Bacterial strains and growth conditions
Streptococcus mutans UA159 (reference strain) from American Type Culture Collection (ATCC 700610) was used in the study. It was maintained on tryptone soya agar (TSA) and cultured regularly in tryptone soya broth (TSB) at 37° C for 24 h. The standard cell suspension of S. mutans for all in vitro experiments was prepared by adjusting the optical density (OD) of overnight culture of 0.4 at 600 nm (1 x 108 CFU/ml) using TSB. Note: In all the assays performed in TSB supplemented with 1 % sucrose.
Vaccenic acid, glutamic acid, sodium oleate, 4-phenyl butyric acid and 3, 5-di-tert-butylphenol (3, 5-DTBP) were dissolved in methanol (10 mg/ml). The aliquots were filtered (using 0.25 µm pore filter sterilized) and used for antibiofilm assay. All the compounds were purchased from Sigma Aldrich (USA) with maximum purity. Methanol was used as vehicle control in all the assays were 3, 5-DTBP was used.
Screening of phytochemicals antibiofilm efficacy against S. mutans
Totally five phytochemicals were screened for their antibiofilm activity using 24 well sterile microtitre plates. Each well contains 1 ml of TSB (supplemented with 1 % sucrose) with 1 % of overnight culture bacterial culture (1 x 108 CFU/ml). For screening, 100 µg/ml of each compound was loaded and assay plate was kept at 37° C for 24 h. After incubation, OD at 600 nm was measured. Then planktonic cells were discarded and each well was washed gentle with sterile distilled water to eliminate loosely bound cells and then the plate was allow to air dried. For quantification, biofilm cells were stained with 0.4 % of crystal violet (CV) for 10 min and excess stain was removed by washing plates thrice with distilled water and air dried for 5 min. Biofilms cells were destained using 20 % glacial acetic acid and OD was read at 570 nm (Vijayakumar and Ramanathan 2020a).
Determination Biofilm inhibitory concentration (BIC) of 3, 5-DTBP
To determine the BIC of 3, 5-DTBP, 24 well MTP assay was performed as mentioned above with various concentrations of 3, 5-DTBP ranging from 20, 40, 60, 80, 100 and 120 µg/ml. Well containing TSB (supplemented with 1 % sucrose) + S. mutans + methanol were maintained as control. The absorbance of control and treated wells was measured at 570 nm.
Microscopic analysis of biofilm
In microscopic analysis, bacterial cells were allowed to form biofilm on glass slides (1 x 1 cm) in the presence and absence of 3, 5-DTBP for 24 h at 37° C. After incubation, the slides were washed with sterile distilled water and stained as needed. For light microscopic analysis, the washed slides were stained with 0.4 % CV and washed to remove excess stain and allow to air dried. Then, the glass slides were visualized at magnification of 400X under light microscope. For confocal laser scanning microscope (CLSM) analysis, the washed slides were stained with 0.1 % of acridine orange for 10 min at dark followed by washing to remove the excess stain and drying. Finally, the glass slides were visualized under CLSM at magnification of 200X (Vijayakumar et al. 2021).
Effect of 3, 5-DTBP on adherence to glass surface
The effect of 3, 5-DTBP on the sucrose-dependent and sucrose independent adherence of S. mutans over the smooth glass surface was performed as described earlier (Hasan et al. 2014). Briefly, S. mutans was grown anaerobically at 37° C at an angle of 30° in boiling test tubes containing 10 ml of TSB with or without 5 % sucrose in the presence and absence of 3, 5-DTBP and incubated for 24 h. After incubation, the planktonic cell suspension was removed and the adhering cells were washed by adding 0.5 M of sodium hydroxide followed by agitation. Then, the adhering cells were washed and suspended in PBS saline and the adherence was quantified using a spectrophotometer at OD 600 nm. The percentage of adherence was calculated according to the formula:
Adherence (%) = (OD of adhered cells/OD of total cells) x 100
Effect of 3, 5-DTBP on cell surface hydrophobicity (CSH) of S. mutans
To evaluate the effect of 3, 5-DTBP on the CSH of S. mutans was performed by microbial adhesion to hydrocarbon (MATH) assay as described earlier with slight modifications (Khan et al. 2010). Briefly, overnight cultures grown in TSB in the presence and absence of 3, 5-DTBP were washed and suspended with sterile saline (0. 85 %) and their optical density (OD) as 0.3 at 600 nm. The cell suspension (3 ml) was taken to the test tubes containing 0.25 ml of toluene. Then, the tubes were agitated uniformly for 2 min and let to equilibrate for 10 min at room temperature. After separation of the toluene phase from the aqueous phase, the OD of the aqueous phase was measured using a spectrophotometer at 600 nm. The percentage of hydrophobicity was calculated according to the formula:
% of hydrophobicity = [Initial OD600nm – Final OD600nm/initial OD600nm] x 100.
