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

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.

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) has evolved as a global health concern due to its multiple drug resistance. In 2017, the World Health Organization (WHO) released the global priority list of antibiotic-resistant bacteria in which MRSA occupied high priority.[1] In addition, MRSA was listed as a serious threat in the antibiotic-resistance threat report published by Centres for Disease Control and Prevention (CDC) in 2019.[2] MRSA is known to cause a wide range of human infections, and the severity of infection increases when the person is immune-compromised. Apart from causing mild superficial skin infections such as sores, boils, abscesses, impetigo, and lesions, MRSA is also responsible for life-threatening systemic infections such as bacteremia, sepsis, toxic shock syndrome, meningo-encephalitis, pneumonia, osteomyelitis, pyomyositis, pericarditis, and endocarditis.[3,4] Chronic infections caused by MRSA are very difficult to treat with commonly available antibiotic therapy, and most of the systemic MRSA infections are persistent in nature because of biofilm formation under in vivo conditions.[5]

Biofilm is the adherent microbial community, and the adherence is mediated by the self-secreted polymeric substances, which provide a hydrated matrix structure, thereby offering a comfortable stay for bacterial cells to reside in the matrix. Polysaccharide intracellular adhesin (PIA) is the predominant extracellular polymeric substance in staphylococcal biofilms, and it is produced by the intracellular adhesion (ica) operon. PIA synthesis is very essential for biofilm formation as it mediates intracellular adherence and provides structural integrity to biofilms. In addition to PIA, extracellular DNA and numerous surface proteins also contribute to stability of the biofilm.[6,7] Once the stable biofilm is formed, it is highly challenging to eradicate as sessile biofilm cells are highly resistant to antimicrobials than free-floating planktonic cells. Biofilms exert antimicrobial resistance via various mechanisms such as poor penetration of antibiotics, genetic adaptation, altered growth rate and metabolism, degradation by matrix enzymes, neutralization of antimicrobials, persister formation, and a higher rate of gene transfer.[8,9] Thus, inhibition of biofilm formation has been considered as an important therapeutic strategy in recent times to counteract persistent bacterial infections.

Apart from biofilm formation, MRSA synthesizes countless virulence factors to defend and survive under unfavorable environmental conditions. Staphyloxanthin is an eponymous virulence trait that is responsible for the golden-yellow color appearance of MRSA.[10] Staphyloxanthin is basically a membrane-bound carotenoid pigment produced from crtMNOPQ operon and provides integrity to the membrane. Especially, this pigment possesses antioxidant property, which protects MRSA from reactive oxygen species (ROS)-mediated stress and also serves as the potential drug target for antistaphylococcal treatment strategies.[11,12] Thus, the present study was designed to identify a drug candidate that has both biofilm and virulence inhibitory potential. In our previous study,[13] we had screened several phytochemicals for antibiofilm activity against MRSA and identified carvacrol as one of the bioactives with potential antimicrobial activity. Furthermore, several previous studies[14−19] have reported the antimicrobial and antibiofilm activities of carvacrol against S. aureus. None of these studies have explored either the staphyloxanthin inhibitory potential or the antivirulence efficacy of carvacrol against MRSA. Hence, the present study has made an attempt to investigate the in vitro antibiofilm, antivirulence, and staphyloxanthin inhibitory potentials of carvacrol and the in vivo efficacy using the model system Galleria mellonella.

Results

Effect of Carvacrol on Growth and Biofilm Formation of MRSA

The impact of carvacrol on the growth and biofilm formation of MRSA was assessed by a broth microdilution assay and a crystal-violet-based biofilm quantification assay, respectively. The result of the broth microdilution assay showed that at 150 μg/mL concentration carvacrol completely inhibited the growth of MRSA and hence the same was considered as the minimum inhibitory concentration (MIC) against MRSA (Figure S1). The antibiofilm activity of carvacrol (25, 50, and 75 μg/mL) against MRSA was analyzed by a biofilm quantification assay. The result revealed the dose-dependent antibiofilm activity of carvacrol against MRSA, and a maximum of 93% biofilm inhibition was observed at 75 μg/mL, and it was fixed as the minimum biofilm inhibitory concentration (MBIC) of carvacrol (Figure A).

Figure 1

Percentage of biofilm inhibition by carvacrol assessed by the crystal violet quantification assay (A). Line and bar graph indicates the effect of carvacrol in the growth and biofilm of MRSA, respectively. Dose-dependent antibiofilm effect of carvacrol on polystyrene and glass surfaces (B). Assays were performed in biological triplicates with three technical replicates. Error bars represent standard deviation (SD), and asterisks indicate the statistical significance value of p ≤ 0.05.

Antibiofilm Potential of Carvacrol against MRSA Biofilms

The effect of carvacrol at increasing concentrations (25, 50, and 75 μg/mL) was assessed against the biofilm formation of MRSA on polystyrene and glass surfaces. Results showed the dose-dependent antibiofilm activity of carvacrol on both tested surfaces (Figure B). Light and confocal laser scanning microscopy (CLSM) analyses were also performed to analyze the antibiofilm potential of carvacrol against MRSA biofilms. Light microscopic images showed dose-dependent inhibition in the biofilm-covered area in carvacrol-treated slides when compared to the MRSA control slide (Figure A). Similarly, CLSM analysis indicated the reduction in the biofilm architecture and also in thickness of the biofilm formed by MRSA in the presence of carvacrol (Figure B).

