Development of a safe formulation that induces biofilm formation in probiotic bacteria for controlling <i>Salmonella enteriditis</i> Heidelberg and avian pathogenic <i>Escherichia coli</i> in wood shavings

1. The aim of this study was to develop a formulation that stimulated the spontaneous formation of biofilms by probiotic bacteria, specifically <i>Bacillus velezensis</i> (BV), <i>Bacillus subtilis</i> (BS), or <i>Lactococcus lactis</i> subspecies lactis (LL) for control of <i>Salmonella enteriditis</i> Heidelberg (SH) and avian pathogenic <i>E. coli</i> (APEC).2. A formulation was developed to induce spontaneous biofilm formation of probiotic bacteria (BV, BS and LL) by testing various media containing minerals and polymers on polystyrene plates. The most potent probiotic strains were identified based on their efficacy in inhibiting SH and APEC. The formulation was tested on wood shavings to hinder APEC and SH. The safety and colonisation of the formulation was assessed in chicken embryos (CE) from 19 d of incubation to the broiler chicken stage.3. After evaluating various media, one containing calcium, magnesium, iron and polymers (dextran, chitosan and xylan) was selected. Notably, xylan, a highly sustainable biopolymer, showed outstanding results at a low concentration (27 µg/ml), which led to its selection for conducting inhibition tests on wood shavings against SH and APEC. The <i>B. velezensis</i> demonstrated superior biofilm formation and efficacy in controlling SH and APEC. Consequently, a BV strain was selected and associated with a strain of LL.4. While drying the formulation, maltodextrin was added and the biofilm formed by BV01 and LLL01 on wood shavings could control both SH and APEC species, reducing them by approximately 92.64 to 99.42%. Inoculating the formulation in CE did not result in a delay in hatching, injury, or death for either the CE or chicks. The probiotic bacteria multiplied and colonised the intestine of CE.5. This work successfully developed a formulation that induced spontaneous biofilm formation in BV and LL, significantly controlling SH and APEC while ensuring safety for birds.