Genomic Investigation of a Bacillus subtilis Strain Sourced from Commercially Available Milk Powder in China Reveals Potential Risk Factors.

Background: Milk powder is a key food source, especially for infants and vulnerable groups. However, Bacillus contamination during production, storage, or handling can cause spoilage, quality issues, or health risks. This study identified and isolated Bacillus subtilis from commercially available Chinese milk powder.

Methods: A pure colony of Bacillus subtilis was isolated from an LB agar plate supplemented with milk powder and identified using mass spectrometry. The genome of this strain was sequenced using third-generation sequencing technology. Following assembly, the genome was functionally annotated and subjected to comprehensive bioinformatic analysis.

Results: Genomic analysis classified the strain as Bacillus subtilis via MALDI-TOF and ANI (98.82% with B. subtilis AMR1). Its genome features a 4.26 Mbp chromosome and 97.6 kbp plasmid encoding 4,539 genes, including virulence factors (209 genes), antibiotic resistance genes (19 genes), and carbohydrate-active enzymes (253 genes). Key virulence mechanisms include immune modulation, stress adaptation, toxin production, and biofilm formation. Antibiotic resistance involves efflux pumps (eg, qacJ, bmr), enzymatic inactivation (eg, FosBx1, aadK), and target modification (eg, vanG cluster, tet(45)). Phylogenetically (LIN78), the strain clusters with foodborne B. subtilis isolates (eg, from Korean gochujang and soybean), diverging from B. cereus and environmental Bacillus clades. Comparative genomics revealed 53 LIN78-specific genes, encompassing defense mechanisms and mobile elements, and synteny in all homologs except B. subtilis ATCC 11774. Genomic islands, CRISPR arrays, and recombination-associated repeats indicate adaptive evolution.

Conclusion: This study characterizes Bacillus subtilis LIN78, a genomically plastic strain isolated from Chinese milk powder. It exhibits adaptation to food environments via horizontal gene transfer, stress tolerance, and spoilage traits, while carrying antimicrobial resistance risks and potential biotechnological applications. The findings necessitate genomic monitoring to manage food safety, resistance spread, and leverage its dual role as both a spoilage organism and source of bioactive compounds..