Figure 2

Concentration-dependent inhibitory effect of carvacrol on MRSA biofilm formation on a glass surface (1 × 1 cm2) as observed from light (A) and CLSM (B) microscopic images. Scale bar indicates 10 and 50 μm for light and CLSM micrographs, respectively. Number of cells present in the MRSA biofilm formed on glass slides in the absence and presence of carvacrol. The MRSA biofilm formed on 1 × 1 cm2 glass slides was enumerated by the colony-forming unit (CFU) assay. Results showed significant variations between the number of MRSA cells (biovolume) in the control (1.3 × 107) sample and carvacrol at 75 μg/mL treated (2.3 × 102) in sample (C).

Assays were performed in biological triplicates with three technical replicates. Error bars represent SD, and asterisks indicate the statistical significance value of p ≤ 0.05.

Nonfatal Effect of Carvacrol on MRSA

An ideal antibiofilm compound is expected to have nonantibacterial activity. Hence, the effect of carvacrol on growth and viability of MRSA was assessed. The growth curve analysis showed that there was no change in the growth curve pattern of the carvacrol-treated sample (75 μg/mL) when compared to the MRSA control sample (Figure A). The results of the Alamar blue assay revealed no significant variance between the amount of viable cells in the MRSA control and carvacrol-treated samples (Figure B).

Figure 3

Nonantibacterial effect of carvacrol at 75 μg/mL as exhibited by the growth curve analysis (A) and no significant change in metabolic viability of MRSA at increasing concentrations of carvacrol as observed from the Alamar blue assay (B). Assays were performed in biological triplicates with three technical replicates. Error bars represent SD.

Effect of Carvacrol on Slime Synthesis and Extracellular Polysaccharide (EPS) Production in MRSA

Biofilm formation in MRSA is highly associated with slime synthesis and EPS production. Therefore, the influence of carvacrol on slime and EPS production was assessed. In the slime synthesis assay, carvacrol showed a concentration-dependent inhibition in black color formation in MRSA (Figure A). In addition, carvacrol was strongly inhibited the EPS production in MRSA and MBIC of carvacrol exhibited 85% of EPS inhibition (Figure B).

Figure 4

Dose-dependent reduction in slime synthesis of MRSA upon treatment with increasing concentrations of carvacrol as revealed by the Congo red agar (CRA) assay (A). Inhibition of EPS production in MRSA in the presence of carvacrol as examined by the phenol–sulfuric acid method of polysaccharide quantification (B). Assays were performed in biological triplicates with three technical replicates. Error bars represent SD, and asterisks indicate a statistical significance value of p ≤ 0.05.

Effect of Carvacrol on Staphyloxanthin Production in MRSA

The influence of carvacrol on staphyloxanthin production in MRSA was examined, and the result showed a concentration-dependent disappearance in golden-yellow pigment production in carvacrol-treated samples when compared to the control sample. The complete appearance of white color colonies was observed in MRSA streaked on a tryptone soya agar (TSA) plate containing 75 μg/mL carvacrol (Figure A). In addition, MRSA cells grown in the tryptone soya broth supplemented with 1% sucrose (TSBS) media containing carvacrol showed a dose-dependent reduction in the golden-yellow color production when compared to MRSA control cells (Figure B).

Figure 5

Qualitative assessment of the staphyloxanthin inhibitory potential of carvacrol on MRSA on the solid medium (A). Quantitative assessment of the dose-dependent inhibitory effect of carvacrol on MRSA staphyloxanthin synthesis in a liquid medium (B). Assays were performed in biological triplicates with three technical replicates.

Effect of Carvacrol on Staphyloxanthin and Its Metabolic Intermediates’ Synthesis in MRSA

Quantitatively, the production of staphyloxanthin and its intermediates in MRSA was analyzed in the absence and presence of carvacrol. The result showed a concentration-dependent reduction in staphyloxanthin and its intermediates. Especially, 75 μg/mL carvacrol showed the maximum inhibition of staphyloxanthin by 72%, 4,4′-diaponeurosporenic acid by 72%, 4,4′-diaponeurosporene by 72%, and 4,4′-diapophytoene by 72% (Figure A–D).

Figure 6

Dose-dependent reduction in metabolic intermediates of the staphyloxanthin biosynthesis pathway such as staphyloxanthin (A), 4,4′-diaponeurosporenic acid (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 technical replicates. Error bars represent SD, and asterisks indicate a statistical significance value of p ≤ 0.05.

Effect of Carvacrol on the Survival of MRSA in H2O2 and Healthy Human Blood

As staphyloxanthin is an antioxidant pigment and supports MRSA survival against oxidants and the host immune system, the effect of carvacrol on MRSA survival in H2O2 and healthy human blood was analyzed. The result of the H2O2 sensitivity assay showed that carvacrol (75 μg/mL) treatment significantly inhibited the survival of MRSA (6 × 107) when compared to the control sample (1.8 × 108) (Figure A). The blood survival assay revealed significant changes between the viability of the MRSA control (5.2 × 107) and carvacrol-treated cells (1.3 × 107) in human blood (Figure B).

Figure 7

Carvacrol treatment increases the susceptibility of MRSA cells toward ROS-mediated killing as observed from the reduced survival of MRSA in H2O2 (A) and healthy human blood (B). Assays were performed in biological triplicates with three technical replicates. Error bars represent SD, and asterisks indicate a statistical significance value of p ≤ 0.05.

