目的: 对某制药企业注射剂无菌检查阳性污染菌A1（葡萄球菌）进行溯源。方法: 采用Illumina 平台高通量测序对A1 和采集自生产和检验环节的16 株葡萄球菌进行全基因组测序,使用Unicycler 软件对序列进行拼接,用Prokka 软件进行菌株基因预测,通过计算基因组序列与模式菌株之间的OrthoANI 值和AF 值进行菌株鉴定,用orthoANI 软件进行两两orthoANI 值比较;用kSNP 软件进行两两SNP 比较,并通过距离矩阵转换进行聚类分析,分析了菌株的基因组大小、GC 含量、编辑基因数量等特征以及A1 与其他收集菌株的同源性关系。结果: 无菌检查阳性污染菌A1 菌株为科氏葡萄球菌,基因组大小为2 765 495 bp,GC 含量32.27%,在调查样本中,A1 菌株的核酸信息与无菌检验环境污染菌A2 菌株的核酸信息具有最大相似性。结论: 全基因组测序简便、快速、稳定、准确,其两两orthoANI 值比较、SNP 比较及聚类分析结果准确显示菌株的同源性关系,适用于对污染菌株进行准确溯源,有利于制药企业实现对生产和检验过程的微生物控制。
Objective： To trace the source of microbial contamination A1（Staphylococcus） isolated from injection sterility test in a pharmaceutical company. Methods： Illumina sequencing was used for genome-wide gene sequencing of A1 and other 16 Staphylococcus strains,which were collected from production and testing process. Unicycler software was used to splice the sequences,Prokka software was used to predict the gene of strains. The OrthoANI value and AF value between the genomic sequence and the model strains were calculated to identify the strains. The OrthoANI value in pairs was also compared. The kSNP software was used to compare SNPs in pairs,and cluster analysis was carried out by distance matrix transformation. Relevant software were used to analyze the sequence for splicing,assembling, gene prediction,gene annotation,cluster of orthologous group and single nucleotide polymorphisms respectively to get the information of the genomic size,GC content,number of editing genes,and the homology of A1 and the other collected strains. Results： Microbial contamination A1 was Staphylococcus cohnii. Its genome size was 2 765 495 bp and GC content was 32.27%. A1 was traceable to environmental bacteria A2,which was introduced during the sterility test. Conclusion： Whole-genome sequence（WGS） method is simple,rapid,stable and accurate.It is suitable for pharmaceutical companies to trace the source of contaminated strains accurately,and is conducive to realize the process control in both production and inspection process.
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