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.

In Vitro and In Vivo Anti-infective Potential of Thymol Against Early Childhood Caries Causing Dual Species Candida albicans and Streptococcus mutans

“…Larva groups were incubated at 37 °C for 5 days. Survival was monitored every 12 h. For in vivo efficacy of thymol in controlling the infection, three larvae from infected and treated groups were cut open with a scalpel; the content was suspended in sterile PBS, and the serial dilutions were plated on a selective medium (HiChrome candida differential medium (HiMedia, India) for C. albicans; Mitis salivarius agar (HiMedia, India) for S. mutans; for dual species, both the plates were used) (Selvaraj et al, 2020).…”

A temperateSiphoviridaebacteriophage isolate from Siberian tiger enhances the virulence of Methicillin-resistantStaphylococcusaureus through distinct mechanisms

“…An increased staphyloxanthin secretion could be indicated by a more intense yellow/orange color of the phage-integration strain during the culture (21). In addition, an increased staphyloxanthin secretion and biofilm formation might also indicate an increased virulence since both of them positively impact the pathogenesis and resistance of MRSA (23). Expectedly, the lysogenic strain SA14 + displayed increased cell adhesion and invasion, anti-phagocytosis and virulence to both Galleria mellonella and mice compared to those of the host strain SA14 ( Figs 3K∼L, Fig.…”

Staphyloxanthin as a Potential Novel Target for Deciphering Promising Anti-Staphylococcus aureus Agents

“…Computational molecular docking unveiled a high affinity of carvacrol towards CrtM gene and SarA regulator in S. aureus. In vivo studies hypothesized the capability of carvacrol to mitigate the propagation of MRSA infection in the model as well as maintain a nontoxic effect on the cells of G. mellonella larvae [25]. Molecular docking analysis showing two-dimensional (2D) and three-dimensional (3D) representation of interaction patterns of carvacrol with CrtM receptor is displayed in Figure 3.…”

“…Carbonyl group is essential for activity, binds to adjacent amino acids of the receptor via hydrogen bonds [21,74]. Hydroxyl moiety enhances the binding affinity to LYS residues through conventional hydrogen bonds [24,25].…”