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

ABSTRACT

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_01 0924.1) 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.