Evaluation of Staphylococcus simulans inhibition of Staphylococcus aureus infection by an in vivo murine mastitis model

ABSTRACT Staphylococcus aureus is a major bovine mastitis pathogen and can result in chronic intramammary infections that are subject to considerable antimicrobial use. In comparison, non- aureus staphylococci (NAS) are common in intramammary infections, but some strains of NAS reduce risk of clinical mastitis and can inhibit S. aureus growth in vitro . This study aims to determine whether in vitro inhibition of S. aureus growth by NAS species translates to in vivo inhibition within a mouse mastitis model and to characterize NAS mammary tissue colonization. Two Staphylococcus simulans strains were selected for in vivo experimentation to compare genetically similar strains that can and cannot inhibit S. aureus in vitro . Inhibition of S. aureus growth in vivo was tested by pre-inoculating mouse mammary glands with S. simulans 24 hours prior to S. aureus inoculation and harvesting mammary gland tissue 24 hours after S. aureus superinfection. Mammary glands were processed for bacterial load quantification, cytokine profiling, and histological processing. Pre-inoculation of either S. simulans strain resulted in reduced S. aureus load in mammary tissue. Superinfection of S. simulans and S. aureus showed no difference in inflammation severity compared to S. aureus alone, although S. simulans pre-inoculation significantly increased expression of IL-10. These results demonstrate that protection against S. aureus mastitis is possible by prior colonization of mammary tissue by S. simulans independent of in vitro growth inhibition capacity. This work will foster future research aiming to fully understand the variety of roles NAS strains play in bovine mastitis, aiding in the development toward alternative mastitis prophylaxis. IMPORTANCE Bovine mastitis is a leading economic concern for dairy production globally, representing the largest reason for antimicrobial use in dairy cattle. Non- aureus staphylococci (NAS) are among the most frequently isolated bacteria from mild, sometimes self-limiting, intramammary infections in cattle and may be associated with a lower risk of infection by major clinical mastitis pathogens such as Staphylococcus aureus . This study investigated the inhibition of S. aureus mastitis by two NAS strains using an in vivo mouse mastitis model. This study demonstrated that when mammary glands are colonized by either one of these NAS strains, the ability of S. aureus to establish within the mouse mammary glands is reduced. These results demonstrate the long-term potential for NAS strains to become an alternative prophylactic treatment for bovine mastitis and support efforts to reduce antimicrobial dependencies in food production.