Bacillus strains inhibit beta-lactamase-producing Gram-negative pathogens isolated from hospital sink drains

<title>Abstract</title> <p> Background Hospital sink drains are increasingly recognized as important environmental reservoirs of multidrug-resistant Gram-negative bacteria and contributors to healthcare-associated infections. Conventional chemical disinfection strategies have limited and transient efficacy and contribute to environmental pollution through the release of chemical biocides. Probiotic-based approaches relying on competitive exclusion represent a promising alternative for the sustainable control of hospital microbial reservoirs. Material and methods In this study, we evaluated the inhibitory activity of two probiotic strains, <italic>Bacillus amyloliquefaciens</italic> NBS31007 and <italic>Bacillus velezensis</italic> S499, against nineteen strains of Gram-negative pathogens isolated from hospital sink drains. They belonged to the species <italic>Klebsiella pneumoniae</italic> , <italic>Enterobacter cloacae</italic> complex, <italic>Citrobacter freundii</italic> , and <italic>Pseudomonas aeruginosa</italic> , and produced extended-spectrum β-lactamases (ESBL) or carbapenemases. Reference strains of each species were used as controls. <italic>Bacillus</italic> strains were pre-cultured under oligotrophic conditions mimicking sink drain environments and subsequently challenged with low inocula of pathogenic strains. Pathogen growth was quantified after 24 hours of co-culture, and compared with growth in the absence of <italic>Bacillus</italic> . Results Both <italic>Bacillus</italic> strains inhibited the growth of all tested drain-related and reference strains, with reductions ranging from 1.0 to 6.1 log₁₀ CFU reduction ( <italic>P</italic> -values &lt; 6.51 10 ^− 4 ) depending on the bacterial species and isolate. Overall, <italic>B. amyloliquefaciens</italic> NBS31007 and <italic>B. velezensis</italic> S499 displayed comparable inhibitory capacities, with only limited strain-dependent differences. Conclusions These findings provide proof of concept that probiotic <italic>Bacillus</italic> pre-cultures can limit the establishment of ESBL- and carbapenemase-producing Gram-negative pathogens in hospital sink drains. This biocontrol strategy may represent a complementary infection prevention approach aligned with antimicrobial resistance mitigation and One Health principles. </p>