Bacterial strain and growth conditions
Methicillin resistant Staphylococcus aureus (ATCC 33591) was purchased from ATCC, Himedia, India and clinical isolate strains MRSAvk9 (GenBank accession number MK757651) and MRSAvk10 (GenBank accession number MK757652) was used in the present study. The strains were streaked on tryptone soya agar (TSA) plates and incubated at 37° C for 24 h. A single isolated colony was picked and cultured in 3 ml of tryptone soya broth supplemented with 1 % of sucrose (TSBS) for enhancing the biofilm formation, incubated at 37° C for 24 h.
Hesperidin was purchased from Sigma-Aldrich (India) and prepared as 10 mg/ml stock solution in methanol and stored at 4° C.
Determination of biofilm inhibitory concentration (BIC)
Biofilm inhibitory concentration (MBIC) of hesperidin against MRSA was assessed by crystal violet (CV) quantification assay was performed in 24 well polystyrene plate as performed earlier (Karuppiah and Thirunanasambandham 2020). Briefly, 1 % of MRSA culture was added to 1 ml of TSBS along with hesperidin at increasing concentrations ranging from 25 to 150 µg/ml. The wells containing 2 ml of TSBS, 1% of overnight MRSA culture with 10 µl of methanol was regarded as vehicle control to evaluate the effect of solvent on MRSA. The wells contain 2 ml of TSBS without hesperidin and 2 ml of TSBS were used as control and blank. The plates were incubated at 37° C for 24 h. After incubation, the planktonic cells were removed and washed twice with sterile distilled water and allow to air dry. The biofilm attached cells were stained with CV (4 % w/v) solution for 10 min and washed twice with sterile distilled water and allow to air dry. Then, the stained biofilms were eluted by 1 ml of 20 % glacial acetic acid. The biofilm biomass was measured using a UV-visible spectrophotometer at 570 nm.
To validate the hesperidin antibiofilm potential against MRSA was assessed by microscopic analysis. Briefly, 1 % of overnight MRSA (ATCC-33591), clinical isolates MRSAvk9 and MRSAvk10 were inoculated into 1 ml of TSBS medium in the 24 well plate, inserting 1 X 1cm glass slides with presence and absence of hesperidin at 100 µg/ml concentrations and allowed to grow for 24 h at 37° C. After incubation, the slides were washed with sterile distilled water to remove debris of medium. For light microscopy analysis, slides were stained with 0.4 % crystal violet (CV) solution and observed at 400X under the light microscope. In confocal laser scanning microscope (CLSM), slides were stained with 0.1 % of acridine orange solution and observed at 200X in CLSM (Karuppiah and Thirunanasambandham 2020).
Growth curve analysis
To evaluate the effect of hesperidin on the growth of MRSA (ATCC-33591), clinical isolates MRSAvk9 and MRSAvk10, 100 ml of TSBS broth was inoculated with 1 % inoculum of MRSA (1.6 x 107 CFU ml-1) in the absence and presence of hesperidin (at its BIC). Initial OD (0 h) was measured at 600 nm and the cultures were kept at 37° C. OD values were taken at 1 h interval for 24 h and CFU/ml was calculated at 3 h interval for 24 h and the growth curve was plotted as OD against time interval along with CFU/ml (Vijayakumar et al. 2020).
Cell viability assay
Further, the cell viability of MRSAs were quantified using the XTT (2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) reduction assay. Briefly, the XTT sodium salt and menadione were dissolved in sterile PBS (1 mg/ml) and acetone (1mM) respectively. For each experiment, XTT –menadione solution was prepared freshly at the ratio of 12.5.1 respectively. After incubation, both biofilm and planktonic cells were collectively removed from the hesperidin treated and untreated wells. The cells were washed twice with sterile PBS and resuspended in the 175 µl PBS with 25 µl of XTT-menadione solution. This mixture was incubated in dark at 37° C for 8 h. The viability of tested strains was measured spectrophotometrically by the reduction of XTT-menadione into the orange colored formation at 490 nm (Vijayakumar et al. 2020).
MRSA was grown in test tubes containing 10 ml of TSBS with presence and absence of hesperidin (at BIC) for 24 h at 37° C. After incubation, the cell pellets were harvested by centrifugation at 8000 rpm for 10 min and washed thrice with PBS. Over, the pellet was resuspended with 10 ml of PBS and kept undisturbed for 24 h. After incubation, 100 µl of cell suspensions were collected from the upper portion of test tubes, the cell density was measured at 600 nm for every 3 h interval up to 24 h (Sorroche et al. 2012).
