Ovine milk-derived non- aureus Staphylococcus and Mammaliicoccus spp. (NASM) isolates and their cell-free supernatants inhibit major mastitis pathogens in vitro

Innovative strategies to control ovine mastitis pathogens and reduce dependence on conventional antimicrobials are globally essential for One Health and sustainable livestock production. Non- aureus Staphylococci and Mammaliicoccus spp. (NASM) are commonly isolated in cases of ovine mastitis and may influence the microbial ecology of the mammary gland. This study evaluated the inhibitory potential of these microorganisms against key ovine mastitis pathogens, Staphylococcus aureus and Escherichia coli , through two experimental approaches. In experiment 1, 30 isolates of NASM were screened for their inhibitory activity using the cross-streaking method, assessing total, partial, or absent inhibition of S. aureus and E. coli . In experiment 2, the antimicrobial effect of cell-free supernatants from non- aureus Staphylococcus cultures (NAS CFS) cultures was tested at varying concentrations (12.5%, 25%, and 50%) under different treatments (heat treatment, pH adjustment, combined heat treatment + pH adjustment, and control). Pathogen growth was quantified by colony forming unit (CFU)/mL counts after incubation, and data were analyzed via ANOVA followed by Tukey’s test. Results from experiment 1 revealed that 36.7% of the isolates partially or totally inhibited S. aureus , while 20% partially inhibited E. coli . In experiment 2, the combined heat treatment and pH adjustment at a concentration of 12.5% significantly reduced S. aureus and E. coli CFU/mL when tested, for example, in the presence of S. xylosus (p < 0.01 for both cases).Results from experiment 1 revealed that 36.7% of the isolates partially or totally inhibited S. aureus , while 20% partially inhibited E. coli . In experiment 2, the combined heat treatment and pH adjustment at a concentration of 12.5% significantly reduced S. aureus and E. coli CFU/mL when tested, for example, in the presence of S. xylosus (p < 0.01 for both cases). These findings highlight the preliminary potential of antimicrobial compounds derived from NASM for ovine mastitis control, while underscoring the need for further studies to confirm their applicability as sustainable strategies alongside conventional approaches.