Estimation of water soluble and water insoluble glucans
The standard phenol/sulfuric acid estimation method was performed to quantify the water soluble and water insoluble glucans as described earlier (Yano et al. 2020). Briefly, S. mutans was cultured in TSB (supplemented with 1 % sucrose) in the presence and absence of 3, 5-DTBP and incubated anaerobically for 24 h at 37° C. After incubation, the cell free culture supernatant (CFCS) was harvested by centrifugation. The supernatant was subjected to ethanol precipitation to obtain extracellular water soluble glucans. The remaining cell pellet was resuspended in 1M NaOH and centrifuged to remove the bacterial cell and, the water insoluble (alkali-soluble) glucan was recovered by ethanol precipitation of the harvested supernatant.
Glycolysis pH drop assay
The level of the glycolysis pH drop of S.mutans was measured as earlier described method (Ban et al. 2012). The cells of S.mutans from the suspension cultures were centrifuged at 15000 g, washed once with salt solution (50 mM KCl and 1 mM MgCl2 ) and resuspended in a salt solution with the presence and absence of 3, 5-DTBP (100µg/ml). The pH was adjusted to 7.2 to 7.4 with 0.2 M KOH solution. Glucose was then added to obtain a concentration of 1% (w/v) and the decrease in pH was assessed over a period of 60 min.
Terminal pH determination assay
To analyze the effect of 3, 5-DTBP on acid production, terminate pH assay was performed. Briefly, an overnight culture of S.mutans was inoculated with TSB (pH 7.4), with presence and absence of 3, 5-DTBP and incubated at 37° C for 24 hours. The pH of bacterial broth was determined at the before and after 24 h incubation. The changes in pH were recorded (Korithoski et al. 2007).
Lactic acid measurement
Briefly, the cells of S. mutans were harvested, washed twice with PBS and resuspended to a final concentration of 1 x 108 CFU/ml in a 24 well plate with 1.5 ml buffered peptone water (BPW) supplemented with 0.2 % sucrose containing BIC of 3, 5-DTBP in each well and incubated for 120 min at 37° C. After incubation, planktonic cells were removed by centrifugation (8,000g, 5 min, 4° C), the supernatant (50 µl) was added to 2 ml of 0.2 % iron (III) chloride, stirred and absorbance was measured at 390 nm (Borshchevskaya et al. 2016).
Lactate dehydrogenase assay
To evaluate the lactate dehydrogenase assay (LDH) activity, the overnight bacterial culture (OD600 = 0.8) was incubated at 37° C with Tris – HCl buffer, pH 7.0 containing 0.5 mg/ml of lysozyme for 1 h. After the incubation, the cell free supernatant (CFS) was collected from centrifugation at 10000g for 20 min at 4° C. Furthermore, the CFS was dialyzed with 10 mM phoaphate buffer saline (PBS) (pH 7.0) at 4° C for overnight. The dialyzed crude LDH protein content was measured by the Bradford method (Bradford 1976). After that, the LDH activity was determined by the previous method described by (Crow and Pritchard 1977). The crude LDH was pretreated with 3, 5-DTBP (at 100 µg/ml) at 30 min at room temperature, and the LDH activity was estimated by measuring the rate of nicotinamide adenine dinucleotide (NADH) oxidation at 340 nm. The standard reaction mixture (for 200 µg/ml) contained 180 µl of 50 mM PBS pH 7.0 with 0.167 mM NADH and 10 mM sodium pyruvate; 10 µl of 1 mM fructose 1, 6-diphosphate (FDP) and 10 µl of pretreated LDH. The results were expressed as enzymatic activity compared to that of the control.
Enolase activity assay
To determine the enolase activity with permeabilized cells of S.mutans were measured by a method described earlier (Belli et al. 1995). Permeabilized cells were pretreated with 100 µg/ml concentration of 3, 5-DTBP for 30 min at room temperature. The enolase activity was observed by the formation of phosphoenolpyruvate (PEP) at 240 nm. The standard reaction mixture (for 200 µl) contained 180 µl of 20 mM KPO4 buffer, pH 6.5 with 2 mM MgSO4; 10 µl of 17.6 mM D- (+)- 2- phosphoglycerate and 10 µl of permeabilized cells already treated with the test concentration of the compounds. The results were expressed as enzymatic activity compared to that of the control.
