Lacticaseibacillus casei Combats Biofilm Formation and Exhibits Antibacterial Activity Against Clinical Isolates of Staphylococcus aureus, Salmonella enterica, and Escherichia coli

Biofilm-forming pathogens are a major cause of persistent infections, showing limited response to antibiotic treatment. The search for alternative strategies has therefore driven extensive research into the antimicrobial potential of beneficial microorganisms. In the present study, the antibacterial and antibiofilm activity of the commercial probiotic strain <i>Lacticaseibacillus casei</i> ATCC 393 (Lc393) was examined against clinical isolates of <i>Staphylococcus aureus</i>, <i>Salmonella enterica</i> subsp. <i>enterica</i> serovar Enteritidis and <i>Escherichia coli</i>. Lc393 reduced pathogen viability and attachment to the colon adenocarcinoma cell line HT-29, with maximal effects recorded against <i>S. aureus</i>. Confocal microscopy visualization of the lactobacilli-pathogens-host interface revealed that Lc393 binds loosely to both host cells and pathogens. The Lc393 cell-free culture supernatant (CFCS) significantly reduced planktonic growth, biofilm mass, and viability of cells in biofilm (>2 logCFU reduction, <i>p</i> < 0.05) and downregulated genes involved in the early stages of biofilm formation in <i>S. aureus</i> (i.e., <i>icaA, fnbpA, eno</i>). In silico analysis of the Lc393 genome identified two bacteriocin clusters, along with genes related to ethanol and organic acid production. Based on in silico predictions and a bacteriocin zymogram, the strain cannot produce functional antimicrobial peptides. Untargeted metabolomics based on UPLC/MS further revealed the presence of putative antimicrobial metabolites. Collectively, our findings highlight the antimicrobial potential of <i>Lc. casei</i> ATCC 393 and support its further investigation for combating clinically relevant human pathogens.