The leaves of C. tamala were collected from Almora, Uttarakhand, India. The leaves were partially shade dried and used for extraction of EO. On hydro-distillation, the partially shade dried leaves yielded 1.2% pale yellow color EO. After distillation, the EO was dried over by adding a pinch of anhydrous sodium sulfate and stored at 4°C until further analysis and experiments. The EO was quantitative and qualitatively analyzed by GC-FID and GC-MS following the methods and conditions used by Sriramavaratharajan et al. (2016). The individual component of the EO such as cinnamaldehyde (#W228613) and linalool (#L2602) was purchased from Sigma–Aldrich.

The Minimum Inhibitory Concentration (MIC) of the EOs were determined as per Clinical and Laboratory Standards Institute (2006) guidelines. Various concentrations of EO were prepared by mixing proportions of EO with 1.25% dimethyl sulfoxide (DMSO). Serial two-fold dilutions of the EO ranging from 1 to 20% v/v were prepared in 96-well titer plate supplemented with test culture and incubated for 24 h at 37°C. Following incubation, microtiter plates were read spectrophotometrically at 620 nm. The lowest concentration inhibiting the growth of the pathogen was determined as the MIC. Similarly, the individual component of EO such as cinnamaldehyde and linalool was tested for their respective MICs against PAO1 and the clinical isolates.

In order to examine that the EO did not have bactericidal effect against P. aeruginosa planktonic cells, growth curve analysis was done. LB broth containing 1% overnight culture of P. aeruginosa was supplemented with Biofilm Inhibitory Concentration (BIC) of the EO and the flask was incubated for 24 h at 37°C. The flask containing overnight culture without EO served as control. The optical density was read spectrophotometrically at every 1 h interval up to 24 h (Packiavathy et al., 2013).

The effect of EO to inhibit the young biofilms was assessed using the crystal violet assay (Nithyanand et al., 2015c). The assay was carried out in 24-well microtiter plate (NEST Biotechnology, Korea) containing 1ml of media, 100 μl of cell suspension (10⁷ cells ml^-1) and EO at its sub-MIC (5% v/v). The well without EO and only DMSO (1.25%) served as a control. Likely, the effect of commercial and MBDs and synergism of EO and the respective DNases were assessed as above to determine the maximum efficacy in inhibiting the biofilm.

To determine the potency of the EO to inhibit mature biofilms, the BIC of EO was added to the preformed biofilms and incubated further for 24 h at 37°C. After incubation, the spent media was discarded and the wells were washed with sterile distilled water. After air drying the wells were stained with 0.4% crystal violet and the absorbance was quantified spectrophotometrically at 595 nm (Nithyanand et al., 2015c). Similarly, the potency of DNases and synergism of EO and DNases over mature biofilm dispersal was also determined.

Biofilms were allowed to form on 1 cm × 1 cm glass slides which were placed into the wells of the 24-well titer plates. The BIC of EO (5%v/v) was added to the wells and the plates were incubated at 37°C for 24 h. After incubation, the biofilm formed on the glass slides was stained using crystal violet dye for 5 min. It was then gently washed with de-ionized water and allowed to dry for 5 min. Then, the slides were viewed under light microscope at a magnification of 40× (Nikon Eclipse Ti 100, Japan) (Thenmozhi et al., 2009).

Confocal Laser scanning microscopy (CLSM) was used to determine the action of EO on mature biofilms. The biofilms were allowed to form on 1 cm × 1 cm glass slide placed in a 24-well titer plates. After 24 h incubation, the BIC of EO was added to the preformed biofilms and the plates were further incubated at 37°C for 24 h. The three dimensional architecture of the preformed biofilm treated with BIC of EO was assessed by CLSM (Nithyanand et al., 2015c). Images were captured and processed by using Zeiss LSM Image Examiner Version 4.2.0.121. The parameters such as biomass, average thickness and surface volume ratio of the control and treated biofilms were evaluated using COMSTAT software (Heydorn et al., 2000).

In order to visualize the reduction in EPS of P. aeruginosa biofilm, SEM analysis was performed. Briefly, P. aeruginosa biofilms were grown in the presence and absence of enzymes (DNaseI and MBD) on the glass slides. The biofilms on the glass slides were fixed with 2.5% glutaraldehyde were washed in 0.1M sodium acetate buffer (pH 7.3). Finally, the dehydrated samples were dried, gold sputtered and examined with VEGA3 TESCAN (Thenmozhi et al., 2009).

In order to investigate the accumulation of eDNA in P. aeruginosa biofilm, eDNA staining was performed. The P. aeruginosa strains were inoculated in LB broth and grown in the presence of DNaseI and MBD onto 1 cm × 1 cm glass slide within a 24-well titer plate and was incubated for 24 h at 37°C. After 24 h, the glass slides were gently rinsed with PBS and stained with 20 μM propidium iodide (PI) (Sigma), which stains the DNA present within a biofilm. Accumulation of eDNA were visualized under a fluorescence microscope (Nikon eclipse Ni, Japan) at excitation and emission wavelengths of 540 and 525 nm for PI and photographed digitally (Rajendran et al., 2013).

