革兰阴性杆菌新德里金属β内酰胺酶基因的筛查  

Screening of New Delhi metallo-β-lactamase 1 for gram negative bacilli

作　　者：周珍文 谢永强 杨镒宇 姚淑雯 关锐黎 钟华敏 周帅 龚四堂 

机构地区：[1]广州市妇女儿童医疗中心检验科,510120

出　　处：《中华检验医学杂志》2013年第10期916-919,共4页Chinese Journal of Laboratory Medicine

基　　金：国家自然科学基金资助项目（30801054）;广州市医药科技重点项目（201102A212013）;广州市医药科技项目（20131A011053）

摘　　要：目的 从耐亚胺培南革兰阴性杆菌中筛查金属β内酰胺酶（MBL）及1型新德里金属蛋白酶（NDM）-1,为NDM-1携带菌感染的治疗及防控提供试验依据.方法 回顾性对广州市妇女儿童医疗中心2010年从7例患者分离的7株耐亚胺培南革兰阴性杆菌进行NDM-1基因筛查,使用亚胺培南-EDTA双协同试验筛查MBL阳性菌株,设计特异性引物PCR扩增NDM-1基因,并将其克隆入pMD-T载体,转化Dh5a,提取重组质粒进行EcoR Ⅰ和Xho Ⅰ双酶切鉴定,并将重组质粒菌进行测序验证.结果 从7株亚胺培南耐药革兰阴性杆菌中筛出2株MBL阳性株,分别为1株鲍曼不动杆菌和1株琼氏不动杆菌,其中MBL阳性琼氏不动杆菌PCR扩增出NDM-1目的产物,将NDM-1成功克隆入pMD-T,双酶切鉴定见目的条带,重组质粒测序验证了其为NDM-1基因,序列同源性分析显示其与已知NDM-1基因序列高度一致（99.9％）,813碱基中仅第468核苷酸G-A突变,其演绎的氨基酸序列与已知NDM-1完全一致,其序列已登录GenBank,登录号为HQ603057.药敏试验显示此琼氏不动杆菌对除氨曲南、头孢哌酮/舒巴坦外的其他所测试的β内酰胺类抗生素均耐药,但对替加环素、氟喹诺酮、氨基糖苷类敏感.结论 在住院儿童血液中筛查出携带NDM-1琼氏不动杆菌,提示临床试验室应加强亚胺培南耐药革兰阴性杆菌NDM-1基因的筛查,为NDM-1携带菌感染的治疗及防控提供试验依据.Objective To screen metallo-β-lactamase （MBL） and New Delhi metallo-β-lactamase 1 （NDM-1） genes among imipenem-resistant gram-negative bacilli, and to provide experimental evidence for the treatment and control of bacteria carrying NDM-1. Methods A total of 7 impenem-resistant gram- negative bacilli isolated from 7 patients in Guangzhou Women and Children＇s Medical Center during 2010 were screened for NDM-1 gene retrospectively. The MBLs were examined by imipenem-ethylenediaminetetra acetic acid （EDTA）double-disc synergy test. NDM-1 gene-specific primers were designed for PCR amplification, and the PCR products were cloned into pMD-T plasmid and then transformed to E. coli Dh5a. Recombinant plasmid was identified by EcoR I ,Xho I double enzyme and sequencing. Results Two MBL positive strains （ Acinetobacter baumannii 1542, Acinetobacter junii 1454 ） were identified, and NDM-1 gene was verified in Acinetobacter junii 1454, and showed high concordance with known NDM-1 gene sequences in Genbank （99. 9% ）. Only one nncleotide G-A mutation among 813 nucleotides（G468A） was found, and the amino acid sequences were exactly the same with known NDM-1. The accession number of the sequence of Acinetobacterjunii NDM-1 under Genbank is HQ603057. Acinetobacterjunii 1454 was resistant to all beta-lactams antibiotics tested except aztreonam and cefoperazone/sulbactam but sensitive to tigecycline, aminoglycosides and quinolones. Conclusion NDM-1 positive Acinetobacter junii was screened from a hospitalized child, suggesting that clinical microbiology laboratories should strengthen imipenem-resistant NDM-1 gene screening.

关 键 词：革兰氏阴性菌 Β内酰胺酶类 亚胺培南 抗药性 细菌 

分 类 号：R440[医药卫生—诊断学]