Carvacrol Targets SarA and CrtM of Methicillin-Resistant Staphylococcus aureus to Mitigate Biofilm Formation and Staphyloxanthin Synthesis: An in Vitro and in Vivo Approach

Abstract

Carvacrol is an essential oil traditionally used in culinary processes as spice due to its aromatic nature and also known for various biological activities. In the present study, the antivirulence efficacy of carvacrol against methicillin-resistant Staphylococcus aureus (MRSA) is explored. MRSA is an opportunistic pathogen capable of causing various superficial and systemic infections in humans. Biofilm formation and virulence factors of MRSA are responsible for its pathogenesis and resistance. Hence, the aim of this study was to explore the antibiofilm and antivirulence efficacy of carvacrol against MRSA. Carvacrol at 75 μg/mL inhibited MRSA biofilm by 93%, and it also decreased the biofilm formation on polystyrene and glass surfaces. Further, microscopic analyses revealed the reduction in microcolony formation and collapsed structure of biofilm upon carvacrol treatment. The growth curve analysis and the Alamar blue assay showed the nonfatal effect of carvacrol on MRSA. Further, carvacrol significantly reduced the production of MRSA biofilm-associated slime and extracellular polysaccharide. In addition, carvacrol strongly inhibited the antioxidant pigment staphyloxanthin and its intermediates' synthesis in MRSA. Inhibition of biofilm and staphyloxanthin by carvacrol enhanced the susceptibility of MRSA to oxidants and healthy human blood. Quantitative polymerase chain reaction (qPCR) analysis unveiled the downregulation of sarA-mediated biofilm gene expression and staphyloxanthin-associated crtM gene expression. The sarA-dependent antibiofilm potential of carvacrol was validated using S. aureus Newman wild-type and isogenic ΔsarA strains. In silico molecular docking analysis showed the high binding efficacy of carvacrol with staphylococcal accessory regulator A (SarA) and 4,4′-diapophytoene synthase (CrtM) when compared to positive controls. Furthermore, the in vivo efficacy of carvacrol against MRSA infection was demonstrated using the model organism Galleria mellonella. The results revealed the nontoxic nature of carvacrol to the larvae and the rescuing potential of carvacrol against MRSA infection. Finally, the current study reveals the potential of carvacrol in inhibiting the biofilm formation and staphyloxanthin synthesis of MRSA by targeting the global regulator SarA and a novel antivirulence target CrtM.