中文摘要：

目的研究我国临床肺炎克雷伯菌和大肠埃希菌对黏菌素的耐药机制。方法回顾性收集2011至2014年我国11个城市15家教学医院中非重复的964株肺炎克雷伯菌和1389株大肠埃希菌。采用琼脂稀释法测定抗菌药物敏感性。PCR和基因测序检测黏菌素MIC〉2mg／L的菌株mcr-1基因携带率和黏菌素耐药相关基因mgrB、pmrB、phoQ是否存在基因突变。采用实时荧光定量PCR方法比较肺炎克雷伯菌黏菌素耐药组（黏菌素MIC值〉2mg／L，n=6）和黏菌素敏感组（黏菌素MIC值≤2mg／L，n=8）二元调控基因pmrB、pmrC、pmrD、pmrK和pmrE的相对表达量；采用RT．qPCR比较大肠埃希菌黏菌素耐药组（n=23）和敏感组（n=24）pmrA、pmrB、pmrC、phoP和phoQ基因的相对表达量。采用接合试验检测携带mcr-1质粒的可转移性。采用SPSSl6．0软件进行统计分析，以P〈0．05为差异具有统计学意义。结果肺炎克雷伯菌、大肠埃希菌对黏菌素耐药率分别为0．62％（6／964）和1．66％（23／1389）。黏菌素耐药的2种菌中，mgrB基因均无突变；6株黏菌素耐药肺炎克雷伯菌phoQ基因均无突变，5株pmrB基因发生点突变。黏菌素耐药大肠埃希菌pmrB和phoQ基因均发生1～4个氨基酸位点突变。肺炎克雷伯菌耐药pmrB、pmrC、pmrD、pmrK和pmrE基因的相对表达量较敏感组差异无统计学意义。耐药组与敏感组：pmrB基因的相对表达量分别为（1．04-4-1．12）与（0．944-0．67），P=0．945；pmrC基因分别为（1．394-2．01）与（0．16±0．27），P=0．101；pmrD基因分别为（1．59±2．43）与（0．884-0．34），P：0．445；pmrK基因分别为（0．64-4-0．62）与（0．04-4-0．10），P=0．051；pmrE基因分别为（3492833388．83-4-8478977986．85）与（20771428．934-38000732．85），P=0．445。而大肠埃希菌耐药组pmrB基因的平均相对表达量较敏感组上升9．5倍。黏菌素耐药的大肠埃希菌、肺炎克雷伯

英文摘要：

Objective To elucidate the resistance mechanisms of clinical colistin-resistant Klebsiella pneumoniae and Escherichia coli isolates in China. Methods A total of 964 K. pneumoniae and 1 389 E. coli isolates were retrospectively collected from national surveillance programs from 2011 to 2014 in China. Antimicrobial susceptibility testing was determined by the microdilution method. The PCR amplification followed by sequencing was used to detect the mcr-1 gene and colistin-resistance genes, including mgrB, pmrB and phoQ. Real-time quantitative PCR was performed to examine the relative transcriptional levels of pmrB, pmrC, pmrD, pmrK and pmrE genes in K. pneumoniae, and pmrA, pmrB, pmrC, phoP and phoQ genes in E. coll. Conjugation experiment was used to detect the transferability of the resistance plasmid carrying the mcr-1 gene. Statistical analyses were performed using IBM SPSS Statistics （version 16. 0） and a P value 〈 0. 05 was considered statistically significant. Results The colistin-resistant rates of K. pneumoniae and E. coli were 0.62% （6/964） and 1.66% （23/1 389）, respectively. No amino acids substitutions were identified in mgrB genes among eolistin-resistant isolates. Among six eolistin-resistant K. pneumoniae isolates, five isolates were identified to have point mutations in pmrB gene, but no point substitution was detected in phoQ gene. One to four point mutations had been found in pmrB and phoQ genes in colistin-resistant E. coli isolates, respectively. The expression level of pmrB, pmrC, pmrD, pmrK and pmrE genes showed no significant difference between colistin-resistant and colistin-susceptible isolates [pmrB, （1.04±1.12） vs. （0.94±0.67）, P=0.945; pmrC, （1.39±2.01） vs. （0.16±0.27）, P= 0.101; pmrD, （1.59±2.43） vs. （0.88 ±0.34）,P=0.445; pmrK, （0.64±0.62） vs. （0.04±0.10）, P = 0. 051 ; pmrE, （3 492 833 388.83 ± 8 478 977 986. 85） vs. （20 771 428. 93± 38 000 732. 85）, P = 0. 445 ]. However, the transcriptional level of pmrB genes in colistin-