Carvacrol Targets Staphylococcal Accessory Regulator A (SarA) and 4,4′-Diapophytoene Synthase (CrtM) of MRSA

Results of molecular docking analysis revealed the ability of carvacrol to interact with SarA and CrtM proteins of MRSA. Significantly, carvacrol interacts with active sites of CrtM with the binding energy of −7.39 kcal/mol and exhibited one hydrogen-bonding interaction (Ala A:134) and a π–π T-shape with Phe A:22. In the case of SarA, carvacrol actively interacts through the π anion with Asp A: 120 and π–π T-shape (Tyr B: 162), and the binding energy of carvacrol with SarA was −6.86 kcal/mol. The strong binding efficacy of carvacrol with SarA and CrtM of MRSA confirmed its antibiofilm and staphyloxanthin inhibitory potential. In addition, the binding efficacy of carvacrol with SarA and CrtM was compared with previously reported compounds that target SarA/CrtM. Docking results revealed that the binding efficacy of carvacrol was found to be more proficient than the positive controls used in this study. Binding energies and the interactions are provided in Table and Figure .

Table 1

Molecular Docking Analysis Reveals Bioactive Compounds’ Binding Efficacy with SarA and CrtM of MRSA

receptor	ligand	key residue/π anion	π–π T-shape	binding energy/docking score (kcal/mol)
SarA	carvacrol	Asp A:120	Tyr B:162	–6.86
	morin	Lys A:172; Ser A:175; Asp A:181		–4.64
	eugenol	Asn B:161; Asp A:120	Tyr B:162	–4.39
	c-di-GMP	Ile A:106, A:103; Thr A:104		–4.47
CrtM	carvacrol	Ala A:134	Phe A:22	–7.39
	lapaquistat acetate	Arg A:265; Lys A:273; Lys A:20		–3.39
	rhodomyrtone	Val A:232; Lys A:231		–4.18
	tripotassium;4-(3-phenoxyphenyl)-1-phosphonatobutane-1-sulfonate	Lys A:20; Lys A:17		–5.75

Figure 8

Molecular docking analysis: two-dimensional (2D) and three-dimensional (3D) representation of interaction patterns of carvacrol and positive controls 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 and positive controls such as lapaquistat acetate, rhodomyrtone, and tripotassium;4-(3-phenoxyphenyl)-1-phosphonatobutane-1-sulfonate with CrtM.

Further, quantitative polymerase chain reaction (qPCR) analysis validated that carvacrol treatment downregulated the expression of biofilm and staphyloxanthin synthesis-associated genes such as sarA, icaA, icaD, fnbA, fnbB, and crtM (Figure ). Overall, molecular docking analysis and qPCR analysis unveiled that carvacrol targets SarA and CrtM of MRSA to inhibit biofilm formation and staphyloxanthin inhibition.

Figure 9

Relative fold change in expression of genes involved in biofilm formation and staphyloxanthin synthesis in MRSA upon carvacrol treatment (75 μg/mL) when compared with the expression of the housekeeping gene gyrB. Assays were performed in biological triplicates with three technical replicates. Error bars represent SD, and asterisks indicate a statistical significance value of p ≤ 0.05.

sarA-Dependent Antibiofilm Efficacy of Carvacrol

To validate the sarA-mediated antibiofilm activity of carvacrol, the effect of carvacrol on biofilm formation of S. aureus wild-type and isogenic ΔsarA strains was examined by surface and ring biofilm analyses. Results revealed that carvacrol was able to inhibit the biofilm formation of S. aureus wild-type at 75 mg/mL. However, the biofilm of ΔsarA was found to be unaffected by carvacrol treatment (Figure ).

Figure 10

Validation of sarA-mediated antibiofilm efficacy of carvacrol on wild-type S. aureus (inhibition in biofilm) (A, B) and isogenic ΔsarA (no biofilm inhibition) (C, D) strains. Line and bar graphs indicate the growth and biofilm of MRSA in the absence and presence of carvacrol, respectively. Biofilm assay on polysterene and glass surfaces evincing the sarA-dependent biofilm inhibition by carvacrol. Assays were performed in biological triplicates with three technical replicates. Error bars represent SD, and asterisks indicate a statistical significance value of p ≤ 0.05.

Evaluation of In Vivo Toxicity of Carvacrol and Protection of Infection Caused by MRSA in G. mellonella Larvae

G. mellonella larvae have been widely used as an alternative nonmammalian animal model to evaluate the in vivo toxicity and efficacy of anti-infective agents.[20−22] Carvacrol administered at a concentration of 250 mg/kg to larvae hemocoel did not result in visible injury or the normal metabolic activity of the larvae. Compared to the control, 90% survival rate was observed in the larvae group that received carvacrol up to 120 h. Thus, the result indicates that the compound is not toxic toward the larvae. Further, it was evidenced by the survival plot that carvacrol could efficiently rescue the larvae from MRSA infection (Figure A,C). This was further validated through the larval bacterial burden counts in which after 24 and 48 h post inoculation, carvacrol protected G. mellonella from MRSA infection to a greater extent (Figure B).

Figure 11

In vivo toxicity and efficacy of carvacrol were assessed through the G. mellonella model system. (A). Kaplan–Meier survival plot displaying the survival of G. mellonella under the influence of various treatments. Carvacrol at 250 mg/kg was found to be nontoxic to the G. mellonella larvae. MRSA infection drastically reduced the survival rate, whereas carvacrol rescued G. mellonella from MRSA infection. (B) Internal MRSA burden at various time points in the absence and presence of carvacrol. (C) Representative image displaying the survival status of G. mellonella at the beginning (0 h) and end (120 h) of the survival experiment. Dead larvae turned completely dark. Assays were performed in biological triplicates with three technical replicates.

