Disrupting Salmonella colonization and resistance: probiotic cell-free supernatants inhibit biofilm formation and invasion in meat and human isolates.

BACKGROUND

Salmonella enterica remains a paramount global public health threat due to the rapid emergence of multi-drug and pan-drug resistant (PDR) strains transmitted through the food supply chain. This study adopted a One Health cross-sectional framework to investigate the occurrence and biocontrol of PDR Salmonella enterica serovars (S. Enteritidis, S. Kentucky, and S. Anatum) isolated from clinical gastroenteritis cases and retail meat products in Egypt.

METHODS

Clinical samples (n=20) from gastroenteritis patients were screened for PDR Salmonella and confirmed via invA gene PCR in addition to six PDR isolates previously from meat samples. Biocontrol efficacy was evaluated using cell-free spent media (CFSM; postbiotic preparations) derived from a consortium of probiotic strains. Chemical profiling of CFSM was executed via High-Performance Liquid Chromatography (HPLC). Antimicrobial activity, minimum inhibitory concentrations, and biofilm formation were quantified. Host cell invasion was assessed using a gentamicin protection assay in human Caco-2 intestinal epithelial cells. Ultrastructural alterations were examined via Transmission Electron Microscopy (TEM). Transcriptomic mechanisms were elucidated via RT-qPCR targeting biofilm regulators (csgD, sdiA) and Salmonella Pathogenicity Island 1 (SPI-1) invasion genes (hilA, invF, prgH, and sipA).

RESULTS

CFSM from Lactobacillus helveticus EMCC 1654 and Lactobacillus plantarum CCUG 30503 demonstrated marked antibacterial and antibiofilm activity. At sub-inhibitory concentrations (10% v/v), CFSM significantly attenuated biofilm formation and reduced host-cell invasion efficiency in S. Kentucky to 0.08% relative to untreated controls. TEM micrographs confirmed severe cell envelope disintegration, membrane detachment, and cytoplasmic leakage in CFSM-treated cells. RT-qPCR analysis revealed significant downregulation of the master biofilm regulator csgD (fold-change: 0.002) and the quorum-sensing receptor sdiA (fold-change: 0.02). Furthermore, key SPI-1 invasion genes were markedly repressed, including hilA (0.188), invF (0.001), prgH, and sipA (0.037). Quantitative HPLC profiling of L. plantarum CCUG 30503 CFSM- the lead candidate for downstream mechanistic analysis- identified a multi-organic acid cocktail dominated by lactic (1.89 mg/mL), succinic (1.53 mg/mL), and acetic (1.11 mg/mL) acids.

CONCLUSION

Lactobacillus-derived postbiotics (CFSM) function as effective antivirulence agents that disrupt both the transcriptional networks governing biofilm synthesis and SPI-1-mediated host invasion, while physically compromising bacterial cell wall integrity. These findings highlight probiotic CFSM as a sustainable, non-antibiotic strategy to mitigate PDR Salmonella persistence across the food-to-clinical continuum.