质粒介导16S rRNA甲基化酶肺炎克雷伯菌临床分离株的检测及分子流行病学研究

目的 调查165 rRNA甲基化酶在肺炎克雷伯菌临床分离株中的流行情况.方法 收集我院2006年1月至2007年9月从临床标本中分离的肺炎克雷伯菌337株,所有菌株均为非重复株.菌种鉴定采用全自动微生物分析仪,庆大霉素、阿米卡星和妥布霉素的药敏试验采用琼脂稀释法.超广谱B内酰胺酶(ESBLs)检测采用美国临床和实验窒标准协会(CLSI)规定的纸片确证法.使用PCR检测16S rRNA甲基化酶基因、整合酶基因和ESBL基因.接合试验检测质粒的可转移性;脉冲场电泳分析菌株的同源性.结果 337株肺炎克雷伯菌对庆大霉素、妥布霉素和阿米卡星的耐药率分别为19.0%(64/337)、8.3%(28/337)和16.3%(55/337).21株16S rRNA甲基化酶基因阳性,阳性率为6.2%(21/337),其中13株rmtB阳性、3株armA阳性、5株rmtB和atmA同时阳性.所有16S rRNA甲基化酶基因阳性株对庆大霉素、妥布霉素和阿米卡星同时耐药且都为高度耐药(MICs≥256μL),21株甲基化酶基因阳性株有19株产ESBLs,ESBL基因主要为CTX-M-14-like、CTX-M-15-like和SHV-12-like.21株均为Ⅰ类整合酶基因阳性.13株通过接合试验把质粒传递给受体菌E. coliJ53.所有接合子Ⅰ类整合酶基因阳性、blaTEM-1基因阳性、产ESBLs及对庆大霉素、妥布霉素、阿米卡星和复方磺胺甲噁唑耐药,对其他抗菌药物敏感.接合子ESBL基因型与供菌一致.21株16S rRNA甲基化酶基因阳性株经脉冲场凝胶电泳(PFGE)分成14个基因型,主要为A型和Ⅰ型.结论 armA和rmtB型16S rRNA甲基化酶基因已经在肺炎克雷伯菌中播散,既可通过克隆株播散也可通过接合性质粒在不同菌株间播散.

Molecular epidemiology and detection of clinical isolates of Klebsiella pneumoniae producing plasmid-mediated 16S rRNA methylases

Objective To investigate the prevalence and molecular epidemiology of clinical isolates of plasmid-mediated 16S rRNA methylases-producing Klebsiella pneumoniae. Methods From January 2006 and September 2007, 337 non-replicate clinical isolates of Klebsiella pneumoniae were consecutively collected from inpatients in a teaching hospital in Wenzhou, China. All of the isolates were identified by the automated microbiology systems. MICs of amikacin, gentamicin and tobramycin were determined by agar dilution method. The isolates were investigated for the presence of ESBLs by the CLSI-recommended confirmatory tests. PCR was used to detect 16S rRNA methylase genes, ESBL genes and class Ⅰ integrase gene. The homology of these isolates was analyzed by pulse-field gel electrophoresis (PFGE). Results Sixty-four ( 19. 0% ), 28 ( 8. 3% ) and 55 ( 16. 3% ) of 337 isolates were resistant to gentamicin, amikacin and tobramycin, respectively. Twenty-one (6. 2% ) isolates carried 16S rRNA methylase genes (3 for armA, 13 for rmtB, 5 for both armA and rmtB) and high-level resistant to gentamicin, amikacin and tobramycin ( MICs ≥256 mg/L). Nineteen of 21 isolates with 16S rRNA methylase genes were ESBL producers, blaCTX-M-14-like, blaCTX-M-like and blaSHV-12-like were predominant genotypes of ESBLs. The plasmids of 13 isolates were transferred into the recipients E. co/iJ53. PCR and sequence analysis revealed that blaCTX-M-14-like,blaCTX-M-15-like and blaSHV-12-like were co-transferred with the armA or the rmtB to the recipients. All transconjugants harbored intll and blaTEN-1. Of the 21 isolates, 14 patterns were obtained by PFGE. Conclusion Both horizontal gene transfer and clonal spread were responsible for the dissemination of the rmtB or the armA gene in Klebsiella pneumoniae.