Potential of Fermented Food-Derived Lactiplantibacillus Cell-Free Supernatants to Control Staphylococcus aureus Growth and Biofilm Development

Staphylococcus aureus biofilms represent a critical healthcare challenge, driving chronic infections and antimicrobial resistance. This study investigates the anti-staphylococcal efficacy of two Lactiplantibacillus strains isolated from traditional Bulgarian pickled vegetables (turshiya): L. plantarum IZITR_24 and L. paraplantarum IZITR_13. Combining whole genome sequencing (WGS) with functional assays, we established a robust genotype-to-phenotype framework to characterize their antimicrobial arsenal. Based on WGS, we identified conserved plantaricin (plnJK, plnEF) clusters in both isolates, with IZITR_13 additionally carrying genes for pediocin and enterolysin A—alongside the confirmed absence of virulence factors. Reconstituted lyophilized cell-free supernatants (LCFSs) were evaluated in dose–response microtiter assays to determine the minimum biofilm inhibitory concentration (MBIC) and minimum inhibitory concentration (MIC). Both strains demonstrated clear, dose-dependent inhibitory activity against the S. aureus growth and biofilm formation. Microscopy (SEM/CLSM) confirmed significant biofilm disruption and cell membrane permeabilization. The observed consistency between genome-inferred capacity and phenotypes highlights the strong predictive value of a genome-first screening approach for selecting bacteriocin-producing lactic acid bacteria (LAB). These findings position IZITR_24 and IZITR_13 as promising postbiotic producers with potent antibiofilm activity against S. aureus. By utilizing their stable postbiotic products rather than relying on live colonization, this study proposes a targeted, antibiotic-sparing strategy to combat persistent staphylococcal biofilms.