Bacterial biofilm consists of self-generated extra polymeric substances which plays a distinct role during biofilm formation. EPS from EO treated and untreated test pathogen was quantified by the method of Nithya et al. (2011). The test pathogen was grown in glass slides placed in 24 well microtiter plates with and without the addition of EO and incubated for 16 h. The glass slides were removed and washed with 0.5 ml of 0.9% NaCl. Equal volume of 5% phenol was added to the cell suspension, to which five volume of concentrated H₂SO₄ was also added. The mixture was incubated for 1 h in the dark and centrifuged at 10,000 rpm for 10 min. The absorbance of the supernatant was measured at 490 nm.

Alginate is an abundant polysaccharide in P. aeruginosa biofilms which aids in the adhesion of EPS that promote cell attachment and shields the cell surface structure. Alginate was quantified from the EO treated and untreated cell suspension. 600 μl of boric acid – H₂SO₄ (4: 1) was added to 70 μl cell suspension and the cell suspension was vortexed for 10 s. Further, 20 μl of 0.2% carbazole solution was added to the cell suspension which was incubated for 30 min at 55°C. After incubation, absorbance was measured spectrophotometrically at 530 nm (Owlia et al., 2007).

Pseudomonas aeruginosa uses las QS system which consists of lasI that interact with the LasR transcriptional activator to activate the lasA virulence gene which leads to the production of LasA protease. The ability of culture supernatant to lyse the boiled Staphylococcus aureus cells was determined by LasA protease activity (Kessler et al., 1993). A 30 ml overnight culture of S. aureus cells were centrifuged at 7,000 rpm for 3 min and the pellet was suspended in 0.02 M Tris-HC1 (pH 8.5). The suspension was boiled for 10 min and then diluted with the same buffer to an OD of 0.8 at 595 nm. To 900 μl of diluted S. aureus suspension, 100 μl of cell free culture supernatant of the strains cultured with or without EO was added. OD was measured at 595 nm after every 15 min interval for 60 min using a UV–visible spectrophotometer. Similarly, the LasA staphylolytic assay was carried out using cinnamaldehyde and linalool against P. aeruginosa and the clinical isolates.

Pseudomonas aeruginosa uses rhl QS system that consists of rhlI which in conjunction with RhlR activates the expression of pyocyanin pigment production. Cell free supernatants of PA01 and the clinical isolates cultured with the presence and absence of EO at their respective sub-MIC concentrations (5 and 7% v/v) was extracted for pyocyanin quantification (Essar et al., 1990). The extracted pyocyanin was quantified spectrophotometrically at OD 520 nm. Cinnamaldehyde and linalool were also tested for their ability to inhibit pyocyanin produced by PA01 and the clinical isolates.

Pseudomonas aeruginosa uses QS to regulate several functions such as biofilm formation, motility, and pigment production. QS regulated swarming motility is characterized as a form of flagella dependent movement on a viscous environment such as semisolid agar surface (Krishnan et al., 2012). Initially, 10 ml of swarming agar [(1% w/v) glucose, (0.5% w/v) agar, (0.5% w/v) peptone and (0.2% w/v) yeast extract] was overlaid on to the sterile petri plate. The 5 ml of swarming media seeded with BIC of EO was poured as an additional layer and was allowed to solidify. The 2 μl of overnight grown P. aeruginosa cells (2 × 10⁷ CFU) was inoculated in the middle of the agar and incubated for 16 h at 37°C (Krishnan et al., 2012). Cinnamaldehyde and linalool were tested for its inhibition of swarming motility by the same method.

To clearly demonstrate the synergized effect of EO and DNases on young biofilms, microscopic examinations were performed in the presence of sub-MIC (5% v/v) of EO. The microscopy analysis revealed maximum reduction of young biofilm upon treatment with EO and DNases. There was also reduction in EO treated biofilm but to a lesser extent on comparison with combination of EO and DNases (Figure 4).

Confocal Laser scanning microscopy was further done to confirm that synergized effect has maximum ability to disrupt matured biofilm than EO and DNases alone and to study the architecture of the biofilm before and after treatment. From the CLSM images (Figure 5), it is evident that there was a disintegration of biofilms in the treated samples on comparing with the control, which showed the presence of a dense biofilm. COMSTAT analysis was carried to determine the three dimensional features like biomass, average thickness and surface volume ratio of matured biofilms before and after treatment. The results show that there was a substantial reduction in different parameters of biofilm architecture such as biomass and average thickness, whereas there was an increase in surface to volume ratio which indicates the detachment of cells from biofilm matrix upon treatment (Table 2). From the Table 2, it is evident that the increase in surface to volume ratio (μm²/μm³) of around 0.043 and decrease in biomass (μm) (32.22) and average thickness (μm) (30.08) is due to the synergistic effect of both EO and DNase.

To analyze the antibiofilm potential of DNaseI and MBD on P. aeruginosa biofilm, SEM was performed. From the SEM results it is depicted that untreated P. aeruginosa (Figure 6A) has a thick layer of EPS, whereas there is a disruption in the EPS matrix of biofilm treated with DNaseI and MBD (Figures 6B,C).

