牙菌斑培养菌群宏基因组文库构建及抗生素耐药基因筛选

Screening antibiotic resistance genes of cultured dental plaque with a metagenomic approach

摘要:

【目的】构建牙菌斑培养菌群宏基因组文库, 筛选牙菌斑生物膜中细菌的抗生素耐药基因。【方法】采集20例无龋健康人的集合牙菌斑并进行厌氧培养。提取牙菌斑培养菌群宏基因组构建Fosmid文库。用卡那霉素、四环素及氨苄西林对文库进行筛选, 并对筛选到的抗性Fosmid克隆进行末端测序、亚克隆构建、亚克隆测序和序列分析。【结果】构建了牙菌斑培养菌群宏基因组Fosmid文库, 插入片段长度在36?48 kb间约有15?120个克隆, 插入片段长度小于36 kb的约有3 360个克隆。筛选获得一个氨基糖苷类双功能修饰酶AacA-AphD基因、一个核糖体保护蛋白型四环素耐药基因tet (M)及一个C家族b-内酰胺酶基因。【结论】证实了可以通过构建宏基因组文库的方法来筛选牙菌斑培养菌群中的抗生素耐药基因。

Abstract:

[Objective] Fosmid library of cultured human dental plaque samples was constructed and screened for antibiotic resistance genes. [Methods] Dental plaques from 20 individuals were obtained and cultured anaerobically in vitro. Bacteria DNA was extracted and Fosmid library was constructed. Antibiotic resistant clones were selected by plating on LB agar containing one of the three antibiotics: kanamycin, tetracycline, and ampicillin. Inserts conferring resistance were sequenced and annotated. [Results] A metagenomic Fosmid library was generated, which contained approximately 18 480 clones. Three antibiotic resistance genes were obtained through functional screening, including one kanamycin resistance gene encoding bifunctional aminoglycoside modifying enzyme AacA-AphD, one tetracycline resistance gene tet (M) and one ampicillin resistance gene encoding class C beta-lactamase. [Conclusion] It turns out that it is possible to construct fosmid libraries using cultivated dental plaque samples to screen antibiotic resistance genes.