Application of mNGS in traceability investigation of foodborne disease outbreaks caused by Salmonella Litchfield.

BACKGROUND: Salmonella is one of the most common pathogens responsible for foodborne outbreaks, posing a serious threat to public health. However, when conventional culture methods fail to isolate the pathogen from food, identifying the contamination source becomes challenging. Here, we report an investigation of a foodborne outbreak caused by Salmonella Litchfield that occurred in Xiamen,China on Sep 22, 2025. Isolation of the pathogen was successful only from clinical specimens, whereas all food and environmental samples tested negative. METHODS: Clinical, food, and environmental surface swab samples were collected. The clinical samples were screened using the 14-plex PCR assay for rapid pathogen detection. Metagenomic sequencing (mNGS) was performed on all samples in parallel. Conventional bacterial culture was also conducted, and the obtained isolates were subjected to whole-genome sequencing (WGS). A SNP-based phylogenetic tree was constructed using WGS data from clinical isolates and reference strains from different geographical regions. RESULTS: This foodborne outbreak was caused by Salmonella Litchfield (sequence type ST124),which was recovered from anal swabs of the six patients, including the chef. Phylogenetic analysis showed that the five patient isolates formed a distinct outbreak clone, whereas the chef's isolate belonged to a separate sublineage. No SNPs differed between the chef and four of the patients; however, the one-SNP difference observed in one patient isolate represented a microevolutionary event during transmission. The chef's isolate was closely related to a strain isolated in Hangzhou 5 years previously, with a 15-SNP difference between them. No pathogens were isolated from any food samples. cgMLSTFinder detected 2,768-2,772 core genes, with completeness >99.71%, and an average GC content of 52.25%. mNGS analysis identified high abundances of Salmonella in food samples at the genus level. Among the virulence genes detected, T3SS2 and T3SS, components of canonical virulence systems in Salmonella, were present at high abundance. Additionally, floR and tet (A) were highly abundant in food samples. CONCLUSIONS: Our integrated approach combining culture, WGS, and mNGS proved effective for rapid outbreak traceability, suggesting that the outbreak most likely originated from a contaminated food source associated with cross-regional dissemination, although the specific vehicle and transmission route remain to be determined.