Interestingly, the hemocytes in the circulating hemolymph were found to be slightly increased in the carvacrol-treated infection group than in the infection control group (Figure ).

Figure 12

Hemocyte density in G. mellonella decreases after challenge with MRSA. Carvacrol rescues G. mellonella from MRSA through increasing the circulating hemocyte count in the hemolymph. (A) Comparison between the hemocyte counts from the hemolymph of infected and treated larvae collected at 24 and 48 h post infection. The graph shows the average and standard deviation of 10 larvae per group. The asterisk indicates statistical significance p ≤ 0.05. (B) Representative microscopic images of hemocytes. Increase in the hemocyte density was observed in the larval group, which received carvacrol treatment post infection with MRSA. Assays were performed in biological triplicates with three technical replicates.

Discussion

Carvacrol, also known as 5-isopropyl-2-methylphenol (PubChem CID: 10364), is a well-known plant essential oil majorly found in Origanum vulgare, commonly known as oregano, and is extensively used as a preservative and food-flavoring agent in food industries and as a fragrant agent in cosmetic preparations because of its aromatic flavor. Carvacrol is also reported to have various biological properties such as broad-spectrum antibacterial, antifungal, acaricidal, antiobesity, anticancer, antioxidant, and anti-inflammatory activities.[23−26] The present study evaluated the biofilm and staphyloxanthin inhibitory potentials of carvacrol against MRSA. Biofilm-associated infections are stubborn and have a huge negative impact on recovery of patients after the course of antibiotic treatment.[27] Antibiofilm agents will inhibit the adherence of the bacterium, and the free living cells can easily be attacked by either antibiotic treatment or the host immune system.[28] Thus, the need for novel antibiofilm agents and research focusing on their molecular mechanism has increased in the recent decade. In the present study, the dose-dependent antibiofilm potential of carvacrol was identified using crystal violet quantification of the MRSA biofilm and 75 μg/mL was determined as MBIC, whereas the MIC of carvacrol was identified as 150 μg/mL. Carvacrol was able to inhibit the adherence of MRSA on a polystyrene surface and also the ring biofilm formed at the air–liquid interface on the glass surface. Further, to check the effect of carvacrol on the structure of the biofilm, microscopic analysis was carried out. Light microscopic images represented the highly organized and aggregated biofilm structure on the control surface, whereas microcolony formation and aggregation were heavily interrupted with carvacrol treatment. In line with light microscopy, CLSM images also evidenced the collapsed architecture, less covered surface, and reduced thickness of the carvacrol-treated biofilm in contrast to the fully covered, dense control biofilm. The CFU analysis of the biofilm cells showed biovolume reduction in carvacrol-treated samples when compared to the control. All of these results clearly confirmed the biofilm inhibitory potential of carvacrol against MRSA. Then, the effect of MBIC of carvacrol on growth and viability of MRSA was examined. Both the growth curve analysis and the Alamar blue assay confirmed the nonantibacterial nature of carvacrol at the tested concentration, and this property is highly favorable to rule out the chances of development of resistance.[29]

Slime synthesis in MRSA plays a significant role in biofilm formation and colonization on various surfaces.[30] Results of the CRA assay showed that carvacrol reduced the slime synthesis of MRSA, and it well correlated with the previous reports, wherein the inhibitory potential of antibiofilm agents was explored on the slime synthesis of MRSA.[13,31,32] Further, EPS is a major component of the bacterial biofilm, and it contains polysaccharide proteins and nucleic acids. It supports the formation of the biofilm architecture and persistence of the bacterial biofilm. Additionally, EPS blocks the penetration of antimicrobials agents and acts as a barrier against the host innate immune system.[33] This hypothesis suggested that inhibiting the EPS production could affect the biofilm architecture and induce bacterial sensitivity to external factors.[34] The same was observed in the EPS quantification assay, and the result revealed the inhibitory efficiency of carvacrol on EPS production in MRSA. Thereby, carvacrol inhibits biofilm formation of MRSA.

In all of the biofilm assays, carvacrol-treated cells consistently appeared in white color, which was very disparate from the control cells with a yellow color. This observation led to studying the efficacy of carvacrol in inhibiting staphyloxanthin, which imparts the yellow color to MRSA. Staphyloxanthin, a carotenoid pigment, stands as the antioxidant defensive mechanism and shields MRSA from host-mediated immunological response.[35] Inhibition of staphyloxanthin will definitely reduce the ROS resistance of MRSA.[36] Streak plate images displayed a concentration-dependent reduction in staphyloxanthin. Interestingly, 75 μg/mL concentration of carvacrol completely inhibited the staphyloxanthin production. Further, quantification of the extracted staphyloxanthin also demonstrated the vast decrease in staphyloxanthin upon carvacrol treatment.