The effect of hesperidin on autolysis of MRSA was evaluated by separating pellets from treated and untreated hesperidin samples. The collected cell pellets were washed thrice with ice-cold PBS and resuspended in auolytic buffer (0.05 % Triton x-100 and 0.05 M Tris-HCL in PBS) and incubated at 30° C. The aliquots were measured at 600 nm for every 30 min interval up to 3 h (Cue et al. 2015).
Effect of hesperidin on lipase production in MRSA was assayed using the method. Briefly, 100 µl of cell free culture supernatant (CFCS) from control and treated MRSA cultures were mixed with 900 µl of lipase substrate buffer containing 1: 9 volume of 0.3 % p- nitro phenyl palmitate in isopropanol and 50mM Na2PO4 buffer (pH 7.0) containing 0.2 % sodium deoxycholate and 0.1 % gumini arabicum and incubated at room temperature in dark for 1h. Following incubation, lipase activity was terminated by adding equal volume of 1M Na2CO3 to the reaction mixture and centrifuged at 12,000 g for 10 min. The absorbance of the supernatant was measured at 410 nm and results are expressed as a percentage of lipase inhibition (Sethupathy et al. 2017).
Hemolysin quantification assay
The efficacy of hesperidin on hemolysin was measured by microdilution method as described earlier (Hollands et al. 2008). Briefly, freshly collected sheep blood was diluted with BHI medium to a final concentration of 2 % (v/v) and divided into three aliquots. The first aliquot, 10 % of standard cell suspension of MRSA was added. To the second aliquot, 10 % of standard cell suspension and hesperidin (at its BIC) were added. The hesperidin alone at its BIC was added to the last aliquot and this was considered as blank. All the tubes were incubated at 37° C for 1 h and subsequently incubated at 4° C for 1 h. After incubation, the tubes were centrifuged at 5000 rpm for 5 min and the absorbance of supernatants was measured at 405 nm.
Staphyloxanthin quantification assay
To evaluate the effect of hesperidin on carotenoid pigment production in MRSA was assessed by previous described method. Briefly, MRSA was grown in 100 ml of TSBS along with hesperidin at 37° C for 24 h at 160 rpm. After incubation, the control and treated samples were centrifuged at 8000 rpm for 10 min, collected the pellets and washed twice with PBS. The pellets were taken for methanolic extraction by resuspending the pellets into methanol and kept in a shaker for 24 h. After incubation, the samples were centrifuged at 10,000 rpm for 10 min and the supernatant was measured at 465 nm (Leejae et al. 2013).
H2O2 sensitivity assay
To determine the sensitivity of MRSA cells on H2O2 in the presence and absence of 5HMF the method was followed described earlier (Valliammai et al. 2019). Briefly, control and hesperidin treated cell suspensions were centrifuged at 8000 x g for 10 min and pellets were resuspended in PBS along with 1 mM H2O2 and incubated at 37° C for 1 h. After incubation, the cells were serially diluted and spread on the TSA plates and incubated at 37° C for 24 h. After incubation, the viable cells were counted manually.
RNA extraction, cDNA conversion and real-time PCR
To evaluate the effect of hesperidin on selected genes responsible for biofilm formation, staphyloxanthin synthesis and adhesion associated genes in MRSA such as sarA, crtM, icaA, icaD, fnbA, fnbB and altA. Total RNA was isolated from hesperidin treated and an untreated sample by the Trizol method and cDNA was converted by using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). As per the manufacturers protocol, the SYBR Green PCR master mix kit (Applied Biosystems) was used to perform qPCR analysis. After normalizing the housekeeping gene expression (gyrB), selected gene expressions were calculated using 2-ΔΔCt values (Chemmugil et al. 2019). The primer sequences of tested genes are listed in Table. 1.
Molecular docking study
To evaluate the hesperidin antibiofilm activity against MRSA, molecular studies were done with SarA protein and CrtM protein which have been reported as major regulator mediated with biofilm formation and staphyloxanthin production in MRSA. Therefore, SarA (ID: 2FNP) and CrtM (ID: 2ZCO) crystal structure was retrived from Protein Data Ban (PDB). Then, the 3D structure of target compound hesperidin (PubChem ID: 10621) were obtained from PubChem database. Autodock Tools v1.5.6 was used to assess the interactions between hesperidin, SarA and CrtM of MRSA. Moreover, BIOVIA Discovery Studio visualizer 2016 v184.108.40.20650 was used to visualize docked complexes (Selvaraj et al. 2019).
Each experiment was performed in triplicate to obtain statistical confidence. Data values of experimental results were recorded as the mean ± Standard Deviation. The significance was determined by using one way ANOVA as
P-value < 0.05 (indicate*). Statistical analyses were performed using SPSS 20.0 statistical software.