肺炎克雷伯菌临床分离株aae（6′）-Ib-cr基因的检测

Prevalence of aac（6′）-Ib.cr in clinical isolates of Klebsiella pneumoniae

目的调查肺炎克雷伯菌临床分离株aac（6′）-Ib—cr基因存在情况。方法收集2006年1月至2007年9月从临床分离的337株非重复的肺炎克雷伯菌。挑选64株对庆大霉素、阿米卡星和妥布霉素任何一种耐药的肺炎克雷伯菌临床分离株进行aac（6′）-Ib-cr基因检测，采用PCR法检测aac（6′）-Ib和Ⅰ类整合酶基因（intll），通过DNA直接测序确定aac（6′）-Ib-cr。抗生素MIC值测定采用琼脂稀释法。ESBLs检测采用双纸片扩散法，通过接合试验检测质粒是否可以发生转移。结果337株肺炎克雷伯菌临床分离株中，对庆大霉素、阿米卡星和妥布霉素的耐药率分别为19．0％（64／337）、8，3％（28／337）和16．3％（55／337）。共有24株细菌经PCR证实aac（6′）-Ib基因阳性，经测序证实全部为aac（6′）-Ib-cr基因，阳性率为37．5％（24／64）。24株aac（6′）-Ib-cr基因阳性株中，有13株对环丙沙星耐药（MIC≥2mg／L）和11株环丙沙星敏感（MIC≤0．25—1．0mg／L）。aac（6′）-Ib-cr基因在环丙沙星耐药菌株和环丙沙星敏感菌株中的检出率分别为54．2％（13／24）和27．5％（11／40），经χ^2检验，差异具有统计学意义（χ^2=11，056，P〈0．01）。24株aac（6′）-Ib-cr基因阳性株中，ESBLs和intll基因的阳性率分别为79．2％（19／24）和91．7％（22／24）。13株aac（6′）-Ib—cr基因阳性菌株的质粒成功转移至受体菌，以受体菌质粒DNA为模板进行aac（6′）-Ib-cr检测，13株受体菌质粒全部为aac（6′）-Ib-cr基因和intl基因阳性，全部为ESBLs阳性株。结论aac（6′）-Ib-cr基因广泛存在于肺炎克雷伯菌临床分离株中，aac（6′）-Ib-cr可能通过Ⅰ类整合子和ESBL基因同时位于可转移的质粒上造成传播。

Objective To investigate the prevalence of aac （6′）-Ib-cr in clinical isolates of Klebsiella pneumoniae. Methods A total of 337 isolates of Klebsiella pneumoniae were isolated from clinical specimens in our hospital from Jan,2006 to Sep, 2007. Gentamycin, amikacin or tobramicin was used to screen the isolated with aac（6′）-Ib-cr, aac（6′）-Ib and class 1 interase gene（ intll ） was determined by PCR. All PCR products of aac （6′）-Ib were sequenced for determination of aac （6′）-Ib-cr. MICs of antibiotics were determined by agar dilution method. The ESBLs-producing isolates were determined by the CISI-recommended confirmatory tests. Conjugation test was used to detect the transfer of plasmid. Results Of the 337 clinical isolates of Klebsiella pneumoniae, 64 （ 19. 0% ） , 28 （8.3 % ） and 55 （ 16.3 % ） isolates were resistant to gentamycin, amikacin and tobramycin, respectively. Among 64 gentamycin-resistant isolates, 24 （37.5%） were positive for aac （6′）-Ib-cr, including 13 ciprofloxacin-resistant isolates and 11 ciprofloxacin- susceptible isolates. The prevalence of aac （6′）-Ib-cr in ciprofloxacin-resistant and -susceptible isolates were 54.2 % （ 13/24 ） and 27.5% （11/40）. The positive rates of ESBLs and intll in the 24 isolates carrying aac （6′）-Ib-cr were 79.2% （19/24）and 91.7% （22/24）. Plasmids carrying aac（6′）-Ib-cr of 13 isolates were successfully transferred to E. coli J53. Plasmids of all transconjugants were positive for aac （6′）-Ib-cr and intll. All transconjugants were ESBL producing strains. Conclusions aac （6′）-Ib-cr exists widely in clinical isolates of Klebsiella pneumoniae, aac （6′）-Ib-cr and ESBL gene usually coexist in a self- transmissible conjugative plasmid by class 1 integron.