Unveiling the chemical composition of a microbiota-derived extracellular polymeric substance and its antivirulence...

Bovine mastitis caused by Staphylococcus aureus represents a major economic and health challenge in the dairy industry due to the pathogen's virulence factors and biofilm-forming ability, which confer resistance to conventional antibiotics. Sustainable alternatives are therefore urgently needed in the context of rising antimicrobial resistance. This study evaluated the chemical composition, surface interaction properties, and antivirulence potential of an extracellular polymeric substance (EPS-H21) produced by commensal Bacillus sp. strain H21, previously isolated from the microbiota of healthy bovine mammary glands and shown to antagonize mastitis-causing S. aureus. Spectroscopic analyses demonstrated that EPS-H21 mainly consists of heteropolysaccharides enriched in N-acetylglucosamine (GlcNAc) and other aldoses, and protein with minor aliphatic components. The biomaterial exhibited high thermal stability (>250 °C), and both amorphous and crystalline domains, as confirmed by differential scanning calorimetry and solid-state ¹ ³C nuclear magnetic resonance (NMR) spectroscopy. Results suggest that EPS-H21 acts as a supramolecular assembly with relevant interfacial activity. Functionally, EPS-H21 inhibited biofilm formation, disrupted mature biofilms, reduced adhesion and internalization of S. aureus into bovine mammary epithelial cells (MAC-T), and impaired intracellular persistence, all without affecting bacterial viability or host cell integrity. Overall, the structural stability and multifunctional biological activity of EPS-H21 highlight its potential as a microbiota-derived biomaterial for developing non-antibiotic therapies or preventive strategies against S. aureus mastitis.