In the present study, healthy human blood was used for blood survival and cytotoxicity assay and sheep blood was used for hemolysin production assay. The human blood sample was taken by a trained person from the healthy individuals and a written informed consent was obtained. The usage of the human blood sample and experimental methodology was assessed and approved by the Institutional Ethical Committee, Alagappa University, Karaikudi (IEC Ref No: IEC/AU/2018/4). The sheep blood used in the study was collected from the Karaikudi municipality slaughterhouse. There is no specific ethical permission since sheep blood is discarded in the slaughterhouse.

The MRSA strains used in this study are listed in Table 1. The clinical strains were isolated and identified by Gowrishankar et al. (2012). All the strains were cultured on tryptic soy agar (TSA, Hi-Media, India) at 37°C for 24 h and stored at 4°C for future use. For in vitro assays, MRSA was cultured in tryptic soy broth with 0.5% sucrose (TSBS) and incubated in an orbital shaker (160 rpm) at 37°C overnight. One percent of overnight MRSA culture was used for all assays.

Stock solutions of phytochemicals were prepared as 10 mg ml^–1 concentration and stored at 4^oC for further use. Myrtenol, α-bisabolol, carvacrol, phytic acid and ethyl linoleate were procured from Sigma-Aldrich, India. Cineole, theophyline, borneol, oleic acid and α-pinene were procured from Alfa Aeser, India. Methanol (Sigma-Aldrich, India) was used to dissolve myrtenol, ethyl linoleate and theophyline. Ethanol (Sigma-Aldrich, India) was used to dissolve α-bisabolol, phytic acid, cineole, theophyline, borneol, oleic acid and α-pinene.

Minimum inhibitory concentration of myrtenol was assessed by micro-broth dilution assay as described by the Clinical and Laboratory Standards Institute method (CLSI, 2018). Briefly, one percent of culture was added into a 24 well sterile micro-titer plate containing TSBS with different concentrations (25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, and 600 μg ml^–1) of myrtenol. TSBS containing 20 μl of methanol and 10 μl of culture was considered as vehicle control and TSBS alone was used as negative control. After incubating at 37°C for 24 h, the plate was read at 600 nm (Spectramax M3, Molecular devices, United States) to determine the MIC of myrtenol. For the determination of MBIC, planktonic cells were discarded and the plate was washed with distilled water to remove unbound cells and air dried. Crystal violet (0.4%) solution was used to stain the biofilm cells for 10 min and washed again with distilled water to remove unbound stains and then allowed to air dry. The biofilm cells were destained using 20% glacial acetic acid and absorbance of crystal violet solution was read at 570 nm. The amount of biofilm was directly proportional to the Optical Density (OD) value of the crystal violet solution in the wells. The percentage of biofilm inhibition was calculated by the following formula: Biofilm inhibition (%) = [(Control OD_570nm - Treated OD_570nm)/Control OD_570nm] × 100 (Singh et al., 2018).

For microscopic visualization, the biofilm was allowed to grow on glass slides (1 cm × 1 cm) in the absence and presence of myrtenol (75, 150, and 300 μg ml^–1) as described in the MBIC assay. Followed by biofilm assay, glass slides were washed with distilled water to remove the unbound cells and biofilm was stained by crystal violet solution (0.4%). Distilled water was used to wash excess stain on glass slides and allowed to air dry. Light microscope (Nikon Eclipse Ti-S, Tokyo, Japan) was used to observe the biofilm on glass slides at the magnification of 400X. For confocal laser scanning microscopy (CLSM) analysis, acridine orange solution (0.1%) was used to stain the biofilm on glass slides and visualized under CLSM (Zeiss LSM-710, Carl Zeiss, Oberkochen, Germany) at the magnification of 200X.