Biosynthesis of staphyloxanthin in MRSA is a well-studied pathway, which is mediated by various enzymes encoded by the crtMNOPQ operon. The first step of this pathway is synthesis of dehydrosqualene, also known as 4,4′-diapophytoene, which is catalyzed by dehydrosqualene synthase (CrtM). Dehydrogenation of dehydrosqualene forms 4,4′-diaponeurosporene by the action of 4,4′-diapophytoene desaturase (CrtN). Then, 4,4′-diaponeurosporene oxidase (CrtP) oxidizes 4,4′-diaponeurosporene to yield 4,4′-diaponeurosporenic acid. Esterification of 4,4′-diaponeurosporenic acid forms glycosyl-4,4′-diaponeurosporenoate, which is mediated by glycosyl transferase (CrtQ). Finally, acyl transferase (CrtO) catalyzes the synthesis of staphyloxanthin.[35,37,38] To precisely find out the molecular mechanism of carvacrol in staphyloxanthin inhibition, metabolic intermediates of the staphyloxanthin synthetic pathway were extracted and quantified. Interestingly, all of the metabolic intermediates were found to be decreased upon carvacrol treatment, and it led to the prediction that carvacrol targets the very initial step of the pathway. As staphyloxanthin is an antioxidant pigment, inhibition of this pigment will have an impact on ROS resistance of MRSA.[39] To validate this factor, ROS resistance of carvacrol-treated MRSA was examined using the H2O2 sensitivity assay. The huge reduction in the survival of carvacrol-treated MRSA confirmed that reduction in staphyloxanthin sensitized the MRSA cells to ROS. Further, the results of the whole-blood survival assay also validated that carvacrol impairs the antioxidant defensive mechanism by staphyloxanthin inhibition, thereby sensitizing the MRSA cells to ROS-mediated killing by immune cells of the ex vivo blood system.

To identify the molecular mechanism behind the biofilm and staphyloxanthin inhibitory potentials of carvacrol, molecular docking and qPCR analysis were performed. Staphylococcal accessory regulator A (SarA) is the global virulence regulatory system in S. aureus and positively regulates biofilm formation. Notably, previous studies reported SarA as the therapeutic target to attenuate the virulence of MRSA.[40−42] In the present study, molecular docking results revealed that carvacrol can interact with SarA through anionic bonding. Further, in comparison with the previously reported SarA inhibitors such as morin,[43] eugenol,[44] and c-di-GMP,[45] carvacrol exhibited better binding efficiency. In addition, carvacrol also decreased the expression of sarA, which could be the central mechanism involved in biofilm inhibition. This result leads to the presumption that the antibiofilm activity of carvacrol could be sarA-dependent. To validate this, the antibiofilm activity of carvacrol was assessed on the ΔsarA strain. As expected, carvacrol was found to be ineffective on ΔsarA, and interestingly, carvacrol efficiently inhibited the biofilm formation of the wild-type strain. The inefficacy of carvacrol on ΔsarA confirmed the sarA-dependent antibiofilm activity of carvacrol. In addition, PIA is a major component of the S. aureus biofilm encoded by the ica operon and expression of the ica operon is under the control of SarA. Apart from PIA, SarA also controls adhesion proteins FnbA and FnbB.[46−51] As a downstream effect of SarA downregulation, expressions of icaA, icaD, fnbA, and fnbA were also decreased by carvacrol treatment. On the other hand, interaction of carvacrol with CrtM, the first enzyme of the staphyloxanthin biosynthetic pathway, was investigated using molecular docking analysis as prior studies reported CrtM as an efficient drug target to inhibit staphyloxanthin synthesis.[31,52,53] Results revealed the strong binding of carvacrol with CrtM through hydrogen bonding. Furthermore, the binding efficacy of previously reported CrtM inhibitors such as lapaquistat acetate,[54] rhodomyrtone,[12] and tripotassium;4-(3-phenoxyphenyl)-1-phosphonatobutane-1-sulfonate[55] was used as positive controls to compare the interaction of carvacrol with CrtM. Binding energies of all three positive controls were found to be lesser than the binding energy of carvacrol. Notably, tripotassium;4-(3-phenoxyphenyl)-1-phosphonatobutane-1-sulfonate, which is a well-known inhibitor of CrtM, was lesser than the binding potential of carvacrol to CrtM, which further validates the staphyloxanthin inhibitory potential of carvacrol. In addition, carvacrol also downregulated the expression of CrtM. This result is in line with the reduction in all of the tested intermediates of the staphyloxanthin synthetic pathway in the presence of carvacrol. Thus, it is confirmed that carvacrol interrupts the activity of CrtM to inhibit staphyloxanthin biosynthesis in MRSA.

The in vivo toxicity and efficacy of carvacrol in preventing MRSA infection were determined using a well-known animal model G. mellonella. Carvacrol was not found to be toxic against the larvae, and the compound had the proficiency to decrease the survival and proliferation of MRSA. The immune response of the larvae against microorganisms has been previously shown to modulate the circulating hemocyte population.[56] From enumerating the hemocyte count from the hemolymph of infected and carvacrol-treated larvae, it was evinced that carvacrol not only rescues G. mellonella infection but also could increase the circulating hemocyte count. Overall, the in vivo efficacy of carvacrol against MRSA infection was revealed through the model organism G. mellonella.

Conclusions

The present study reveals antibiofilm and staphyloxanthin inhibitory potentials of carvacrol through interacting with the target molecules SarA and CrtM of MRSA. Interestingly, carvacrol did not affect the growth and metabolic viability. Furthermore, reduction in the survival of MRSA in the presence of ROS and healthy human blood portrays the medicinal value of carvacrol against MRSA infections. In vivo analysis using the G. mellonella model system further validated the anti-infective potential of carvacrol against MRSA. Overall, this holistic study depicts the efficacy of carvacrol as a therapeutic regimen for the treatment of MRSA biofilm-associated infections.

Materials and Methods

Ethical Approval

In this study, healthy human blood was used to perform the whole-blood killing assay and the blood sample was collected from a healthy person with the help of a technically trained person. Written informed consent was obtained from the donor (one of the authors of the manuscript). The protocol of the whole-blood killing assay and human blood sample usage was evaluated and permitted by the Institutional Ethical Committee (IEC), Alagappa University, India (IEC Ref no: IEC/AU/2018/4). The experiment was performed by following the guidelines and regulations of the IEC of Alagappa University.

