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.2020 Nov 24;5(48):31100-31114.
doi: 10.1021/acsomega.0c04252. eCollection 2020 Dec 8.

Carvacrol Targets SarA and CrtM of Methicillin-ResistantStaphylococcus aureus to Mitigate Biofilm Formation and Staphyloxanthin Synthesis: AnIn Vitro andIn Vivo Approach

Affiliations

Carvacrol Targets SarA and CrtM of Methicillin-ResistantStaphylococcus aureus to Mitigate Biofilm Formation and Staphyloxanthin Synthesis: AnIn Vitro andIn Vivo Approach

Anthonymuthu Selvaraj et al. ACS Omega..

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-resistantStaphylococcus 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 ofsarA-mediated biofilm gene expression and staphyloxanthin-associatedcrtM gene expression. ThesarA-dependent antibiofilm potential of carvacrol was validated usingS. 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, thein vivo efficacy of carvacrol against MRSA infection was demonstrated using the model organismGalleria 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.

© 2020 American Chemical Society.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Percentage of biofilm inhibition by carvacrolassessed by the crystalviolet quantification assay (A). Line and bar graph indicates theeffect of carvacrol in the growth and biofilm of MRSA, respectively.Dose-dependent antibiofilm effect of carvacrol on polystyrene andglass surfaces (B). Assays were performed in biological triplicateswith three technical replicates. Error bars represent standard deviation(SD), and asterisks indicate the statistical significance value ofp ≤ 0.05.
Figure 2
Figure 2
Concentration-dependent inhibitory effect of carvacrol on MRSAbiofilm formation on a glass surface (1 × 1 cm2) asobserved from light (A) and CLSM (B) microscopic images. Scale barindicates 10 and 50 μm for light and CLSM micrographs, respectively.Number of cells present in the MRSA biofilm formed on glass slidesin the absence and presence of carvacrol. The MRSA biofilm formedon 1 × 1 cm2 glass slides was enumerated by the colony-formingunit (CFU) assay. Results showed significant variations between thenumber of MRSA cells (biovolume) in the control (1.3 × 107) sample and carvacrol at 75 μg/mL treated (2.3 ×102) in sample (C).
Figure 3
Figure 3
Nonantibacterial effectof carvacrol at 75 μg/mL as exhibitedby the growth curve analysis (A) and no significant change in metabolicviability of MRSA at increasing concentrations of carvacrol as observedfrom the Alamar blue assay (B). Assays were performed in biologicaltriplicates with three technical replicates. Error bars representSD.
Figure 4
Figure 4
Dose-dependent reduction in slime synthesis of MRSA upon treatmentwith increasing concentrations of carvacrol as revealed by the Congored agar (CRA) assay (A). Inhibition of EPS production in MRSA inthe presence of carvacrol as examined by the phenol–sulfuricacid method of polysaccharide quantification (B). Assays were performedin biological triplicates with three technical replicates. Error barsrepresent SD, and asterisks indicate a statistical significance valueofp ≤ 0.05.
Figure 5
Figure 5
Qualitative assessment of the staphyloxanthininhibitory potentialof carvacrol on MRSA on the solid medium (A). Quantitative assessmentof the dose-dependent inhibitory effect of carvacrol on MRSA staphyloxanthinsynthesis in a liquid medium (B). Assays were performed in biologicaltriplicates with three technical replicates.
Figure 6
Figure 6
Dose-dependent reduction in metabolicintermediates of the staphyloxanthinbiosynthesis pathway such as staphyloxanthin (A), 4,4′-diaponeurosporenicacid (B), 4,4′-diaponeurosporene (C), and 4,4′-diapophytoene(D) in the absence and presence of increasing concentrations of carvacrol.Assays were performed in biological triplicates with three technicalreplicates. Error bars represent SD, and asterisks indicate a statisticalsignificance value ofp ≤ 0.05.
Figure 7
Figure 7
Carvacrol treatment increases the susceptibility of MRSA cellstoward ROS-mediated killing as observed from the reduced survivalof MRSA in H2O2 (A) and healthy human blood(B). Assays were performed in biological triplicates with three technicalreplicates. Error bars represent SD, and asterisks indicate a statisticalsignificance value ofp ≤ 0.05.
Figure 8
Figure 8
Molecular docking analysis: two-dimensional(2D) and three-dimensional(3D) representation of interaction patterns of carvacrol and positivecontrols with SarA and CrtM. Left panel: interaction among carvacrol,morin, eugenol, and 3′-5′-cyclic diguanylic acid (c-di-GMP)with SarA of MRSA. Right panel: interaction between carvacrol andpositive controls such as lapaquistat acetate, rhodomyrtone, and tripotassium;4-(3-phenoxyphenyl)-1-phosphonatobutane-1-sulfonatewith CrtM.
Figure 9
Figure 9
Relative fold change in expression of genes involved in biofilmformation and staphyloxanthin synthesis in MRSA upon carvacrol treatment(75 μg/mL) when compared with the expression of the housekeepinggenegyrB. Assays were performed in biological triplicateswith three technical replicates. Error bars represent SD, and asterisksindicate a statistical significance value ofp ≤0.05.
Figure 10
Figure 10
Validation ofsarA-mediatedantibiofilm efficacyof carvacrol on wild-typeS. aureus (inhibition in biofilm) (A, B) and isogenic ΔsarA (no biofilm inhibition) (C, D) strains. Line and bar graphs indicatethe growth and biofilm of MRSA in the absence and presence of carvacrol,respectively. Biofilm assay on polysterene and glass surfaces evincingthesarA-dependent biofilm inhibition by carvacrol.Assays were performed in biological triplicates with three technicalreplicates. Error bars represent SD, and asterisks indicate a statisticalsignificance value ofp ≤ 0.05.
Figure 11
Figure 11
Invivo toxicity and efficacy of carvacrol wereassessed through theG. mellonella modelsystem. (A). Kaplan–Meier survival plot displaying the survivalofG. mellonella under the influenceof various treatments. Carvacrol at 250 mg/kg was found to be nontoxicto theG. mellonella larvae. MRSA infectiondrastically reduced the survival rate, whereas carvacrol rescuedG. mellonella from MRSA infection. (B) Internal MRSAburden at various time points in the absence and presence of carvacrol.(C) Representative image displaying the survival status ofG. mellonella at the beginning (0 h) and end (120h) of the survival experiment. Dead larvae turned completely dark.Assays were performed in biological triplicates with three technicalreplicates.
Figure 12
Figure 12
Hemocyte density inG. mellonella decreases after challenge with MRSA. Carvacrol rescuesG. mellonella from MRSA through increasing the circulatinghemocyte count in the hemolymph. (A) Comparison between the hemocytecounts from the hemolymph of infected and treated larvae collectedat 24 and 48 h post infection. The graph shows the average and standarddeviation of 10 larvae per group. The asterisk indicates statisticalsignificancep ≤ 0.05. (B) Representativemicroscopic images of hemocytes. Increase in the hemocyte densitywas observed in the larval group, which received carvacrol treatmentpost infection with MRSA. Assays were performed in biological triplicateswith three technical replicates.
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