Cell-Free but potent: Antimicrobial potential of Lactobacillus supernatants against multidrug-resistant pathogens

Antimicrobial resistance poses a major threat to public health and the food industry, driving the search for novel biologically derived antimicrobial alternatives. This study evaluated the antimicrobial activity of cell-free supernatants (CFS) obtained from 30 Lactobacillus strains against 15 bacterial and yeast pathogens, including multidrug-resistant isolates. A species-independent antimicrobial response was observed, with 10 CFS classified as effective, showing predominant MIC and MBC/MFC values of 20 mg/mL, in contrast to 5 ineffective CFS that had higher values despite some being of the same species. Effective CFS caused marked reductions in microbial viability, reaching 2-4 log (CFU/mL) decreases, and residual viable counts between 104 and 105 cells/cm2, as determined by fluorescence microscopy. Neutralization of CFS pH and enzymatic treatments resulted in a partial loss of antimicrobial activity, indicating that both acidic and non-acidic metabolites contribute to the observed effects. Cytotoxicity assays revealed no significant impact on HepG2 cell viability, whereas RAW 264.7 macrophages exhibited a reduction in MTT signal, which may reflect altered metabolic activity or increased cellular sensitivity rather than definitive cytotoxicity. Overall, Lactobacillus-derived CFS exhibit broad-spectrum antimicrobial activity associated with a diverse chemical composition, supporting their prioritization for further chemical characterization and translational studies.