Evaluation of <i>Staphylococcus simulans</i> inhibition of <i>Staphylococcus aureus</i> infection by an <i>in vivo</i> murine mastitis model

<i>Staphylococcus aureus</i> is a major bovine mastitis pathogen and can result in chronic intramammary infections that are subject to considerable antimicrobial use. In comparison, non-<i>aureus</i> staphylococci (NAS) are common in intramammary infections, but some strains of NAS reduce risk of clinical mastitis and can inhibit <i>S. aureus</i> growth <i>in vitro</i>. This study aims to determine whether <i>in vitro</i> inhibition of <i>S. aureus</i> growth by NAS species translates to <i>in vivo</i> inhibition within a mouse mastitis model and to characterize NAS mammary tissue colonization. Two <i>Staphylococcus simulans</i> strains were selected for <i>in vivo</i> experimentation to compare genetically similar strains that can and cannot inhibit <i>S. aureus in vitro</i>. Inhibition of <i>S. aureus</i> growth <i>in vivo</i> was tested by pre-inoculating mouse mammary glands with <i>S. simulans</i> 24 hours prior to <i>S. aureus</i> inoculation and harvesting mammary gland tissue 24 hours after <i>S. aureus</i> superinfection. Mammary glands were processed for bacterial load quantification, cytokine profiling, and histological processing. Pre-inoculation of either <i>S. simulans</i> strain resulted in reduced <i>S. aureus</i> load in mammary tissue. Superinfection of <i>S. simulans</i> and <i>S. aureus</i> showed no difference in inflammation severity compared to <i>S. aureus</i> alone, although <i>S. simulans</i> pre-inoculation significantly increased expression of IL-10. These results demonstrate that protection against <i>S. aureus</i> mastitis is possible by prior colonization of mammary tissue by <i>S. simulans</i> independent of <i>in vitro</i> 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.IMPORTANCEBovine mastitis is a leading economic concern for dairy production globally, representing the largest reason for antimicrobial use in dairy cattle. Non-<i>aureus</i> 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 <i>Staphylococcus aureus</i>. This study investigated the inhibition of <i>S. aureus</i> mastitis by two NAS strains using an <i>in vivo</i> mouse mastitis model. This study demonstrated that when mammary glands are colonized by either one of these NAS strains, the ability of <i>S. aureus</i> 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.