The effect of myrtenol on air-liquid interface biofilm formation was qualitatively determined by ring biofilm assay for which glass tubes containing one percentage of MRSA in 2 ml of TSBS in the absence and presence of myrtenol with increasing concentrations (75, 150, and 300 μg ml^–1) were incubated for 24 h at 37°C. Then, the planktonic cells in test tubes were discarded and washed thrice with sterile distilled water and air dried. Further, the test tubes were stained by crystal violet (0.4%) and washed thrice with distilled water to remove excess stain. Finally, test tubes were allowed to air dry in inverted position and then visually observed and photographed (Mathur et al., 2006).

The Alamar blue [Resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide)] assay was performed to assess the metabolically active cells in control and treated samples (Gargotti et al., 2018). Stock solution of Alamar blue (Sigma-Aldrich, India) at 6.5 mg ml^–1 in 1X Phosphate buffered saline (PBS) was prepared separately. The control and myrtenol (75, 150, and 300 μg ml^–1) treated cells were harvested and washed twice with PBS and resuspended in PBS. Cell suspension (0.9 ml) and Alamar blue substrate (0.1 ml) were mixed and incubated in the dark at 37°C for 4 h. Sterile PBS with Alamar blue substrate was used as blank. After incubation, the samples were centrifuged at 8000 rpm for 10 min and the fluorescent intensity of the supernatant containing the reduced Alamar blue was observed at 590 nm emission and 560 nm excitation wavelengths.

CFU assay was performed to determine the cell count variation between control and myrtenol treated cells (75, 150, and 300 μg ml^–1) of MRSA. Briefly, MRSA was grown in the absence and presence of myrtenol for 24 h at 37°C. After incubation, control and treated MRSA cells were serially diluted up to 10^–8 and spread on a TSB agar plate.

Growth curve assay was performed to assess the influence of myrtenol (300 μg ml^–1) on the growth of MRSA. MRSA was grown in the absence and presence of myrtenol at 37°C. At a regular interval of 1 h, OD was observed at 600 nm up to 24 h.

In order to assess the slime production, congo red agar (CRA) plates were prepared with TSB (3%), sucrose (3.6%), and agar (1.8%). The congo red dye (0.08%) was prepared separately and added to the agar medium. Control and myrtenol (75, 150, and 300 μg ml^–1) treated MRSA culture were streaked on CRA plates and incubated for 24 h at 37°C (Gowrishankar et al., 2016).

The para-nitro phenyl palmitate (pNPP) substrate was used to assess the lipid hydrolytic activity of cell free culture supernatant (CFCS) of control and myrtenol treated MRSA. Briefly, the lipase substrate solution was prepared by adding one volume of pNPP [0.3% pNPP in 2-propanol] to nine volumes of 50 mM Tris–HCl buffer (pH 8.0) containing sodium deoxycholate (0.2%) and gummi arabicum (0.1%). CFCS was obtained by centrifugation of MRSA cells cultured in the absence and presence of myrtenol (75, 150, and 300 μg ml^–1). To 100 μl of CFCS, 900 μl of lipase substrate was added and incubated at 37°C for 1 h and centrifuged at 10000 rpm for 10 min. Then, the supernatant was collected and absorbance was read at 410 nm to calculate lipase inhibition using following formula: Lipase inhibition (%) = [(Control OD_410nm - Treated OD_410nm)/Control OD_410nm] × 100 (Patel et al., 2018).

Cell free culture supernatant was collected from control and myrtenol treated (75, 150, and 300 μg ml^–1) MRSA strains as described earlier. Hemolytic activity was determined by incubating the CFCS with equal volume of sheep red blood cells (2% v/v in PBS) for 1 h at 37°C. After incubation, tubes were centrifuged at 3000 rpm for 10 min and the OD of supernatant was measured at 405 nm to determine the hemolytic activity using the following formula: Hemolysin inhibition (%) = [(Control OD_405nm - Treated OD_405nm)/Control OD_405nm] × 100 (Kannappan et al., 2017).

