Antagonistic activity of Bacillus velezensis L09 isolated from crocodile gut against Aeromonas dhakensis: Mechanisms and potential for aquaculture applications

The global aquaculture industry is facing increasing threats from multidrug-resistant pathogens, particularly Aeromonas dhakensis , which causes septicemia in crocodiles, causes severe infections in aquatic species, and poses zoonotic risks to humans. To address this challenge, we isolated Bacillus velezensis L09, a novel probiotic, from the gut of Crocodylus siamensis in Hainan Province, China, and evaluated its antimicrobial efficacy. Whole-genome sequencing identified seven antimicrobial biosynthesis gene clusters and diverse carbohydrate-active enzymes, highlighting its metabolic versatility and environmental adaptability. In vitro co-culture experiments revealed a clear time-dependent reduction of A. dhakensis burden—approximately 65 ​% at 24 ​h and 95.7 ​% at 48 h—quantified by Colony-forming unit (CFU) counts and supported by scanning electron microscopy, which showed progressive envelope wrinkling, surface pitting, and overt lysis in treated bacteria. In zebrafish infection models, L09 exhibited dual protective effects against A. dhakensis : competitive exclusion—blocking 78.58 ​% of pathogen adhesion—and immunomodulation. Specifically, L09 upregulated intestinal barrier genes ( tjp1 , muc2 ) and activated tlr1/tlr7/tlr22–nfkb–tnfa/il6 signaling axis, thereby enhancing intestinal barrier integrity and activate the host immune response. Histopathological analysis confirmed the restoration of the villus structure and goblet cell repopulation. Safety assessments showed no hemolytic activity and susceptibility to 27 clinically relevant antibiotics. These findings demonstrate that B. velezensis L09 effectively suppresses pathogens, repairs intestinal barriers, and mitigates inflammation, establishing it as an functional adapted probiotic for sustainable aquaculture and a promising alternative for A. dhakensis -associated disease management. • This study is the first to report that Bacillus velezensis L09 from crocodile gut exhibits potent antagonistic activity against Aeromonas dhakensis. • Genomic and in vivo analyses revealed that L09 produces diverse antimicrobials, restores intestinal barrier integrity, and attenuates inflammation. • L09 significantly reduced pathogen load and improved host survival, highlighting its potential as a next-generation probiotic for sustainable aquaculture.