing. PROVEAN platform was used to predict changes in the biological functions of proteins related to drug resistance. Expression ofpmrH, pmrC, mgrB and phoP genes was meas- ured using quantitative real-time PCR. LPS silver staining and conjugation assay were performed to analyze the three clinical colistin-resistant isolates. Results Amino acid substitutions in PmrA （G53V）, PmrB （T157P, R256G） , MgrB （F44C） and CrrB （E189K） were detected. ISkpnl4 and IS5-1ike insertion se- quences were detected in FK713R and FK729R, respectively. FK1149, FK1920 and FK1934 were negative for mcr genes. Compared with the wild-type strain, expression ofpmrH and pmrC genes at the transcriptional level was increased in all investigated isolates. Changes in the expression of phoP and mgrB genes were also observed. A partial deletion of LPS was identified in FK1149. Conclusion LPS modification induced by inactivation of PmrAB or MgrB is the main molecular mechanism of eolistin resistance in K. pneumoniae iso-lates in this study. Mutations in PmrA （G53V）, MgrB （F44C） and CrrB （E189K） that might be related to colistin resistance are detected for the first time in clinical isolates of K. pneumoniae.