Bacterial Strain and Growth Condition

The MRSA strain (ATCC-33591) used in the study was obtained from ATCC, Himedia, India. S. aureus Newman wild-type and isogenic ΔsarA strains were provided by Dr. Christiane Wolz, Professor, Institute for Medical Microbiology and Hygiene, University of Tubingen, Germany.[57] Initially, bacterial strains were streaked on tryptone soya agar (TSA) plates, incubated at 37 °C for 24 h, and stored at 4 °C for further experiments. A single isolated colony was cultured in 2 mL of tryptone soya broth supplemented with 1% of sucrose (TSBS) to enhance biofilm formation and grown for overnight at 37 °C and 160 rpm.

Phytocompound

Carvacrol was purchased from Sigma-Aldrich (India) and prepared as 10 mg/mL stock solution in ethanol and stored at 4 °C.

Minimum Inhibitory Concentration (MIC) Assay

MIC was determined by following the protocol of Clinical and Laboratory Standards Institute (CLSI) to test bacterial susceptibility by performing the broth microdilution assay in a 96-well microtiter plate (CLSI, 2015). Briefly, 1% of an overnight culture of MRSA (1×108) was used to inoculate in 200 μL of TSBS containing carvacrol (25, 50, 75, 100, 125, and 150 μg/mL) and incubated at 37 °C for 24 h. After incubation, the optical density (OD) was measured at 600 nm using a microplate reader-spectrophotometer (SpectraMax M3, Molecular Devices).

Crystal-Violet Biofilm Quantification Assay

To determine the minimum biofilm inhibitory concentration (MBIC) of carvacrol against MRSA, the crystal-violet-based biofilm quantification assay was performed in a 24-well polystyrene plate as previously reported.[58] Briefly, each well of the 24-well plate was filled with 2 mL of TSBS medium, 1% MRSA overnight culture, and added with increasing concentrations of carvacrol (25, 50, 75, 100, 125, and 150 μg/mL). The wells without inoculum and the wells without carvacrol were considered as blank and control, respectively. The wells containing 2 mL of TSBS medium, 1% MRSA overnight, and 10 μL of ethanol were regarded as vehicle controls to assess the influence of solvent on MRSA. Then, the plate was incubated for 24 h at 37 °C and planktonic cells were discarded and washed thrice with phosphate-buffered solution (PBS). To quantify biofilm formation, a 24-well plate was stained with 0.4% crystal violet solution for 10 min, washed with PBS to remove excess stain, and the biofilm bound stain was removed by dissolving it in 2 mL of 30% glacial acetic acid. Then, the plate absorbance was measured at 570 nm.

Ring Biofilm Assay

The biofilm assay was performed in glass test tubes as described. Briefly, 1% MRSA overnight culture was added to test tubes containing 2 mL of TSBS medium without and with carvacrol (25, 50, and 75 μg/mL). Then, the tubes were incubated in the standing condition for 24 h at 37 °C. After incubation, the biofilm was stained with crystal violet solution (0.4%).[31]

Microscopic Analyses

To assess the biofilm architecture of MRSA, 1% of overnight MRSA was inoculated into 1 mL of TSBS medium in a 24-well plate containing 1 × 1 cm2 glass slides without and with carvacrol at 25, 50, and 75 μg/mL concentrations and grown for 24 h at 37 °C. For light microscopy analysis, 0.4% crystal violet solution was used to stain the glass slides and observed at 400× under light microscopy (Nikon Eclipse 80i). To visualize the biofilm under confocal laser scanning microscopy (CLSM), slides were stained with acridine orange solution (0.1%) and observed at 200× in CLSM (LSM 710, Carl Zeiss, Germany).[59,60]

Biovolume Quantification Assay

The number of cells present in the biofilm of the MRSA control and carvacrol-treated samples was assessed through CFU analysis. Briefly, the biofilm assay was performed as described earlier in 1 × 1 cm2 glass slides in the absence and presence of carvacrol (25, 50, and 75 μg/mL) and incubated at 37 °C for 24 h. Following incubation, the glass slides were dip-washed by immersing into sterile PBS to remove unbound cells. Then, adhered cells were collected by scrapping out the biofilm, followed by repeated aspiration to disintegrate the cells. Cells were finally suspended in 1 mL of PBS. Number of cells in the MRSA control and carvacrol-treated samples were enumerated by the serial dilution method followed by spread-plating on TSA plates. CFU was calculated, and the graph was plotted.[61]

Growth Curve Analysis

To determine the effect of MBIC (75 μg/mL) of carvacrol on MRSA growth, the growth curve analysis was performed. Briefly, 200 mL of TSBS medium containing 1% MRSA overnight culture without and with 75 μg/mL carvacrol was incubated at 37 °C for 24 h. The OD at 600 nm of control and carvacrol-treated samples was measured at 1 h interval and continued up to 24 h.[51]

Alamar Blue Assay

To assess the metabolically active cells present in the MRSA control and carvacrol-treated cultures, the Alamar blue assay was performed. MRSA was grown without and with carvacrol (25, 50, and 75 μg/mL) for 24 h at 37 °C. Then, cells were collected by centrifugation at 8000 rpm for 10 min and cells were separately resuspended in freshly prepared PBS. Alamar blue (Sigma-Aldrich, India) was prepared as 6.5 mg/mL in PBS and mixed with a bacterial cell suspension in the ratio of 1:9. The samples were incubated at 37 °C in the dark for 4 h, and then, the fluorescent intensities were measured at 560 nm excitation and 590 nm emission wavelengths.[13]