To determine the effect of myrtenol on the autolysis property of MRSA, control and myrtenol (75, 150, and 300 μg ml^–1) treated MRSA cells were harvested by centrifugation at 8000 rpm for 10 min and washed twice with ice cold PBS. Then, the cell pellet was resuspended in PBS containing 0.02% (v/v) Triton X-100. The cell suspensions were incubated at 37°C and lysis of cells was monitored by measuring the OD at 600 nm every 30 min for 3 h (Rowe et al., 2010).

The amount of eDNA in MRSA biofilm was qualitatively analyzed by agarose gel electrophoresis. Briefly, MRSA was allowed to form biofilm in the absence and presence of myrtenol (75, 150, and 300 μg ml^–1) on a 6 well polystyrene plate for 24 h at 37°C. Then, the planktonic cells were removed and biofilm was washed three times with PBS. The biofilm cells were scraped, resuspended in TE buffer (10 mM Tris, 1 mM EDTA) and vigorously vortexed for 1 h then centrifuged at 8000 rpm for 10 min. The supernatant was collected and the amount of eDNA present in the supernatant was separated by 1.5% (w/v) agarose gel electrophoresis (Kaplan et al., 2012).

MRSA culture was grown in 10 ml of TSB medium with and without myrtenol (75, 150, and 300 μg ml^–1) for 24 h at 37°C. The cell pellet was harvested by centrifugation at 8000 rpm for 10 min, washed thrice with PBS and resuspended in 10 ml of PBS. Bacterial suspensions were transferred to glass tubes and allowed to stand without any disruption for 24 h and 200 μl of the upper portion was collected at 0, 3, 6, 12, and 24 h time points for measuring the absorbance at 600 nm. For visual observation, tubes were photographed (Tareb et al., 2013).

To assess the staphyloxanthin production, one percentage of MRSA was used to inoculate in 10 ml of TSBS with and without the myrtenol (75, 150, and 300 μg ml^–1). After 24 h incubation, MRSA culture was centrifuged at 10000 rpm for 10 min. Then, the pellet was washed twice with PBS and resuspended in 2 ml of methanol. For methanol extraction, the tubes were kept in a shaking incubator for 24 h. After the methanolic extract, the samples were centrifuged at 10000 rpm for 10 min and the OD of the supernatant was measured at 465 nm. The percentage of staphyloxanthin inhibition was calculated by following formula: staphyloxanthin inhibition (%) = [(Control OD_465nm - Treated OD_465nm)/Control OD_465nm] × 100 (Lan et al., 2010).

Control and myrtenol (75, 150, and 300 μg ml^–1) treated MRSA cells were pelleted by centrifugation at 8000 rpm for 10 min and resuspended in PBS containing 1 mM H₂O₂ and incubated at 37°C for 1 h. Then, the cells were serially diluted, spread on the TSB agar plate and incubated at 37°C for 24 h. After incubation, viable cells were counted to determine the sensitivity of cells to H₂O₂ (Tatsuno et al., 2014).

The control and myrtenol (75, 150, and 300 μg ml^–1) treated cell suspensions were prepared in PBS. The cell suspensions were mixed gently with healthy human blood at a ratio of 1:4 and incubated at 37°C for 1 h. After incubation, the samples were serially diluted and spread over TSB agar plate. The plates were incubated for 24 h at 37°C and colonies were manually counted to identify the sensitivity of cells to healthy human blood (Subramenium et al., 2015).

