In Vitro Inhibition of Methicillin-Resistant Staphylococcus aureus (MRSA) by Different Species of Non-aureus Staphylococci

One of the main pathogens involved in bovine mastitis is methicillin-resistant Staphylococcus aureus (MRSA), which poses a significant challenge to animal health due to its persistence and pathogenic risk. In this context, non-aureus staphylococci (NAS) have been investigated for their potential to inhibit pathogens through the production of antimicrobial compounds. This study evaluated the inhibitory action of isolates of Staphylococcus simulans, S. warneri, and S. haemolyticus against MRSA strains isolated from the milk of cows with mastitis and healthy cows. Five MRSA isolates carrying the mecA gene and five NAS isolates were used: two S. haemolyticus (NAS35 and NAS36), one S. warneri (NAS43), and two S. simulans (NAS50 and NAS95). A quantitative assay was performed on TSA plates using increasing concentrations of NAS isolates (1.5×10² to 1.5×10⁷ CFU·mL⁻¹), followed by the inoculation of MRSA (1.5×10¹ to 1.5×10⁶ CFU·mL⁻¹). After 18 hours of incubation at 37 °C, colony-forming units (CFUs) of MRSA were counted, and the point of total inhibition was determined. Statistical analysis was conducted using Cox Regression, with the log₁₀ concentration of the different NAS as the time variable until the event (total inhibition of pathogen growth). Qualitative observations revealed complete inhibition of pathogens by at least one isolate from each NAS species at concentrations below 1.5×10⁷ CFU·mL⁻¹. All NAS isolates promoted total inhibition from 1.5×10⁴ CFU·mL⁻¹, and for some MRSA isolates, 1.5×10³ CFU·mL⁻¹ of NAS was sufficient to reduce their viability by approximately 2 log₁₀ compared to the control. The statistical analysis, on the other hand, did not show a significant association among NAS species (p > 0.05). These findings contribute to the understanding of NAS as potential biocontrol agents for bovine mastitis and reinforce their in vitro potential against MRSA. Due to variability among isolates and the possible influence of genetic and phenotypic factors, continued research using genomic and functional approaches may elucidate the underlying mechanisms and support future biotechnological applications in mastitis prevention.