Data regarding anti-quorum sensing and antimicrobial activity of Melaleuca alternifolia and Salvia sclarea essential oil against Pseudomonas aeruginosa

To combat the increasing number of multi-drug resistant bacteria, researchers are now looking for alternatives that reduce the virulence and pathogenic potential of the bacteria without killing it. It can be accomplished by interfering with the quorum sensing (QS) system of bacteria. In this article, we aim to determine the antimicrobial and anti-QS activity of Salvia sclarea and Melaleuca alternifolia essential oils (EOs) against Pseudomonas aeruginosa. The sub-lethal concentration of these EOs was found with the help of a growth curve, and further experiments were carried out below this concentration. To check for their anti-quorum activity, a bioreporter strain E. coli pJN105LpSC11 (to measure the concentration of 3-oxo-C12—HSL) and Chromobacterium violaceum CV026 (to check for the reduction in the formation of violacein pigment) was used. Several virulence phenotype assays like pyocyanin, alginate, and protease production, along with swarming motility, were done. The effect of these EOs on biofilm formation was also checked. The results were confirmed by checking the expression of genes by real-time PCR.


a b s t r a c t
To combat the increasing number of multi-drug resistant bacteria, researchers are now looking for alternatives that reduce the virulence and pathogenic potential of the bacteria without killing it. It can be accomplished by interfering with the quorum sensing (QS) system of bacteria. In this article, we aim to determine the antimicrobial and anti-QS activity of Salvia sclarea and Melaleuca alternifolia essential oils (EOs) against Pseudomonas aeruginosa . The sub-lethal concentration of these EOs was found with the help of a growth curve, and further experiments were carried out below this concentration. To check for their anti-quorum activity, a bioreporter strain E. coli pJN105LpSC11 (to measure the concentration of 3-oxo-C 12 -HSL) and Chromobacterium violaceum CV026 (to check for the reduction in the formation of violacein pigment) was used. Several virulence phenotype assays like pyocyanin, alginate, and protease production, along with swarming motility, were done. The effect of these EOs on biofilm formation was also checked. The results were con-

Objective
The National Institute of Health (NIH) reports that almost 65% of microbial infections are related to the formation of biofilm. Due to the excessive use of antibiotics, the number of multi-drug resistant bacteria is increasing. So, to overcome this problem, newer strategies are being adopted that exert low selection pressure on the bacteria. This study aims to examine the possibility of Salvia sclarea (common name is Clary sage) and Melaleuca alternifolia (common name is Tea tree) EOs to show antimicrobial and anti-quorum sensing activity against Pseudomonas aeruginosa , which is responsible for a large percentage of nosocomial infections and diseases in immuno-compromised patients due to the formation of biofilm. Salvia sclarea essential oil (EO) has been known for its anti-inflammatory, antiviral, antibacterial, and antimalarial properties. On the other hand, Melaleuca alternifolia EO has been reported to have antiprotozoal activity in addition to antibacterial, antifungal, and antiviral properties.

Data Description
Herbal extracts and essential oils are gaining worldwide attention for their therapeutic properties against a wide species of bacteria. In this regard, we explored the antibacterial and ant-QS activity of Melaleuca alternifolia and Salvia sclarea EOs against a multi-drug resistant bacteria Pseudomonas aeruginosa PA01. The determination of MIC of EOs was done using a 96-well microtiter plate with each well having different concentrations ofEOs ranging from 80 μl/ml to 0.125 μl/ml. The MIC calculated for these EOs is mentioned in Table 1 . The growth curve was plotted at different concentrations; however, only that concentration of EO was selected for further experiments that did not significantly affect the growth kinetics (displayed in Fig. 1 ) of P. aeruginosa and mimicked the growth pattern of the control. The anti-QS inhibition activity of Salvia sclarea and Melaleuca alternifolia , EO against Pseudomonas aeruginosa was assessed using CV026 biosensor strain as depicted in Fig. 2 . DMSO was used as a control here. β-Galactosidase assay was performed to determine the effect of EO on the production of 3-oxo-C 12 -HSL by P. aeruginosa as presented in Fig. 3 . Here, the control was an untreated PA01 culture, and the result was expres sed in the form of miller units. To check for the effect of EOs on the virulence phenotypes under the control of the QS system, several assays were performed like pyocyanin Table 1 Minimum inhibitory concentration (MIC) and concentration of essential oils that doesn't affect the growth kinetics of    Fig. 4(B) . To estimate the level of protease activity in the treated and untreated culture of P. aeruginosa , protease assay was performed as presented in Fig. 5 and the data regarding the zone of protease inhibition is mentioned in Table 2 . Swarming motility of the treated culture and control (untreated culture) was assessed and the result is  presented in Fig. 6 and Table 3 . The data regarding potential of EOs at different concentrations to inhibit biofilm formation is given in Fig. 7 . Fig. 8 depicts the gene expression studies, which reflect the effect of EOs on quorum sensing (QS) system genes of P. aeruginosa like lasI, lasR, rhlI, rhl .

