Probiotic strain identification and safety assessment of unlabeled bacteria in infant products

Probiotics are increasingly incorporated into infant and toddler nutritional products for their health benefits; however, concerns regarding the accuracy and safety of commercial probiotic product labeling remain unresolved. This study aimed to establish strain-level identification standards through whole-genome sequencing (WGS) and to evaluate the microbial composition and safety of 23 infant and toddler probiotic products. By integrating culturomics and WGS, analysis of probiotic strains Lacticaseibacillus rhamnosus HN001 (L. rhamnosus HN001), Bifidobacterium breve M-16V (B. breve M-16V) and Bifidobacterium animalis subsp. lactis HN019 (B. animalis subsp. lactis HN019) revealed significant diversity among isolates, with median single nucleotide polymorphism (SNP) distances ranging from 5 to 8, alongside other genomic variations such as plasmid loss. Furthermore, we identified 198 antibiotic resistance genes and 131 virulence factors in unlabeled strains. Particularly high levels of virulence factors were detected in Bacillus cereus (B. cereus) and Enterobacter hormaechei (E. hormaechei), These strains exhibited acute toxicity in mouse experiments. Live bacterial counts in some products failed to meet declared values. These findings underscore the urgency of strengthening regulatory oversight and establishing standardized genomic tools to ensure the safety and quality of probiotics.