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

Abstract

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. 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 ). 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. 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.