Slime Synthesis Assay

To evaluate the slime synthesis in MRSA control and carvacrol-treated cells, Congo red agar (CRA) plates (3% TSB, 3.6% sucrose, 2% agar, and 0.08% Congo red dye) were prepared without and with carvacrol (25, 50, and 75 μg/mL). Then, MRSA culture was streaked on CRA plates and incubated at 37 °C for 24 h.[13]

Extracellular Polysaccharide (EPS) Quantification Assay

The phenol–sulfuric acid method was performed to quantify the total amount of EPS production in the MRSA control and carvacrol-treated samples. MRSA was grown in the absence and presence of carvacrol (25, 50, and 75 μg/mL) in a 24-well plate for 24 h at 37 °C. Afterward, plates containing planktonic cells and biofilm cells were vicariously aspirated and collected for centrifugation at 8000 rpm for 10 min. After centrifugation, cells were resuspended in PBS, in which an equal volume of 5% phenol and five volumes of H2SO4 solution were added. Finally, the mixture was incubated for 1 h in the dark and then the absorbance of sample supernatants was measured at 490 nm.[62]

Qualitative Analysis of Staphyloxanthin Production

To qualitatively assess the influence of carvacrol on staphyloxanthin production in MRSA, TSA plates were prepared without and with carvacrol at 25, 50, and 75 μg/mL concentrations. Then, the MRSA culture was streaked on control and carvacrol-added plates and then incubated at 37 °C for 24 h. The staphyloxanthin production in control and carvacrol-treated plates was visually observed and photographed.[63]

Staphyloxanthin Quantification Assay

To assess the effect of carvacrol on carotenoid pigment production and its intermediates’ synthesis, MRSA was grown in 100 mL of TSBS supplemented without and with carvacrol (25, 50, and 75 μg/mL) at 37 °C for 24 h at 160 rpm. Afterward, pellets were collected by centrifugation at 8000 rpm for 10 min and washed twice with PBS. The pellets were taken for methanolic extraction by resuspending the pellets into methanol and kept in an orbital shaker for 24 h. After that, methanolic extracts were filtered by Whatman filter paper and dried under a vacuum. Then, the methanolic extracts were dissolved in 1 mL of methanol and measured at 462, 455, 435, and 286 nm to assess production of staphyloxanthin [β-d-glucopyranosyl 1-O-(4,4′-diaponeurosporene-4-oate)-6-O-(12-methyltetradecanoate)], 4,4′-diaponeurosporenic acid, 4,4′-diaponeurosporene, and 4,4′-diapophytoene.[31]

Hydrogen Peroxidase (H2O2) Susceptibility Assay

To evaluate the susceptibility of MRSA to H2O2, MRSA was grown without and with carvacrol (25, 50, and 75 μg/mL) for 24 h at 37 °C. Then, the cell pellets were collected by centrifugation for 10 min at 8000 rpm and resuspended in PBS containing 20 mM H2O2 and incubated for 1 h at 37 °C. After incubation, the samples were serially diluted and spread on TSA to enumerate the viable cells.[31]

Whole-Blood Killing Assay

The effect of carvacrol on the viability of MRSA was assessed by the whole-blood killing assay. Control and carvacrol (25, 50, and 75 μg/mL)-treated samples were grown for 24 h at 37 °C. The cell pellets were separated by centrifugation for 10 min at 8000 rpm, and the bacterial cell suspension was prepared in PBS. Then, one volume of bacterial cell suspension (100 μL) was mixed with three volumes of freshly drawn heparinized healthy human blood (300 μL) and the samples were incubated at 37 °C for 3 h with 160 rpm agitation. For the enumeration of surviving CFU, the samples were serially diluted and spread over the TSA plates.[64]

Quantitative Real-Time PCR Analysis

To evaluate the effect of carvacrol (75 μg/mL) on candidate genes responsible for biofilm formation and staphyloxanthin synthesis in MRSA such as sarA, icaA, icaD, fnbA, fnbB, and crtM, qPCR was performed. Briefly, MRSA cells grown in the absence and presence of carvacrol (75 μg/mL) were incubated at 37 °C for 24 h. Cells were collected by centrifugation and the Trizol method was followed to isolate the total RNA. Then, RNA samples were converted into cDNA by following the protocol of the high-capacity cDNA reverse transcription kit (Applied Biosystems). As per the manufacturer’s instruction, the SYBR Green PCR master mix kit (Applied Biosystems) was used to perform qPCR analysis (7500 Sequence Detection System, Applied Biosystems Inc., Foster, CA). After normalizing the housekeeping gene expression (gyrB), candidate gene expressions were calculated using 2–ΔΔCt values.[13] The Primer sequences of tested genes are listed in Table . Assays were performed in biological triplicates with three technical replicates.

