Chuwatthanakhajorn S.Wedley A.Watts A.Slater R.Green K.Probert C.Wiriyarat W.Wigley P.Campbell B.J.Mahidol University2026-04-012026-04-012026-01-01Probiotics and Antimicrobial Proteins (2026)18671306https://repository.li.mahidol.ac.th/handle/123456789/115946With the trend shifting towards reducing antibiotic usage, the utilization of lactobacilli as a probiotic is emerging as an alternative, effective method for mitigating Salmonella infection in the poultry industry. Here, we investigated the antimicrobial activity of cell-free culture supernatants (CFCS) of three lactobacilli (Lactobacillus (Lact.) johnsonii, Lact. reuteri and Lact. crispatus) isolated from caecal microbiota from mature, healthy broiler chickens, against five Salmonella enterica serovars (Salm. Typhimurium 4/74, Salm. Enteritidis P125109, Salm. Enteritidis AviPro^®, Salm. Gallinarum 9 and Salm. Gallinarum 287/91). Lactobacilli CFCS demonstrated potent bactericidal activity against all Salmonella serovars although the degree of inhibition varied amongst the three strains, where MIC ranged from 8.98 to 18.58 mg/mL, and MBC from 12.62 to 25.54 mg/mL. In support, co-culture assays confirmed all serovars as being completely inhibited by lactobacilli CFCS within 3–6 h. In agar well diffusion assays, Lact. crispatus demonstrated highest inhibitory activity, with Salm. Gallinarum showed highest susceptibility. Lactobacilli CFCS significantly suppressed Salmonella invasion of caecal enterocytes and proinflammatory cytokine release from macrophages. Molecular characterisation of inhibitory moieties within lactobacilli CFCS established they were ≤ 3 kDa, pH dependant, heat tolerant and resistant to proteases. Head-space solid-phase microextraction-gas chromatography/mass spectrometry identified volatile organic short- and medium-chain fatty acids, aldehydes, alcohols, ketones and pyrazines as candidate anti-Salmonella post-biotics generated by lactobacilli. In conclusion, lactobacilli isolated from the caecal microbiota of healthy broiler chickens exhibit excellent potential as probiotic candidates to control salmonellosis in poultry and warrant testing in flock intervention studies.Biochemistry, Genetics and Molecular BiologyImmunology and MicrobiologyArticleSCOPUS10.1007/s12602-026-10980-52-s2.0-10503328863118671314