Pre-Colonization of Bacillus siamensis on Ocular Surface Mitigates Fusarium keratitis Through Direct Antifungal Activity and Pre-Activation of NF-κB Pathway

Purpose: Fungal keratitis (FK) is a severe ocular disease that leads to corneal ulceration and permanent vision loss. The ocular surface microbiota comprises beneficial symbiotic and pathogenic bacteria. Therefore, this study aimed to isolate beneficial bacterial strains from the ocular surface and evaluate their effect on Fusarium infections. Methods: Alterations in the ocular surface microbiota of patients with FK were analyzed using 16S rRNA sequencing. Candidate bacteria were isolated from mouse eyeballs and evaluated for antifungal activity. Quantitative PCR (qPCR) and culture were used to determine the colonization efficiency of Bacillus siamensis (B. siamensis). In addition, its biological safety was assessed. The effects of B. siamensis pre-colonization on Fusarium keratitis were evaluated using slit-lamp examination, clinical scoring, optical coherence tomography, hematoxylin and eosin staining, and RNA sequencing. Western blotting and RT-qPCR were used to assess its effects of on NF-κB signaling and inflammation, whereas flow cytometry was used to measure changes in immune cell populations following B. siamensis pre-colonization. Results: Ocular surface microbiota of patients with FK had significantly low levels of Bacilli. B. siamensis exhibited significant direct antifungal activity with minimal toxicity. Pre-colonization with B. siamensis mitigated FK-associated corneal edema and opacity, structural damage, inflammation, and fungal burden. Additionally, it enhanced ocular surface immunity into a "pre-immune" state by activating NF-κB pathway in healthy mice cornea. Conclusions: B. siamensis on the ocular surface can directly engulf Fusarium hyphae and secrete antifungal substances to exert antifungal effects. Moreover, it enhanced ocular surface immunity into a "pre-immune" state by activating NF-κB pathway to facilitate rapid immune response.