Inhibitory effects of cell-free supernatant of <i>Bifidobacterium bifidum</i> on biofilm formation and virulence gene expression in Group B <i>Streptococcus</i> clinical isolates

Group B Streptococcus (GBS) is a major cause of maternal and neonatal infections, complicated by increasing antimicrobial resistance and high virulence. In this study, from 235 vaginal swabs, 45 GBS isolates were identified and screened; 12 representative isolates (strong biofilm producers with the complete virulence gene profile) were selected for downstream analyses. Molecular analysis showed high prevalence of virulence genes (fbsA 95.5%, lmb 91.1%, pavA 88.8%, fbsB 86.6%) and biofilm-related genes (pil-1 88.8%, pil-2a 91.1%, pil-2b 84.4%). Subsequently, a Bifidobacterium bifidum (designated B. bifidum BB-6; GenBank accession number PX474696) isolated from human breast milk was used to prepare a cell-free supernatant (CFS). Gas chromatography-mass spectrometry analysis (GC-MC) of the CFS of B. bifidum identified several bioactive compounds, including acetic acid, propionic acid, and lactic acid. Checkerboard assays indicated synergism between CFS and penicillin/vancomycin, with a fractional inhibitory concentration index (FICI) of ≤ 0.5 in most cases. Sub-minimum inhibitory concentration (sub-MIC) of CFS significantly inhibited biofilm formation (P < 0.01), and quantitative real-time PCR (qRT-PCR) revealed downregulation of virulence (fbsB down -4.40-fold) and biofilm genes (pil-2b down -5.49-fold). These results highlight the therapeutic potential of B. bifidum CFS against GBS, warranting further studies to isolate active compounds and evaluate safety and efficacy in vivo.