Total RNA was extracted from MRSA grown for 24 h in the absence and presence of myrtenol (300 μg ml^–1) by Trizol method of RNA isolation and converted to cDNA using High-capacity cDNA Reverse Transcription Kit (Applied Biosystems Inc., United States) according to the manufacturers’ protocol. qPCR analysis was performed on thermal cycler (7500 Sequence Detection System, Applied Biosystems Inc., Foster, CA, United States) for the genes such as sarA (transcriptional regulator), icaA and icaD (involved in poly-beta-1,6-N-acetyl glucosamine biosynthesis), agrA (response regulator), and agrC (transmembrane protein), fnbA and fnbB (fibronectin binding proteins), clfA (clumping factor), cna (collagen binding protein), hla (alpha-hemolysin), hld (delta-hemolysin), geh (lipase), altA (autolysin), and crtM (involved in staphyloxanthin biosynthesis) using PCR mix (SYBR Green kit, Applied Biosystems, United States) at a predefined ratio. Cycle threshold (Ct) values of all the tested genes were normalized using that of housekeeping gene gyrB (gyrase) and expression was quantified by 2^(–ΔΔCt) method (Livak and Schmittgen, 2001; Gowrishankar et al., 2015). Primer sequences of the genes taken for study and qPCR conditions are given in Supplementary Tables S1 and S2, respectively.

To determine the effect of myrtenol on mature biofilm, MRSA was allowed to form biofilm on glass slides and polystyrene surface of 24 well plate for 24 h at 37°C as mentioned previously. After the formation of mature biofilm, the planktonic cells were discarded and washed with sterile PBS to remove non-adhered cells. Fresh TSBS media and various concentrations of myrtenol (75, 150, 300, and 600 μg ml^–1) were added to the specific wells and incubated for 24 h at 37°C. After incubation, glass slides were washed thrice with sterile PBS and stained for observation under light microscope and CLSM as described previously. For biofilm quantification, the plate was washed with distilled water to remove unbound cells and biofilm cells were stained with crystal violet (0.4%) solution and washed again with distilled water and then allowed to air dry. The biofilm cells were destained using 20% glacial acetic acid and absorbance of crystal violet solution was read at 570 nm (Chen et al., 2018).

The blood sample (5 ml) was collected from a healthy human volunteer in a tube containing EDTA. Equal volume of lymphocyte separation medium (Histopaque-HiMedia, India) was taken in a fresh tube upon which the freshly collected blood was layered carefully and subjected to centrifugation at 2200 rpm for 30 minutes at 20°C. After removing the upper layer, the buffy coat mononuclear layer was carefully transferred to a fresh tube and washed using RPMI-1640 (Roswell Park Memorial Institute) medium at 2200 rpm for 15 min at 20°C. The pellet was resuspended in the complete medium [RPMI medium (HiMedia, India), 10% fetal bovine serum (HiMedia, India) and 1% Anti-Anti (Antibiotic-Antimycotic) solution (Invitrogen, United States)] and the cell viability was assessed through trypan blue exclusion assay. PBMCs were adjusted to 1 × 10⁶ cells ml^–1 in complete medium for further experiments (Syad and Kasi, 2014).

Cytotoxicity was assessed using PBMC by Alamar blue assay and trypan blue exclusion assay. PBMCs were incubated in the presence of different concentrations of myrtenol (75, 150, 300, and 600 μg ml^–1) at 37°C with 5% CO₂ incubator for 24 h. Appropriate control, positive control (1mM H₂O₂) and vehicle controls (0.05% DMSO) were also maintained. The cell viability was assessed after 24 h by trypan blue exclusion assay (Strober, 1997) using the formula: Percentage of cell cytotoxicity = (Number of viable cells/Total number of cells) X 100 (Rajavel et al., 2017).

Alamar blue was prepared in PBS solution at 1 mg ml^–1 concentration. The control and treated PBMCs were collected by centrifugation and washed twice with PBS and resuspended in PBS. To 90 μl of cell suspension, 10 μl of Alamar blue solution was added and incubated in dark at 37°C for 4 h. After incubation, the samples were centrifuged at 8000 rpm for 10 min and the fluorescent intensity of the supernatant was observed at 590 nm emission and 560 nm excitation wavelengths (Menezes et al., 2016).

All the experiments were carried out in three biological replicates with at least two technical replicates and values are presented as Mean ± standard deviation (SD). To analyze the significant difference between the value of control and treated samples, one-way analysis of variance (ANOVA) and Duncan’s post hoc test was performed with the significant p-value of <0.05 by the SPSS statistical software package version 17.0 (Chicago, IL, United States).

