Background: The vaginal microbiota of healthy women is predominantly composed of lactic acid bacteria (LAB), particularly species from the lactobacilli group, which contribute to the maintenance of vaginal health through multiple protective mechanisms. Objectives: In the present study, vaginal LAB isolated from healthy premenopausal Algerian women were evaluated for their probiotic potential. Methods: A total of 259 LAB isolates were initially screened for antimicrobial activity against vaginal pathogens, among which 164 strains exhibited inhibitory effects. Based on antimicrobial capacity and biofilm formation ability, 38 strains were selected for further characterization. A refined subset of 15 strains belonging to Lactiplantibacillus (Lpb.) plantarum, Limosilactobacillus (Lmb.) fermentum, and Ligilactobacillus (Lgb.) salivarius were subsequently identified according to their superior antimicrobial and probiotic-related properties. The selected strains were evaluated for several functional and safety characteristics, including antimicrobial activity, production of lactic acid and hydrogen peroxide (H₂O₂), biofilm formation, auto-aggregation, cell surface hydrophobicity, antibiotic susceptibility, hemolytic activity, and tolerance to acidic conditions. These studies were complemented with comparative genomics using the complete genome sequences of selected strains. Results: The isolates demonstrated significant inhibitory activity against vaginal pathogens associated with aerobic vaginitis, and vulvovaginal candidiasis. In addition, strong correlations were observed between metabolite production and antimicrobial activity. Several strains also exhibited enhanced biofilm formation, aggregation capacity, and favorable safety profiles. Among the tested isolates, Lmb. fermentum 70BJ2, Lpb. plantarum 87JG8, and Lpb.plantarum N17 emerged as particularly promising probiotic candidates due to their combined antimicrobial, metabolic, and adhesion-related properties. Conclusions: Overall, these findings demonstrate that the vaginal microbiota of healthy Algerian women represents a valuable source of potential probiotic strains and provide a strong basis for future in vivo validation and clinical application in the prevention and management of vaginal infections.

The vaginal microbiota contributes to mucosal immune homeostasis, yet the immunomodulatory properties of individual vaginal lactic acid bacteria (LAB) strains remain poorly characterized. In this study, eight LAB strains previously isolated from healthy Algerian women and selected based on their probiotic-related properties were evaluated for their capacity to modulate macrophage immune responses. Seven Lactiplantibacillus plantarum strains (N1, N2, N7, N11, N17, 87JG8, and 87BG2) and one Limosilactobacillus fermentum strain (70BJ2) were initially screened using porcine alveolar macrophages (PAM cells) under basal conditions and following lipopolysaccharide (LPS) challenge. Distinct strain-dependent cytokine profiles were observed. Hierarchical clustering separated the strains into two major groups: N1, N2, N7, and N11 were characterized mainly by stronger inflammatory responses, whereas N17, 87BG2, 87JG8, and 70BJ2 displayed comparatively lower inflammatory responses together with enhanced IL-6 and IL-27 expression at later time points. Based on these profiles, N17, 87JG8, and 70BJ2 were selected for further characterization in porcine intestinal macrophages (IPIM cells) and RAW 264.7 murine macrophages (RAW cells). In IPIM cells, 70BJ2 reduced LPS-induced TNF-α and IL-6 expression, while N17 enhanced IL-10 and IL-27 expression following LPS challenge. In RAW cells, N17 and 87JG8 enhanced several inflammatory and chemokine responses, whereas all three strains increased IFN-γ, IL-12, CSF3, IL-10, and IL-27, with 70BJ2 inducing the strongest IL-10 and IL-27 responses and uniquely increasing CSF2. Overall, vaginal LAB displayed distinct strain- and macrophage-dependent immunomodulatory profiles rather than uniformly suppressing or enhancing LPS-induced inflammation. Among the selected strains, Lmb. fermentum 70BJ2 exhibited particularly prominent regulatory properties across macrophage models. These findings extend the functional characterization of vaginal LAB and support their strain-level evaluation for the identification of candidate immunobiotics capable of modulating mucosal immune responses.