Acid tolerance assay
Acid tolerance ability of S. mutans in the presence and absence of 3, 5-DTBP, was assessed by acid tolerance assay. S. mutans was grown till mid logarithmic phase and pelleted by centrifugation. The pellet was divided into two aliquots, one aliquot was directly resuspended in TSB at a killing pH of 3.5 and incubated at 37° C for 2 h. These cells were considered as unadapted cells. Then, the other aliquot was first suspended in TSB at a pH of 5.5 and incubated at 37° C for 1 h to ease adaptation. The resulting adapted cells were then transferred to TSB at a lethal pH of 3.5 and incubated for 2 h at 37° C. Cell fractions were removed from these unadapted and adapted cultures at regular intervals (at 0, 1 and 2 h) and their viability was tested by TSB agar plates at favorable pH (pH 7.5) for 48 h at 37° C (Viszwapriya et al. 2017).
F1F0 – ATPase activity assay
The F1F0 – ATPase activity was determined by using permeabilized cells of S.mutans by adding the cells to 10 % toluene followed by series of freezing and thawing as described earlier method. The F1F0 – ATPase activity was evaluated by the release of inorganic phosphate in the following reaction mixture 75mM of Tris – maleate buffer (pH 7.0) containing 5mM adenosine – 5 – triphosphate (ATP), 10mM of MgCl2, permeabilized cells along with BIC concentration of 3, 5-DTBP. The release phosphate was determined as previously described method (Bencini et al. 1983).
Auto aggregation assay
S.mutans was grown in the presence and absence of 3, 5-DTBP (100 µg/ml) and incubated at 37° C for 24 h. The bacterial suspensions were then centrifuged and the pellet was resuspended in PBS (pH 7.4). The suspensions were kept undisturbed and visually observed for aggregation at the interval of 10 min to 30 min. After incubation, 200µl of the upper layer was read at 600 nm (Sorroche et al. 2012). The percentage of autoaggregation was calculated using the formula
% aggragation = [(ODinitial – ODfinal)/ODinitial)] x 100
H2O2 sensitivity assay
S. mutans survival ability was analyzed using H2O2 sensitivity assay as reported earlier. Briefly, S. mutans control and 3, 5-DTBP treated cells were centrifuged at 8000 rpm for 10 min and pellets were suspended in 1ml of phosphate buffered saline (PBS), subsequently treated with 0.2 % of H2O2 and incubated at 37° C for 3 h and then plated on TSB agar and incubated at overnight, to analyze the viable bacterial cells. After incubation, colonies formed were counted to plot the graph (Van Sorge et al. 2013).
Growth curve and viability of S. mutans
To evaluate the effect of 3, 5-DTBP on growth of S. mutans, 100 ml of TSB was inoculated with 1 % of overnight culture of S. mutans in the presence and absence of BIC of 3, 5-DTBP. The zero hour OD was measured at 600 nm and the culture was incubated at 37° C. The OD values were taken at 3 h interval for 24 h and the growth curve was plotted as OD against time interval (Subramenium et al. 2015).
Further, the cell viability of S. mutans 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 3, 5-DTBP 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. 2020b).
RNA extraction and cDNA preparation
The total RNA of S. mutans grown in the presence and absence of 3, 5-DTBP for 24 h was isolated using a method described earlier (Oh and So 2003). The isolated RNA samples were reversed transcribed using High capacity cDNA reverse transcription kit (Applied Biosystems) as per the manufacturer’s instructions. Then cDNA samples were used for subsequent real-time polymerase chain reaction (qRT-PCR) analysis.
Real-Time PCR (qRT- PCR) analysis
The qRT- PCR was performed using 7500 thermal cycler system (Applied Biosystems, USA) with the SYBR Green chemistry kit (Applied Biosystems, USA) following the manufacturer’s instructions. The oligonucleotide primers were designed with Primer3 software and are listed in Table. 1. The cycling parameters were as follows: initial denaturation of 5 min at 95° C, followed by 40 cycles of denaturation at 95° C for 30 s, annealing at 56° C for 30 s, extension at 72° C for 30 s and a final extension at 72° C for 1 min. The data was acquired at each cyclic extension step. All the experiments were performed and analyzed in triplicate. The 16S rRNA was used as calibrator to normalize the gene expression. The relative expression levels of target genes were calculated using the 2-ΔΔCt method (Livak and Schmittgen 2001).
All the experiments were performed in triplicate at least three times. The data were expressed as mean ± standard deviation (SD). One way ANOVA, 5-DTBP was performed using SPSS 20.0 software (SPSS, Inc., Chicago, IL, USA). Results with *P <0.05 and was indicated statistically significant.