The antibiofilm activity of ten phytochemicals was tested against MRSA at 300 μg ml^–1. Out of ten phytochemicals, myrtenol showed strong antibiofilm activity (72%) at 300 μg ml^–1. Among the remaining phytochemicals, cineole, carvacrol, α-pinene and α-bisabolol exhibited antibacterial activity whereas the rest of the phytochemicals at 300 μg ml^–1 concentrations showed insignificant changes in cell density as well as biofilm formation of MRSA (Supplementary Figure S1).

To determine the MIC and MBIC of myrtenol against MRSA ATCC strain and clinical isolates, micro-broth dilution assay and biofilm assay was performed. The result exhibited myrtenol at 600 μg ml^–1 which completely inhibited the growth of MRSA ATCC strain and clinical isolates. Therefore, MIC of myrtenol was fixed as 600 μg ml^–1. Biofilm inhibition was assessed by crystal violet quantification assay. The results demonstrated the concentration dependent antibiofilm activity of myrtenol against MRSA strains. At 300 μg ml^–1 concentration, myrtenol showed a maximum of 72% (MRSA ATCC-33591), 79% (MRSA-395), 85% (MRSA-410), and 82% (MRSA-44) of biofilm inhibition in MRSA strains. Inhibition of the growth was observed at concentrations higher than 300 μg ml^–1 of myrtenol. An ideal antibiofilm agent should not affect the growth of the bacteria. Hence, 300 μg ml^–1 was fixed as MBIC of myrtenol (Figures 1A–D).

Microscopic technique was used to qualitatively analyze biofilm architecture of MRSA strains. In light microscopic analysis, highly aggregated biofilm formation was observed in control samples. Whereas in treated samples, concentration dependent reduction in the biofilm covered surface area was observed (Figure 2A). The CLSM was used to assess the three-dimensional biofilm architecture of MRSA strains. Reduction in the thickness of biofilm formation was noticed in myrtenol treated samples when compared to that of control samples (Figure 2B).

MRSA ATCC strain was taken for further studies. Hence, the effect of myrtenol on biofilm reference strain was assessed. Myrtenol showed concentration dependent biofilm inhibitory activity against MRSA. At a concentration of 300 μg ml^–1, myrtenol exhibited a maximum of 72% (p ≤ 0.05) biofilm inhibition in MRSA (Figure 3A). Growth OD values of control and treated samples did not reveal any significant difference up to 300 μg ml^–1 concentrations of myrtenol (Figure 3A). In addition, myrtenol showed a concentration dependent reduction in ring biofilm formation on glass test tubes (Figure 3B) and surface biofilm formation on polystyrene plate (Figure 3C).

In S. aureus, slime production is linked with biofilm formation. Hence the slime production was qualitatively analyzed by the CRA plate method. The results exhibited a reduction in the black coloration around the colonies of treated samples when compared to the colonies of control sample (Figure 3D).

To determine the effect of myrtenol on growth and viability of MRSA, Alamar blue assay, CFU assay and growth curve analysis were performed. In Alamar blue assay, the fluorescent intensity of resazurin dye was found to be unchanged for control and myrtenol treated MRSA cells which directly confirms the non-antibacterial effect of myrtenol (Figure 4A). The CFU of MRSA control (4.8 × 10⁸ cells) and myrtenol treatment at MBIC (4.7 × 10⁸ cells) was found to be significantly unchanged (Supplementary Figure S2). Furthermore, the growth curve analysis of MRSA also showed no significant change in myrtenol treated sample compared to control culture (Figure 4B).

Extra-cellular lipase and hemolysin are important virulence factors produced by S. aureus to degrade the phospholipid bilayer of host cells and erythrocytes of host blood respectively. The results indicated significant reduction up to 64% of lipase and 62% of hemolysin production upon treatment with myrtenol at MBIC (p ≤ 0.05) (Supplementary Figure S3). In addition to ATCC strain, the effect of myrtenol on hemolysin production was assessed for clinical isolates and results showed a concentration dependent reduction in hemolysin production (Supplementary Figure S4).

