产ESBLs大肠埃希菌和肺炎克雷伯菌的检测及其基因分析

Detection of Escherichia coli and Klebsiella pneumoniae producing extended-spectrum beta-lactamases and analysis of their genetypes

目的了解临床分离菌中产超广谱β内酰胺酶(ESBLs)大肠埃希菌和肺炎克雷伯菌发生率,分析其耐药表型,探讨其ESBLs基因类别。方法采用美国国家临床实验室标准化委员会(NCCLS)推荐的纸片扩散法,对364株大肠埃希菌和71株肺炎克雷伯菌进行产ESBLs菌株初筛试验,并用双纸片确证试验和双纸片协同试验进行产ESBLs菌株确证。按照NCCLS文件标准,用KirbyBauer纸片扩散法进行产ESBLs株21种抗生素药敏试验。用PCR方法扩增168株ESBLs表型阳性菌中blaTEM1、blaSHV1、CTXM1组、TOHO1组等4种基因。结果(1)大肠埃希菌和克雷伯菌中产ESBLs的检出率分别为46.7%和32.4%。(2)产ESBLs菌对青霉素类100%耐药;对第1、2代头孢菌素耐药率达85%～100%;对除头孢他定以外的第3代头孢菌素,产ESBLs大肠埃希菌耐药率为80%以上,产ESBLs肺炎克雷伯菌耐药率为65%～75%以上;产ESBLs菌对复方新诺明的耐药率为75%～85%;产ESBLs大肠埃希菌对环丙沙星耐药率为80%～90%、庆大霉素耐药率为50%～75%;产ESBLs菌对阿米卡星、头孢替坦、亚胺培南有较好的敏感性(80%～90%以上)。(3)产ESBLs大肠埃希菌中,blaTEM1、blaSHV1、CTXM1组等3种基因扩增阳性率分别为93.9%、7.4%和53.4%。51.4%的菌株同时携带2种耐药基因,2.7%的菌株同时携带3种耐药基因。产ESBLs肺炎克雷伯菌中blaTEM1、blaSHV1、CTXM1组等3种基因扩增阳性率分别为50.0%、95.0%和20.0%。同时携带2种耐药基因的菌株占35.0%,同时携带3种耐药基因的菌株占15.0%。两种细菌中均未检出TOHO1组基因。结论大肠埃希菌和肺炎克雷伯菌中产ESBLs的检出率较高,大肠埃希菌中产ESBLs菌比例有逐年上升趋势。产ESBLs菌对头孢噻肟的耐药率远高于头孢他定,且多数菌株对青霉素类,第1、2、3代头孢菌素、喹诺酮类、磺胺类、氨基糖甙类等抗生素呈多重耐药。大肠埃希菌中以TEM型和CTXM型基因为主。肺炎克雷伯菌以SHV型基因为主,同时存在TEM型和CTXM型基因。

Objective To determine the prevalence, multidrugs resistance, and the genetypes of extended-spectrum beta-lactamases （ESBLs） among clinical isolates of Klebsiella pneumoniae and Escherichia coli. Methods 364 isolates of Escherichia coli and 71 isolates of Klebsiella pneumoniae isolated from a hospital in Kunming during the period from January 2002 to June 2004 were screened for ESBL production by disc diffusion methods as described by the NCCLS. ESBL-producing isolates were confirmed by double-disk synergy test and disc diffusion confirming test as recommended by the NC-CLS. The in vitro activities of 21 antimicrobial agents in the ESBL- producing isolates were determined by Kirby-Bauer method according to the NCCLS guidelines. PCR analysis was used for blaTEM-1, blaSHV-1 ,CTX-M1 group and TOHO-1 group genes present in 20 isolates Klebsiella pneumoniae and 148 isolates Escherichia coli. Results （1） The ESBL phenotype was detected in 170 （46.7%） E. coli and 23（32.4%） K. pneumoniae isolates. （2） The percentage of ESBL- producing organisms that were resistant against antibiotic was 100% for penicillins, 85%- 100 for the first and the second products of cephalosporin, 65 %-75 % for the third products of cephalosporin excluding ceftazidime, 75 %- 85% for trimethoprin/sulfamethoxozol, 50% - 75% for gentamicin, and 80% - 90% for ciprofloxacin. 80%-90 % of ESBL-producing isolates showed susceptibility to amikacin, cefotetan and imipenem. （3） The genes of blaTEM-1, blaSHV-1 and CTX-M-1 group were detected in 93.9% ,7.4% and 53.4% of ESBL-producing E. coil respectively, and in 50.0% ,95.0% and 20.0% of ESBL-producing K. pneumoniae respectively. The ESBL-producing organisms carried two of these genes were 51.4% of E. coil and 35.0% of K. pneumoniae. 15.0% of ESBL-producing K. pneumoniae carried all of these genes. The TOHO-1 group gene was not detected among both of E. coil and K. pneumoniae. Conclusion The prevalence of ESBLs among clinical isolates of Klebsiella pneumoniae and Escherichia coli was high, and increased during the period from January 2002 to June 2004 for Escherichia coll. The ESBL-producing organisms showed multidrug resistance. The ESBL-gene possibly were the CTX-M-type in Escherichia coil, and the SHV-type in Klebsiella pneumoniae.