Table 2

List of Primers Used for qPCR Analysis

genes	forward primer	reverse primer
gyrB	5′-GGTGCTGGGCAAATACAAGT-3′	5′-TCCCACACTAAATGGTGCAA-3′
sarA	5′-CAAACAACCACAAGTTGTTAAAGC-3′	5′-CAAACAACCACAAGTTGTTAAAGC-3′
icaA	5′-ACACTTGCTGGCGCAGTCAA-3′	5′-TCTGGAACCAACATCCAACA-3
icaD	5′-ATGGTCAAGCCCAGACAGAG-3′	5′-AGTATTTTCAATGTTTAAAGCA-3′
fnbA	5′-ATCAGCAGATGTAGCGGAAG-3′	5′-TTTAGTACCGCTCGTTGTCC-3′
fnbB	5′-AAGAAGCACCGAAAACTGTG-3′	5′-TCTCTGCAACTGCTGTAACG-3′
crtM	5′-ATCCAGAACCACCCGTTTTT-3′	5′-GCGATGAAGGTATTGGCATT-3′

Molecular Docking Analysis

Molecular docking was performed to evaluate the binding energy and interactions of carvacrol with SarA and CrtM of MRSA. The 3D structures of SarA (PDB ID: 2FNP) and CrtM (PDB ID: 2ZCO) of MRSA were retrieved from the Protein Data Bank, and the carvacrol chemical structure (C10H14O; PubChem ID: 10364) was obtained from PubChem (NCBI; pubchem.ncbi.nlm.nih.gov/compound/Carvacrol). The 3D structures of previously reported SarA targeted compounds such as morin (PubChem ID: 5281670), eugenol (PubChem ID: 3314), and 3′-5′-cyclic diguanylic acid (c-di-GMP; PubChem ID: 135440063) were obtained from the bchem database. Lapaquistat acetate (PubChem ID: 9874248), rhodomyrtone (PubChem ID: 12050020), and tripotassium;4-(3-phenoxyphenyl)-1-phosphonatobutane-1-sulfonate (PubChem ID: 25244957) were used as positive controls for CrtM. Molecular docking analysis was performed using Autodock Tools v1.5.6,[65] and the 3D and 2D structures were visualized through BIOVIA Discovery Studio visualizer 2016 v16.1.0.15350.

Biofilm Assay with S. aureus Newman Wild-Type and Isogenic ΔsarA Mutant Strains

The biofilm assay was performed as described previously. Briefly, 1% overnight culture of S. aureus wild-type and ΔsarA strains was added to a 24-well polystyrene plate containing 1 mL of TSBS with increasing concentrations of carvacrol (25, 50, and 75 μg/mL) and incubated for 24 h at 37 °C. Then, the percentage of biofilm inhibition was calculated as mentioned previously. For ring biofilm formation, bacterial strains were added to glass tubes containing 2 mL of TSBS with increasing concentrations of carvacrol (25, 50, and 75 μg/mL) and kept under constant shaking for 24 h at 37 °C. After discarding planktonic cells, biofilm cells were stained with 0.4% crystal violet, photographed, and read at 570 nm.

Larvae Culture and Inoculation

The greater wax moth larvae killing assay was performed as described by Dong et al.[66] with minor modifications. Briefly, larvae weighing between 0.2 and 0.4 g were taken for all of the experiments. Ten randomly chosen healthy larvae of the required weight were used per group in each experiment. Each group was segregated and placed in sterile Petri dishes layered with Whatman filter paper. The MRSA culture was enumerated spectrophotometrically to a concentration of (5 × 108) ± 0.3, with the resulting final inoculum concentration for each group as (5 × 106) ± 0.3 cells/larvae. Injection was performed with a U-100 insulin syringe (Dispovan, HMD, India) in the last proleg. The larvae were incubated at 37 °C after inoculation, and the survival was monitored every 12 h. Larvae were considered to be dead when they turned black and did not respond to any physical stimulus. The groups were allocated as follows.

Group I—untouched control group in which the larvae were not handled but incubated at the same conditions as other larval groups.

Group II—PBS control.

Group III—vehicle control, PBS + 2% dimethyl sulfoxide (DMSO).

Group IV—carvacrol (250 mg/kg), to analyze the toxic effect of the compound, if any, on the survival of larvae.

Group V—infection group, inoculated with a final concentration of (5 × 106) ± 0.3 MRSA cells/larvae.

Group VI—treatment group, inoculated with MRSA culture and carvacrol.

Survival Assay

For the survival assay, each larva received injection as per the above-mentioned group and maintained at 37 °C. Survival was monitored every 12–120 h, and the dead larvae, if any, were removed at each time point of survival. Percentage of survival was calculated, and the graph was plotted.

In Vivo Efficacy of Carvacrol

To determine the in vivo efficacy of carvacrol in eliminating the MRSA, two groups, viz. the infection group and the treatment group, were considered. The infection group received the MRSA culture alone, and the treatment group received the MRSA culture and carvacrol (250 mg/kg). In vivo survival of MRSA in the absence and presence of carvacrol was calculated by performing the CFU assay at 0, 24, and 48 h post inoculation for which three larvae/group were randomly chosen, surface-sterilized with ethanol, and cut opened with a scalpel. The material was suspended in sterile PBS, serially diluted, and plated onto mannitol salt agar plates.[67]

Determination of Hemocyte Density in the Hemolymph

Hemolymph was collected from the infection and treatment groups at 24 and 48 h post inoculation into the microcentrifuge tube on ice to prevent melanization. Hemolymph was diluted in PBS, and the density was determined using an automated cell counter (Countess II FL, Invitrogen).[68] Equal proportion of hemolymph and 0.1% trypan blue was mixed and observed under a fluorescent microscope (Nikon Eclipse Ts2R, Japan).

Statistical Analysis

All of the assays were done in biological triplicates with three technical replicates, and data are given as mean ± standard deviation (SD). The statistical analysis was carried out through one-way analysis of variance (ANOVA) as well as Duncan’s post hoc test using SPSS version 17.0 (SPSS Inc., Chicago, IL). p-Value of <0.05 was regarded as significant, and asterisks are used to indicate significance.