First report of food-associated Enterococcus bulliens CFF05 from Thai cooked fermented fish: Genomic insights and safety assessment

Enterococci are frequently detected in fermented foods, where they contribute to microbial ecology but may also pose food safety concerns due to their potential to harbor antimicrobial resistance and virulence-associated traits. Enterococcus bulliens remains a poorly characterized species, with limited genomic data available and no prior reports from fermented fish products. In this study, we report the isolation and comprehensive characterization of E. bulliens strain CFF05 from Thai cooked fermented fish, representing the first food-associated genome of this species. Phenotypic analyses revealed resistance to chloramphenicol and erythromycin, susceptibility to ampicillin, tetracycline, vancomycin, and high-content gentamicin and streptomycin, and a beta-hemolytic phenotype on blood agar. Long-read whole-genome sequencing generated a complete 2.24 Mb chromosome with 38.4% GC content and 2581 predicted coding sequences (CDSs). Comparative genomic analysis confirmed species assignment, with an average nucleotide identity of 99.4% to the reference strain E. bulliens SS1972. In silico screening identified vanW and vanY homologs; however, strain CFF05 remained phenotypically susceptible to vancomycin, and no complete vancomycin resistance operon was detected. The genome also contained the biocide tolerance-associated gene qacJ and the putative hemolysin A gene tlyA, whereas no plasmids or complete high-risk antimicrobial resistance operons were identified. Mobile genetic element (MGE) analysis revealed a predominance of chromosomally encoded recombination, repair, and prophage-associated genes, with no evidence of transferable virulence or resistance determinants. In addition, pairwise gene presence or absence comparison highlighted strain-specific metabolic adaptations, including enhanced amino acid, carbohydrate, and riboflavin biosynthesis pathways in CFF05, consistent with adaptation to a fermented fish environment. Although PathogenFinder classified CFF05 as a potential human pathogen, this prediction was driven exclusively by conserved housekeeping genes rather than true virulence factors. E. bulliens CFF05 exhibits a conserved genomic backbone with limited safety-relevant traits; however, the presence of beta-hemolysis, antimicrobial resistance phenotypes, and MGEs underscores the importance of strain-level genomic and phenotypic evaluation when assessing enterococci from traditional fermented foods.