Biocontrol potential of Staphylococcus Simulans against Escherichia coli isolated from bovine mastitis

Mastitis is one of the most common diseases in dairy herds, significantly impacting global milk production. Among the pathogens involved, Escherichia coli stands out as a major causative agent, alongside Staphylococcus aureus and Streptococcus spp. Recently, non-aureus Staphylococcus (NAS) species have been recognized not only as a potential mastitis pathogen but also as possible protective agents against pathogenic bacteria. Aiming to explore this inhibitory activity, the present study evaluated 96 NAS strains isolated from both healthy cows and cows with subclinical mastitis. An initial screening was conducted to assess the ability of each NAS strain to inhibit the growth of five E. coli isolates obtained from clinical mastitis cases. Based on these results, ten NAS strains with the highest inhibitory potential were selected. Subsequently, survival curve analyses were performed to evaluate the prolonged effectiveness of this inhibition over time. Serial dilutions of the NAS strains (at concentrations of 10³, 10⁴, 10⁵, 10⁶, 10⁷, and 10⁸ CFU/mL) were prepared in TSA agar and incubated for 24 hours at 37°C. After initial incubation, 10 μL of an E. coli culture was spotted onto the plates containing the predetermined NAS concentrations. All plates were incubated under aerobic conditions for 18 hours at 37°C. After incubation, the number of E. coli colony-forming units (CFU/mL) was determined to quantify the degree of inhibition.The obtained CFU counts were then used to generate survival curves representing the cumulative survival of E. coli at each NAS concentration. The analyses revealed that Staphylococcus simulans strains exhibited the highest inhibitory capacity against E. coli. The cumulative survival curve of E. coli in the presence of S. simulans began to decline at lower NAS concentrations (approximately 2 log₁₀ CFU/mL), reaching near-complete inhibition (cumulative survival close to zero) at around 4 log₁₀ CFU/mL. S. haemolyticus strains were also effective but required higher concentrations (4 log₁₀ CFU/mL) to inhibit the growth of E. coli. In contrast, S. chromogenes and S. warneri strains demonstrated lower inhibitory capacity, requiring concentrations of approximately 5 log₁₀ CFU/mL to fully inhibit E. coli. These results indicate that S. simulans is highly effective, capable of inhibiting most E. coli isolates at relatively low concentrations, reinforcing its potential as a biocontrol agent against mastitis.