制药企业生产环境凝固酶阴性葡萄球菌多位点序列分型方法的建立

Development of Multilocus Sequence Typing Method for Dominant Coagulase-negative Staphylococci from Pharmaceutical Manufacturing Enterprises

凝固酶阴性葡萄球菌是药品生产环境的主要污染微生物。本研究以凝固酶阴性葡萄球菌为研究对象,基于泛基因组学筛选单核苷酸多态性看家基因序列,建立与评价目标靶基因PCR扩增方法,并通过目标靶基因序列多样性分析等步骤,研发了包含femA、ftsZ、gap、pyrH、rpoB和tufA共6个看家基因序列且同时适用于多种常见凝固酶阴性葡萄球菌的多位点序列分型(MLST)方法。应用本研究建立的MLST 方法,可将分离自制药企业生产环境中的118株凝固酶阴性葡萄球菌(27 株表皮葡萄球菌、25株溶血葡萄球菌、23株科氏葡萄球菌、16株人葡萄球菌、14株沃氏葡萄球菌和13株头状葡萄球菌)分为73个不同的序列型,辛普森指数为0.989。本研究建立的MLST方法对多种凝固酶阴性葡萄球菌均具有良好的分型能力,为药品生产过程污染凝固酶阴性葡萄球菌的溯源调查提供了有力工具。

Coagulase-negative Staphylococci is the dominant contaminating microorganism in the pharmaceutical manufacturing environment. In this study, a comprehensive multilocus sequence typing (MLST) scheme including femA, ftsZ, gap, pyrH, rpoB and tufA genes for coagulase-negative Staphylococci was developed through screening target housekeeping genes with single nucleotide polymorphism, establishing and evaluating PCR method of target genes, and analyzing nucleotide sequence diversity of target genes. The 118 coagulase-negative Staphylococci strains (27 Staphylococcus epidermidis, 25 S. haemolyticus, 23 S. cohnii, 16 S. hominis, 14 S. warneri, and 13 S. capitis) isolated from pharmaceutical manufacturing environment were tested by the MLST scheme. It was shown that the 118 strains could be divided into 73 different sequence types, and the Simpson's index was 0.989. The results indicated that the MLST scheme developed in this study had a good discrimination power for coagulase-negative Staphylococci and provided a powerful tool for the traceability investigation of coagulase-negative Staphylococci.