二代测序技术在一起食源性疾病事件病原鉴定中的初步应用

Preliminary application of next generation sequencing technique in pathogen identification of foodborne disease

目的 分析一起食源性疾病事件中病原的基因组学特征.方法 选取事件中死亡病例血样和洗胃前胃内容物,以及其就餐食物作为样品,共11份,对金黄色葡萄球菌和蜡样芽胞杆菌以及有毒物质进行检测,并对食物中检测到的细菌进行计数.采用实时荧光PCR方法检测分离得到的10株蜡样芽胞杆菌的16 S rDNA保守区基因和呕吐毒素ces基因,并采用二代测序技术对分离株进行了基因组测序,初步分析质粒携带情况,并与公共数据库中参考序列进行比较基因组学分析.结果 所有样品中,仅蜡样芽胞杆菌检测阳性,其中蛋炒饭中蜡样芽胞杆菌计数为1.9×10^7 CFU/g,干炸鱼和卤猪头肉中均为3.0×10^3 CFU/g.10株分离株均携带hlyⅢ、nheA、nheB、inlA、inhA毒力基因;携带plcR和nheC基因的菌株均为9株.分离株16 S rDNA保守区基因和ces基因均为阳性,而且均携带完整的ces基因簇序列,并且与分离自英国的AH187株携带的呕吐毒素质粒pCER270（NC_010924.1）和分离自日本的NC7401株携带的质粒pNCcld（NC_016792.1）的序列完全一致.质粒比对结果显示,分离株序列与炭疽杆菌的pXO1和pXO2质粒差异较大,但携带含ces基因的pNCCld质粒.10株分离株基因组系统发生树显示,10株蜡样芽胞杆菌高度相似（系统发生树上距离为2.0×10^-6～9.0×10^-6）且与AH187株和NC7401株均高度相似（均属ST26型,系统发生树上距离均为3.8×10^-5～4.5×10^-5）.结论 本次食源性疾病事件由不携带pXO1、pXO2质粒的呕吐型蜡样芽胞杆菌污染蛋炒饭引起.提示蜡样芽胞杆菌引起呕吐型食物中毒可能与携带ces基因的ST26、ST164等型别菌株相关.

Objective To analyze genomic features of pathogens based on next generation sequencing technique in a food-borne disease event. Methods A total of 11 blood samples, stomach contents before gastric lavage from the death and patients＇ foods were collected. S. aureus, B. cereus and toxic substances were detected. B. cereus detected in foods were counted. The conserved region of 16 S rDNA gene and ces gene（cereulide） of B. cereus isolates were detected by real-time PCR. Next Generation Sequencing （NGS） technology was applied to acquire genome sequences of isolates. Different plasmids distribution and comparative genomics analysis with reference sequences in public databases were analyzed. Results Only B. cereus tested positive in all samples. The counts of B. cereus in Egg fried rice, one food samples, were 1.9× 10^7 CFU/g, and the counts of B. cereus in dried and fried fish and brine pork head meat samples were 3.0×10^3 CFU/g both. Ten isolates were carrying hlyⅢ, nheA, nheB, inlA and inhA genes, and nine isolates carried the plcR gene and nine isolates carried the nheC gene. The PCR result of 16 S rDNA gene and ces gene of all isolates were positive. All carried the complete ces genes cluster sequence which were identical to the sequence of plasmid pCER270 （NC_010924.1） from strain AH187 in United Kingdom and pNCcld （NC_016792.1） from NC7401 in Japan. The alignment of plasmids turned out the sequence of the isolate differed from the pXO1 and pXO2 plasmids of B. anthracis, but carried the pNCcld plasmid containing the ces genes cluster. The phylogenetic tree based on genomic sequences of ten isolates showed high similarity （distances in phylogenetic tree from 2.0×10^-6-9.0×10^-6） to each other and to the B. cereus strains AH187 and NC7401 （MLST ST26 type, distances in phylogenetic tree from 3.8 × 10^-5-4.5 × 10^-5）. Conclusion The foodborne disease event was caused by vomiting type Bacillus cereus without plasmid pXO1 and pXO2 contaminated egg fried rice. The vomiting-type food poisoning caused by B. Cereus globally is probably associated with ST26, ST164 and other strains harboring ces gene.