Essential Oils
Both the Melaleuca alternifolia and Salvia sclarea EOs were purchased from Moksha Lifestyle products. The manufacturer provided its certificate of analysis, and GC-MS report containing the list of chemical compounds. Being hydrophobic, they were dissolved in DMSO, purchased from SRL, India.

Microorganisms and Culture Conditions
Pseudomonas aeruginosa PA01 was purchased from the MTCC. Chromobacterium violaceum CV026, a mini-Tn5 mutant (ATCC31352), was purchased from CTET, Spain, and was grown in Luria-Bertani (LB) broth at 28 °C broth in the presence of kanamycin (25 μg/ml). N-hexanoylhomoserine lactone (HHL) was added to the CV026 culture when we needed to screen EOs for anti-QS activity. It was purchased from Sigma-Aldrich. Dr. Peter Greenberg (University of Washington), USA, gave a reporter strain of E. coli pJN105LpSC11 that was gentamycinand ampicillinresistant. The concentration of gentamycin and ampicillin that was used to culture E. coli pJN105LpSC11 was 10 μg/ml and 100 μg/ml respectively. All these bacterial cultures were grown overnight in an incubator at 120 rpm.

Antimicrobial Activity
The antimicrobial activity of EOs was tested against Pseudomonas aeruginosa PA01. Briefly, the bacteria were grown overnight at 37 °C, 120 rpm and used for the experiment when its OD reached 0.5 at 600 nm. Then the culture was spread uniformly on the LBA plates, and the wells were punched into which 100 μl of different concentrations of EOs was pipetted. Then the plates were kept in an incubator for overnight at 37 °C. The antimicrobial potential of both Salvia sclarea and Melaleuca alternifolia EOs was measured by the diameter of the zone of inhibition (ZOI) around the well.

Calculation Of Minimum Inhibitory Concentration (MIC) of EOs
Micro-broth dilution method was used to determine the MIC of Salvia sclarea and Melaleuca alternifolia EO against PA01 [1] . The culture was grown overnight, which, when it reached an OD of 1 at 600 nm, was mixed with new media in a 96-well plate, and allowed to grow in the presence of different concentrations of EOs. The plate was incubated overnight at 37 °C, and the minimum concentration that inhibited the growth of bacteria completely was taken as the MIC of that EO.

Growth Curve Analysis of EOs
It was done to find the sub-lethal concentration of EOs. In brief, the overnight grown PA01 culture (OD 600nm = 1) was inoculated into fresh media containing different concentrations of EOs for 24 h; the plate was kept in an incubator at 37 °C, and OD was taken regularly at an interval of 2 h.

Qualitative Screening for QS Inhibition by Essential Oils
The anti-QS activity of EOs was determined by a bioreporter strain, Chromobacter violaceum CV026. Briefly, LB agar plates were made that contain hexanoyl homoserine lactone (0.125 μg/ml) and antibiotic kanamycin (20 μg/ml), and 100 μl of an overnight grown culture of C. violaceum was spread on the plate uniformly, and 100 μl of EO was added into the wells, and the plates were kept overnight at 28 °C and observed for colorless colonies [2] .

Quantitative Estimation of 3-oxo-C 12 -HSL
To assess the effect of EOs on the level of autoinducer 3-oxo-C 12 -HSL, reporter strain E coli DH5 α pJN105LpSC11 was cultured overnight in LB media containing ampicillin and gentamycin.
Then this culture was inoculated into fresh LB media, and at OD 600nm 0.2, LasR expression was induced by 0.2% (w/v) l -arabinose and left for incubation until the OD 600nm reached 0.5. The treated culture was centrifuged for 10 min at 10,0 0 0 rpm, and the supernatant was filtered and mixed with 1:10 0 0 dilution of E coli DH5 α pJN105LpSC11 and kept for incubation at 37 °C for 2 h. The concentration of 3-oxo-C 12 -HSL was then determined by miller assay [3 , 4] .

