基于全基因组测序分析1起产气荚膜梭菌导致的腹泻暴发事件

Application of whole genome sequencing in a Clostridium perfringens diarrhea outbreak

目的对1起产气荚膜梭菌导致腹泻暴发事件进行病原学分析。方法采集病例肛拭子样本和环境涂抹样本,肛拭子样本增菌前后分别进行产气荚膜梭菌plc和cpe基因荧光PCR检测和产气荚膜梭菌分离培养,对分离菌落进行全自动生化鉴定和飞行时间质谱鉴定。对鉴定为产气荚膜梭菌的分离株进行全基因组测序,分析菌株携带毒力基因和耐药基因情况,基于全部分离株核心基因组单核苷酸多态性进行遗传聚集性分析。结果未增菌的病例肛拭子cpe和plc基因检出率分别为46.15%(6/13)和53.85%(7/13),基于BHI厌氧增菌后病例肛拭子cpe和plc基因检出率分别为38.46%(5/13)和53.85%(7/13);10名病例肛拭子分离到产气荚膜梭菌,均为F生物型(携带毒力基因分布plc+/cpb-/etx-/iA-/cpe+/cpb2+/netB-),10个分离株基于cgSNP构建的聚类树形成2个相互独立且遗传距离较远的分支,Lineage 1仅分布1株,为ST589,携带耐药基因为erm(Q),Lineage 2分布9株,为ST149,携带耐药基因为tetB(P),为一组高度克隆化的菌株。结论本次暴发事件由F型产气荚膜梭菌所导致,且多数病例感染一组cpe+高度克隆化菌株。全基因组测序技术可应用于产气荚膜梭菌暴发事件病原学分析,基于肛拭子样本的产气荚膜梭菌增菌方法和分子筛查方法有待开发和应用。

An etiological analysis of a diarrhea outbreak attributed to C.perfringens was conducted.Anal swab and environmental smear samples were collected and subjected to fluorescence PCR detection of C.perfringens plc and cpe,as well as isolation and culture of C.perfringens before and after enrichment culture.The isolated colonies underwent fully automated biochemical identification and time-of-flight mass spectrometry analysis.Whole genome sequencing of isolates identified as C.perfringens was performed to analyze the strain carrying virulence and resistance genes,and the genetic aggregation based on single nucleotide polymorphisms in the core genome for all isolates.The positivity rates for plc and cpe genes without bacterial enhancement were 46.15%(6/13)and 53.85%(7/13),respectively.The positivity rates for plc and cpe genes after 24 h anaerobic bacterial enhancement in BHI were 38.46%(5/13)and 53.85%(7/13).All ten isolated CP belonged to the F biotype,with virulence gene characteristics of plc+/cpb-/etx-/iA-/cpe+/cpb2+/netB-.The phylogenomic tree indicated that all ten case-patient isolates except P1 isolate(lineage 1)were closely related and clustered together in a single clade(lineage 2).Lineage 1 belonged to ST589 and carried the macrolide resistance gene erm(Q),whereas lineage 2 belonged to ST149 and carried the tetracycline resistance gene tetB(P).The outbreak was caused by type F C.perfringens,and most cases were infected with a group of highly clonogenic cpe+colonies.Whole genome sequencing technology can be applied to etiology analysis of C.perfringens outbreak events,and the enrichment culture and molecular screening methods for C.perfringens based on anal swab samples should be further developed and applied.