Molecular Identification and Forensic Characterization of Bacillus velezensis Isolated from Tuna Grills: A Systematic Review

Abstract

Bacillus velezensis, an integral taxon within the Bacillus subtilis species complex, displays a distinct dual functionality in food microbiology by operating simultaneously as an effective biological control agent and a persistent food spoilage organism. Adhering to PRISMA 2020 guidelines, this systematic review evaluates 84 peer-reviewed studies published between 2020 and 2026 to elucidate the taxonomic evolution, molecular diagnostic constraints, whole-genome sequencing (WGS) parameters, and microbial forensic tracking of B. velezensis recovered from thermally processed seafood, particularly grilled tuna (Thunnus spp.). Conventional 16S rRNA gene sequencing lacks the resolution required to separate B. velezensis from sibling taxa such as B. amyloliquefaciens and B. subtilis due to sequence similarities exceeding 99%, thus requiring Multi-Locus Sequence Analysis (MLSA) alongside hybrid short- and long-read WGS platforms. Genomic demarcation indices, notably Average Nucleotide Identity (ANI >95–96%) and digital DNA-DNA Hybridization (dDDH >70%), provide definitive species boundaries, while antiSMASH bioinformatic mining uncovers extensive non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) clusters encoding surfactin, iturin, fengycin, macrolactin, bacillaene, and difficidin. Within protein-dense grilled tuna tissues, thermally resilient endospores withstand cooking, germinate, and drive structural tissue liquefaction through aggressive protease and lipase secretion, while concurrently promoting biogenic amine synthesis and recalcitrant biofilm establishment. Furthermore, high-precision genomic forensics utilizing core-genome Single Nucleotide Polymorphism (cgSNP, <5 SNPs threshold) and core-genome Multi-Locus Sequence Typing (cgMLST) enables accurate contamination source attribution across seafood production lines. Ultimately, comprehensive genomic screening against enterotoxin homologues (nhe, hbl) and mobile antimicrobial resistance (AMR) determinants remains mandatory before deploying candidate strains in food biopreservation.