Pyocyanin Production
PA01 culture was grown in the presence of EOs individually. The culture was then centrifuged, and the supernatant was separated and filtered. Chloroform (1:1 vol) was used to extract pyocyanin and, the brownish upper part was discarded, the blue-colored pyocyanin pigment was visible, which was further extracted by 0.2 N HCL, and yielded pink color. The OD was measured at 520 nm [5] .

Protease Assay
Protease production by P. aeruginosa was estimated by skim milk agar assay. The PA01 culture was treated overnight with EOs and then centrifuged at 10,0 0 0 rpm for 10 min; the supernatant was then filter sterilized. Next, 100 μl of this supernatant was added to wells cut on a skim milk agar plate. This plate was then incubated overnight at 37 °C, and the zone of clearance around each well was observed [6] .

Swarming Motility Assay
Briefly, swarm agar plates containing yeast extract, bacto agar (0.4% (w/v)), bacto peptone, and glucose were made and its pH was maintained at 7.0. P. aeruginosa culture was treated overnight in the presence of EOs. 10 μl of overnight culture was pipetted onto the middle of the swarm plate, and these plates were kept in an incubator at 37 °C for overnight [7] .

Alginate Assay
PA01 culture was grown overnight and inoculated in fresh media in the presence and absence of EOs, 500 μl of 1 M NaCl was mixed with an equal volume of treated culture and subjected to centrifugation. Then, 500 μl of 0.1% (w/v) cetylpyridinium chloride (CPC) was mixed into the supernatant, and the tubes were centrifuged at 10,0 0 0 rpm for 10 min at room temperature. Then 500 μl chilled isopropanol was used to re-suspend the pellet. After that, the tube was centrifuged again, and the supernatant was discarded; then, 1 M NaCl was added to the tube to re-suspend the pellet, which was left overnight. Carbazole assay was further carried out to estimate the alginate concentration in culture [8] . Briefly, 50 μl of sample was added to 200 μl of 25 μM sodium tetraborate (in H 2 SO 4 ), the plate was then heated for 10 min at 100 °C in an oven. After cooling at room temperature, we added 50 μl of 0.1% carbazole (in absolute ethanol) to it and then again heated the plate for 15 min at 100 °C. The final absorbance was then taken at 550 nm.

Inhibition of Biofilm Formation by EOs
P. aeruginosa PA01 culture was adjusted to OD 600nm = 1 and then inoculated into fresh LB media, and 100 μl of this culture was poured into individual wells of a 96-well microtiter plate. Then the sub-lethal concentration of EOs was mixed with the culture, and the plate was kept overnight at 37 °C. Fresh LB media was prepared and poured into a new 96-well plate, and 1 μl of culture was dispensed into wells. The culture in the wells was pipetted out, and phosphate buffer saline (PBS) was used to wash the wells. Next, the wells were stained for 20 min with crystal violet dye (0.4% (w/v)). The dye was discarded, and the wells were rinsed thrice with distilled water till all the unbound dye was removed from the wells. Next, the stain was solubilized by adding 100 μl DMSO, and the optical density (OD) of the wells was taken at 590 nm and 600 nm [9 , 10] .

Gene Expression Analysis Through RT-QPCR
In brief, the bacterial culture was treated with EOs, and mRNA was extracted from it, which was further used to synthesize cDNA using RevertAid First Strand cDNA synthesis kit. The reaction mixture included 5 μl SYBR green, 500 ng cDNA, 10 μM forward and reverse primers, and 2 μl distilled water. The plate was then subjected to real-time PCR analysis, and the reaction was completed in 2 steps; the first step is initial denaturation for 30 s, while the second step included denaturation for 60 s and annealing for 30 s at 60 °C; the cycle was repeated 40 times. The results obtained from RT-PCR were in the form of C t values, which were used to calculate fold change with the help of C t method. The result was reported in the form of relative gene expression [11] .

Statistical Analysis
All the experiments in this study were conducted thrice, and the results are expressed in terms of average values and their standard mean error. The significance of the values was checked by one-way analysis of variance (ANOVA) using GraphPadPrism, and only values at p < 0.05 were taken to be significant.

Ethics Statements
This work does not involve any human subject or any trial on animal. Social media was not used to collect any data. This data article adheres to all the ethics necessary for publishing.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability
Data regarding anti-quorum sensing and antimicrobial activity of Melaleuca alternifolia and Salvia sclarea essential oil against Pseudomonas aeruginosa (Original data) (Mendeley Data).