The mechanistic role of natural antimicrobials in preventing Staphylococcus aureus invasion of MAC-T cells using an in vitro mastitis model.

Abstract

Starting primarily as an inflammation of the mammary gland, mastitis is frequently driven by infectious agents such as Staphylococcus aureus. Mastitis has a large economic impact globally, which includes diagnostic, treatment, and the production costs not to mention the potential milk contamination with antimicrobial residues. Currently, mastitis prevention and cure depends on intramammary infusion of antimicrobials, yet, their overuse risks engendering resistant pathogens, posing further threats to livestock. In our study we aimed to investigate, in vitro, using bovine mammary epithelial cells (MAC-T), the efficacy of the AuraShield an antimicrobial mixture (As) in preventing S. aureus attachment, internalisation, and inflammation. The antimicrobial mixture (As) included: 5% maltodextrin, 1% sodium chloride, 42% citric acid, 18% sodium citrate, 10% silica, 12% malic acid, 9% citrus extract and 3% olive extract (w/w). Herein we show that As can significantly reduce both adherence and invasion of MAC-T cells by S. aureus, with no impact on cell viability at all concentrations tested (0.1, 0.2, 0.5, 1%) compared with untreated controls. The anti-apoptotic effect of As was achieved by significantly reducing cellular caspase 1, 3 and 8 activities in the infected MAC-T cells. All As concentrations were proven to be subinhibitory, suggesting that Ac can reduce S. aureus virulence without bacterial killing and that the effect could be dual including a host modulation effect. In this context, we show that As can reduce the expression of S. aureus clumping factor (ClfB) and block its interaction with the host Annexin A2 (AnxA2), resulting in decreased bacterial adherence in infection of MAC-T cells. Moreover, the ability of As to block AnxA2 had a significant decreasing effect on the levels of pro inflammatory cytokine released upon S. aureus interaction with MAC-T cells. The results presented in this study indicate that mixtures of natural antimicrobials could potentially be considered an efficient alternative to antibiotics in treating S. aureus induced mastitis.