Transcriptomic reprogramming and displacement of mastitis-associated Staphylococcus aureus biofilms by lactic acid bacteria

Staphylococcus aureus is one of the main etiological agents of acute mastitis during lactation, and its ability to persist in biofilms can compromise conventional antimicrobial treatments. Lactic acid bacteria (LAB) have emerged as promising probiotic candidates for mastitis management, but the molecular mechanisms underlying their interaction with S. aureus remain poorly understood. This study aimed to evaluate the ability of three LAB strains ( Limosilactobacillus fermentum I7, Limosilactobacillus reuteri 7SNG3–30 and Ligilactobacillus salivarius 22SNG3-30) to interfere with mature biofilms formed by two mastitis-associated S. aureus strains, SA4 and SA55, and to characterize the associated transcriptional responses. The LAB strains were able to colonize preformed 24-h S. aureus biofilms, eventually dominating the mixed-species communities and reducing the attached staphylococcal population. RT-qPCR showed limited transcriptional changes after 4 h of LAB exposure, followed by a broader 16-h response characterized by induction of the stress/adaptation marker uspA and repression of genes related to quorum sensing, virulence, biofilm matrix production and iron acquisition. RNA-seq analysis of SA4 biofilms exposed to LAB for 16 h confirmed extensive transcriptional remodeling in S. aureus and revealed a conserved response across the three LAB treatments, including activation of stress, proteostasis, energy metabolism and arginine catabolism functions, together with repression of surface-associated, matrix-related, metal acquisition and virulence-associated pathways. In contrast, LAB transcriptomic responses were more moderate and strain-dependent, suggesting different metabolic, nutrient-acquisition and envelope-adaptation strategies during biofilm-associated interactions. Overall, these findings suggest that these LAB strains can displace mastitis-associated S. aureus from mature biofilms and promote a coordinated transcriptional shift toward stress adaptation and attenuation of virulence- and persistence-associated functions.