多重PCR快速检测金黄色葡萄球菌中氨基糖苷类抗生素耐药基因及其临床应用  

Development of multiplex PCR system for rapid detection of genes encoding aminoglycoside resistance in Staphylococcus aureus

作　　者：黄革[1,2] 周晓红[2] 蒋文玲[1] 李运雄[1] 荣卡彬[1] 罗宪玲[1] 赵莹[2] 

机构地区：[1]广东省人民医院医学研究中心广东省医学科学院,广州510080 [2]南方医科大学公共卫生与热带医学学院病原生物学系

出　　处：《中国感染与化疗杂志》2009年第4期244-247,共4页Chinese Journal of Infection and Chemotherapy

基　　金：广东省重大专项子课题(2006B33761002)

摘　　要：目的建立金葡菌氨基糖苷类抗生素耐药基因的多重PCR快速检测体系,了解耐药基因acc(6′)-Ie+aph(2″)、aph(3′)-Ⅲat ant(4′)-Ia在广州地区的流行分布。方法采用VITEK-60或PHOENIX-100微生物自动鉴定系统鉴定菌株及药敏试验,利用琼脂扩散法检测4种氨基糖苷类抗生素的耐药表型。通过优化PCR体系建立多重PCR并应用于检测金葡菌氨基糖苷类抗生素耐药基因。结果构建了四重PCR快速检测体系,124株金葡菌临床分离株中,acc(6′)-Ie+aph(2″)、aph(3′)-Ⅲa和ant(4′)-Ia的检出率分别为62.1%、32.3%和1.6%;所有庆大霉素、奈替米星以及阿米卡星耐药的金葡菌菌株均检测到上述3个耐药基因中的1个或1个以上基因;74株mecA阳性菌株中72株(97.3%)检测到acc(6′)-Ie+aph(2″)基因。结论所构建的多重PCR检测体系为临床实验室提供快速而有效的菌株耐药基因检测手段。编码AAC(6′)-APH(2″)双功能酶的acc(6′)-Ie+aph(2″)基因是金葡菌最重要的氨基糖苷类抗生素耐药基因,其次为aph(3′)-Ⅲa,ant(4′)-Ia则少见。Objective The rapid multiplex PCR （MPCR） system for detection of genes encoding aminoglycoside resistance in Staphylococcus aureus was developed. The distribution of antibiotic resistant genes acc （6′） -Ⅰe ＋ aph （2＂）, aph （3′） - Ⅲa and ant（4′）-Ⅰa in Guangzhou was analyzed using the established system. Methods S. aureus strains were identified and susceptibili- ty tests were performed using VITEK-60 or PHOENIX-100 system as recommended by the manufacturer. Aminoglycoside resistance was determined by disk diffusion method. MPCR system for detection of antibiotic resistance genes was optimized. Results The MPCR assay was established successfully. The prevalence of acc（6′） -Ⅰe ＋ aph（2′1） , aph（3′）-Ⅲa and ant （41） -Ⅰa in the 124 clinical S. aureus isolates was 62.1%, 32.3% and 1.6%, respectively as analyzed by MPCR. Good correlation between antibiotic resistance phenotypes and genotypes was observed. One or more of the genes encoding aminoglycoside modifying enzymes could be detected in all gentamicin- or netilmicin- or amikacin-resistant isolates. The acc（6′）-Ⅰe ＋ aph（2＂） gene was identified in 72 of 74 mecA-positive isolates. Conclusions This MPCR system could be used for rapid and reliable analysis of the antibiotic-resistant genotypes of clinical S. aureus isolates. The gene acc（6′）-Ⅰe ＋ aph（2＂） may be the predominant determinant of aminoglycoside resistance, followed by gene aph（3′）-Ⅲe. The prevalence of ant（4′）-Ⅰa gene is relatively low.

关 键 词：金葡菌 多重聚合酶链反应 氨基糖苷类抗生素耐药基因 

分 类 号：R450[医药卫生—治疗学]