To detect the presence of eDNA, P. aeruginosa biofilms were stained with PI (which specifically stains eDNA) and was visualized under a fluorescent microscope. From the fluorescent micrograph results it is evident that untreated biofilm has high accumulation of eDNA (Figure 7A) whereas treated has lesser eDNA accumulation (Figures 7B,C).

Essential oil inhibits the production of EPS, a maximum reduction in OD was observed with P. aeruginosa treated with sub-MIC (5% v/v) (Figure 8). Similarly, EO exhibited a concentration dependent inhibitory effect in the production of alginate by PAO1. At sub-MIC (5% v/v) the alginate production was reduced to the maximum of 71% ± 4.38 whereas 1% and 2.5% v/v had lesser inhibitory effect on alginate production (Figure 9A). The EO at its sub-MIC (7% v/v) inhibited the alginate production of clinical isolate AU07 by 65% ± 3.49 and 61% ± 3.58 for AU09 (Figures 9B,C). Likely, cinnamaldehyde inhibits the alginate production at its sub-MIC (0.18 mg/ml) of about 69% ± 2.75 for P. aeruginosa, 63.5% ± 4.63 for AU07 PACI-01 at its sub-MIC (1 mg/ml) and 59.5% ± 2.43 for AU09 PACI-02 at its sub-MIC (1 mg/ml) (Supplementary Figure S1). Linalool, another component of EO, inhibits alginate of about 67% ± 2.99 for P. aeruginosa, 63% ± 1.99 for AU07 PACI-01 and 60% ± 3.12 for AU09 PACI-02 (Supplementary Figure S2).

Supernatant of PAO1 treated with EO showed significant reduction in LasA staphylolytic activity in a concentration dependent manner. A maximum reduction of about 76% ± 8.06 was observed at sub-MIC (5% v/v) treated (Figure 10A). The EO at its sub-MIC (7% v/v) reduces the LasA staphylolytic activity for AU07 by 68% ± 4.95 and 75% ± 7.93 for AU09 (Figures 10B,C). The reduction of LasA staphylolytic activity was found to be 70% ± 5.26 for P. aeruginosa at its sub-MIC (0.18 mg/ml), 62.5% ± 4.22 for AU07 PACI-01 and 60% ± 3.99 for AU09 PACI-02 at its sub-MIC (1 mg/ml) (Supplementary Figure S3). For Linalool the reduction was found to be 67% ± 4.56 for P. aeruginosa, 63% ± 3.65 for P. aeruginosa and 60% ± 3.42 for AU09 PACI-02 (Supplementary Figure S4).

The production of pyocyanin a blue green phenazine pigment in P. aeruginosa is regulated by QS system. P. aeruginosa biofilm treated with EO showed a maximum reduction of pyocyanin production of about 72% ± 4.65 at its sub-MIC (5%v/v) when compared to the control (Figure 11A). The EO at its sub-MIC (7% v/v) reduces the pyocyanin production by 68% ± 3.95 for AU07 and 62% ± 3.90 for AU09 (Figures 11B,C). Pyocyanin was reduced significantly of about 78% ± 4.70 at its sub-MIC (0.18 mg/ml) for P. aeruginosa treated with cinnamaldehyde and 67% ± 0.87 for AU07 PACI-01 at its sub-MIC (1 mg/ml) and 63% ± 2.01 for AU09 PACI-02 at its sub-MIC (1 mg/ml) (Supplementary Figure S5). Likely, linalool significantly reduces pyocyanin pigment of about 70% ± 2.94 for P. aeruginosa, 67% ± 1.30 for AU07 PACI-01 and 65% ± 2.63 for AU09 PACI-02 (Supplementary Figure S6).

To explore the anti-QS potential of the EO of C. tamala, swarming assay was done against P. aeruginosa in a concentration dependent manner. Maximum reduction of swarming motility was observed in the swarming plates treated with EO at its sub-MIC (5% v/v) concentration (Figure 12). From the results, it is envisaged that EO at its BIC (5% v/v) has the ability to interfere in the Las and Rhl mediated QS system. The individual component of EO such as cinnamaldehyde (Supplementary Figure S7) and linalool (Supplementary Figure S8) inhibit swarming motility of P. aeruginosa and two clinical isolates such as AU07 PACI-01 and AU09 PACI-02 at its sub-MIC. Earlier studies which report about the anti-QS activity of cinnamon oil cite that cinnamaldehyde was the major component of the oil and it was responsible for the anti-QS activity (Brackman et al., 2008; Yap et al., 2015). However, a recent study which report about the anti-QS activity of Ceylon-type whole cinnamon oil had eugenol as its major component (Kalia et al., 2015). In similar lines, the EO from C. tamala had linalool (42%) as its major component followed by cinnamaldehyde (31%). So, we fully concur with the view that the exhibited QSI activity might be due to the synergistic action of the major components present in the EO (Kalia et al., 2015). The potency of an EO form an underexplored Cinnamomum sp. in attenuating QS dependent swarming motility provides a new insight into the anti-virulent potential of the EO and opens up a new avenue for bioprospecting EOs from several underexplored wild Cinnamomum sp. present in the Himalaya range.