Anti-biofilm and anti-infection activities of Lynronne-1 against major bovine mastitis pathogens

Mastitis, an inflammatory condition affecting the udders of dairy cattle, is one of the most prevalent and costly diseases in the global dairy industry. This study investigated the efficacy of the antimicrobial peptide Lynronne-1 (lyn-1) against established biofilms formed by key mastitis-causing pathogens, including Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Staphylococcus chromogenes. A panel of 44 mastitis-associated bacterial strains comprising 25 Gram-positive and 19 Gram-negative species was screened for their biofilm-forming capacity, and seven strains were selected as representative targets to assess the ability of lyn-1 to prevent pathogen adhesion and invasion of bovine mammary alveolar cells (MAC-T). The minimal inhibitory concentration (MIC) of lyn-1 against polymicrobial cultures of the seven biofilm-forming strains was achieved at 128 μg/mL. On average, lyn-1 at 1x MIC reduced pre-formed biofilms of Gram-positive and Gram-negative strains by 22 % and 11 %, respectively. Lyn-1 significantly disrupted established biofilms of K. pneumoniae strain 160, S. aureus strains 184 and 186, and the polymicrobial culture (p < 0.05). Moreover, lyn-1 prevented or reduced new biofilm formation by already established biofilms in four out of seven tested strains (p < 0.05). Lyn-1 also significantly decreased (p < 0.05) both adhesion and invasion of the tested pathogens (n = 7) to MAC-T cells at two multiplicities of infection of 1:1 and 1:100 (cell:bacteria). Fluorescence microscopy confirmed the specificity of the peptide by demonstrating its antibacterial activity without detectable damage to the MAC-T cell line, corroborating previous studies that reported lyn-1 to be non-cytotoxic to MAC-T cells at concentrations up to 256 μg/mL. These results illustrate that lyn-1 is effective against polymicrobial cultures of mastitis bacteria and shows promise as a targeted and safe agent for preventing and treating mastitis, particularly infections involving biofilm-forming Gram-positive bacteria.