Facing Antimicrobial Resistance and Virulence in Streptococcus agalactiae During Late Pregnancy: Evaluation of Lactobacilli as a Supportive Approach.

Streptococcus agalactiae (Group B streptococcus, GBS) remains a major cause of neonatal sepsis and meningitis, despite the success of intrapartum antibiotic prophylaxis. However, the rising trend of antimicrobial resistance and the emergence of hypervirulent lineages highlight the need for complementary, innovative prophylactic strategies. This study aimed to characterize the resistance and virulence repertoire of colonizing GBS isolates and to assess the antagonistic potential of selected Lactobacillus strains under conditions mimicking the vaginal niche. We performed integrated phenotypic, genotypic, and functional analysis of 31 clinical GBS isolates collected during routine pre-partum screening, focusing on antimicrobial susceptibility, hemolytic and aggregation phenotypes, capsular serotypes, pilus island profiles, and virulence genes. The collection included MDR isolates, strains exhibiting inducible clindamycin resistance and high-level gentamicin resistance, as well as hypervirulent ST-17 related lineages, with marked heterogeneity in β-hemolytic activity and aggregation phenotypes linked to specific combinations of capsule, pilus, and virulence genes. In co-culture experiments with HeLa cells, individual Lactobacillus strains exerted strong, strain-dependent inhibition of GBS, preserving epithelial cell viability, and impairing pathogen adhesion by up to 98%. Importantly, the multi-strain Lactobacillus combination provided broader and more consistent antagonistic activity, including against MDR, inducible clindamycin-resistant, hypervirulent ST-17-associated, and strongly hemolytic GBS isolates. Although these findings are preliminary, being derived from a limited number of isolates and an in vitro model, they support the promising potential of rationally selected multi-strain Lactobacillus formulations as a strategy to reduce colonization by high-risk GBS lineages and warrant further investigation.