Bacillus spp. have emerged as pivotal sources of probiotic preparations, garnering considerable attention in recent years owing to their vigorous bacteriostatic activity and antimicrobial resistance. This study aimed to investigate these probiotic characteristics in depth and verify the safety of Bacillus velezensis K12, a strain isolated from broiler intestine. The K12 strain was identified as Bacillus velezensis based on its morphology and 16S rDNA sequence homology analysis. Subsequently, B. velezensis K12 was evaluated for acid resistance, bile salt resistance, gastrointestinal tolerance, drug sensitivity, and antimicrobial activity. Additionally, whole-genome sequencing technology was employed to dissect its genomic components further, aiming to explore its potential applications as a probiotic strain. B. velezensis K12 was sensitive to six antibiotics and had acid tolerance. Furthermore, it showed potent antimicrobial activity against a wide range of pathogenic bacteria, including Escherichia coli (E. coli), Staphylococcus aureus, Salmonella, Clostridium perfringens, Bacillus cereus, and Vibrio parahaemolyticus. The complete genome sequencing of B. velezensis K12 revealed a genomic length of 3,973,105 base pairs containing 4123 coding genes, among which 3973 genes were functionally annotated. The genomic analysis identified genes associated with acid and bile tolerance, adhesion, antioxidants, and secondary metabolite production, whereas no functional genes related to enterotoxins or transferable antibiotic resistance were detected, thereby confirming the probiotic properties of B. velezensis K12. B. velezensis K12 exhibits broad-spectrum bacteriostatic activity and in vitro safety, positioning it as a potential candidate strain for developing probiotic Bacillus preparations.

Bacillus species represent promising alternatives to antimicrobial growth promoters, offering potential benefits for productivity and gut health in broilers. This study aimed to isolate Bacillus strains with inhibitory activity against Clostridium perfringens and evaluate their probiotic potential through in vitro and in vivo approaches. In Experiment 1 (in vitro), five strains—B. siamensis C66, B. tequilensis Y7, B. velezensis L15, B. amyloliquefaciens C271, and B. siamensis C377—were isolated and assessed for stress tolerance, digestive enzyme production, and antimicrobial activity. All strains demonstrated high survival rates under acid and bile stress, produced multiple digestive enzymes, and significantly inhibited the growth of C. perfringens. In Experiment 2 (in vivo), 630 day-old male Arbor Acres broilers were randomly assigned to one of seven dietary treatments for 42 days: a negative control (CON, basal diet), a positive control (ANfT, basal diet supplemented with 6.4 g/t virginiamycin), and five groups receiving basal diet supplemented with one of the Bacillus strains at 1 × 10¹¹ CFU/kg. Among these, B. amyloliquefaciens C271 significantly increased breast muscle yield (p < 0.05), improved jejunal morphology—evidenced by increased villus height and villus height-to-crypt depth ratio (p < 0.05)—and positively modulated cecal microbiota composition compared to the CON group. These findings demonstrate that the newly isolated B. amyloliquefaciens C271 possesses strong probiotic properties in vitro and promotes growth performance and gut health in broilers, suggesting its viability as an antibiotic growth promoter substitute.

Type II toxin–antitoxin (TA) systems are significantly expanded in the Mycobacterium tuberculosis complex; however, the functional role of the VapBC40 system in Mycobacterium bovis (M. bovis) pathogenesis remains poorly characterized. This study aimed to investigate the role of VapBC40 in mycobacterial virulence and evaluate its potential as a target for rational vaccine attenuation. We performed evolutionary analysis and yeast two-hybrid assays to characterize VapBC40 system specificity, conducted in vitro macrophage infection models and in vivo murine studies to assess virulence contribution, and evaluated the immunoprotective efficacy of a VapC40 knockout strain. Evolutionary analysis revealed progressive sequence conservation and stringent homologous pairing specificity within the VapBC40 system. The VapC40 toxin correlates with enhanced intracellular bacterial survival, increased host cell death, and more severe pulmonary pathology with systemic dissemination. Based on these findings, we evaluated the vaccine potential of a vapC40 knockout strain. Immunization with this attenuated strain elicited a Th1 cellular immune response, characterized by enhanced IFN-γ production and increased frequency of CD4⁺IFN-γ⁺ T cells. Upon challenge with virulent M. bovis, the knockout strain conferred superior protection compared to the conventional BCG vaccine, significantly reducing lung pathology and restricting extrapulmonary bacterial dissemination. Although the molecular mechanisms underlying VapC40-mediated effects remain to be fully elucidated, our findings suggest an important role of the VapBC40 system in mycobacterial-host interactions and support its potential as a target for next-generation tuberculosis vaccine development.