Autolysin has been reported as an essential factor for S. aureus biofilm formation. Hence, the effect of myrtenol on autolysis of MRSA was spectrophotometrically measured. The results showed the inhibition in autolysis activity of myrtenol (75, 150, and 300 μg ml^–1) treated cells, whereas in the control cells, highly active autolysis was observed (Figure 5A).

The quantity of eDNA released by MRSA was assessed in the absence and presence of myrtenol (75, 150, and 300 μg ml^–1). The decrease in the amount of eDNA release was observed in myrtenol treated cells when compared to the amount of eDNA released by control cells (Figure 5B).

Autoaggregation is an important process in S. aureus biofilm formation. Thus, the effect of myrtenol on the autoaggregation property of MRSA was evaluated. Interestingly, the autoaggregation of MRSA was reduced due to the addition of myrtenol in the growth medium. The significant reduction in autoaggregation was observed in myrtenol (75, 150, and 300 μg ml^–1) treated samples compared to the control sample (Figure 5C). Autoaggregation was visually observed after incubating tubes for 24 h in static condition (Figure 5D).

Staphyloxanthin synthesis of myrtenol treated and untreated cells was evaluated quantitatively and qualitatively. Staphyloxanthin production was inhibited in a concentration dependent manner up to 20−65% (p ≤ 0.05) in treated cells (Figure 6A) when compared to the control. The staphyloxanthin inhibition was also observed visually in myrtenol treated cells (Figure 6B).

The sensitivity of MRSA to healthy human blood and H₂O₂ was assessed between myrtenol (75, 150, and 300 μg ml^–1) treated cells and the untreated cells by blood survival assay and H₂O₂ sensitivity assay. The results demonstrated that the myrtenol (300 μg ml^–1) treated cells were highly susceptible to H₂O₂ (1.7 × 10⁷ cells) (Figure 6C) and healthy human blood (5 × 10⁶ cells) (Figure 6D) when compared to the control sample (3.5 × 10⁷ cells). Altogether, the resistance of MRSA against human blood and H₂O₂ was reduced upon treatment with myrtenol.

Real-time PCR was performed to assess the effect of myrtenol at 300 μg ml^–1 on genes involved in the virulence factors production and biofilm formation in MRSA. The results revealed down regulation in the expression of sarA, agrA, icaA, icaD, fnbA, fnbB, clfA, cna, hla, hld, geh, altA, and crtM and up regulation in the expression of agrC (Figure 7).

Mature biofilm disruption assay was performed to assess the antibiofilm efficacy of myrtenol on the preformed MRSA biofilm. Growth of MRSA was found to be unaltered whereas no significant reduction in biofilm was observed at MBIC. Interestingly, at 600 μg ml^–1 concentration, myrtenol disrupted 40% of the preformed biofilm of MRSA (Figure 8A). Spectrometric results were also confirmed by the light microscopic and CLSM analysis as well, in which the biofilm matrix was found to be reduced at 600 μg ml^–1 concentration of myrtenol (Figure 8B).

The cytotoxic effect of myrtenol on PBMCs was assessed by trypan blue exclusion and Alamar blue assay. The fluorescent intensity of resazurin dye observed in the PBMCs with or without myrtenol (75, 150, 300, and 600 μg ml^–1) was found to be unchanged (Supplementary Figure S5A). Trypan blue exclusion assay result revealed that myrtenol did not affect the viability of human PBMCs even at a concentration higher than MBIC for 24 h (Supplementary Figure S5B). Similar results were observed in the microscopic visualization of untreated and treated PBMCs where no significant reduction in viability or changes in morphology were observed when compared to the H₂O₂ treated positive control cells (Supplementary Figure S6). Hence, from the results it is apparent that myrtenol has no cytotoxic effect at the tested concentrations.