Anti-Bacterial and Anti-Biofilm Activities of Anandamide against the Cariogenic Streptococcus mutans

Abstract

Streptococcus mutans is a cariogenic bacterium in the oral cavity involved in plaque formation and dental caries. The endocannabinoid anandamide (AEA), a naturally occurring bioactive lipid, has been shown to have anti-bacterial and anti-biofilm activities against Staphylococcus aureus. We aimed here to study its effects on S. mutans viability, biofilm formation and extracellular polysaccharide substance (EPS) production. S. mutans were cultivated in the absence or presence of various concentrations of AEA, and the planktonic growth was followed by changes in optical density (OD) and colony-forming units (CFU). The resulting biofilms were examined by MTT metabolic assay, Crystal Violet (CV) staining, spinning disk confocal microscopy (SDCM) and high-resolution scanning electron microscopy (HR-SEM). The EPS production was determined by Congo Red and fluorescent dextran staining. Membrane potential and membrane permeability were determined by diethyloxacarbocyanine iodide (DiOC2(3)) and SYTO 9/propidium iodide (PI) staining, respectively, using flow cytometry. We observed that AEA was bactericidal to S. mutans at 12.5 µg/mL and prevented biofilm formation at the same concentration. AEA reduced the biofilm thickness and biomass with concomitant reduction in total EPS production, although there was a net increase in EPS per bacterium. Preformed biofilms were significantly affected at 50 µg/mL AEA. We further show that AEA increased the membrane permeability and induced membrane hyperpolarization of these bacteria. AEA caused S. mutans to become elongated at the minimum inhibitory concentration (MIC). Gene expression studies showed a significant increase in the cell division gene ftsZ. The concentrations of AEA needed for the anti-bacterial effects were below the cytotoxic concentration for normal Vero epithelial cells. Altogether, our data show that AEA has anti-bacterial and anti-biofilm activities against S. mutans and may have a potential role in preventing biofilms as a therapeutic measure.

Anti-bacterial and anti-biofilm activities of arachidonic acid against the cariogenic bacterium Streptococcus mutans

“…Streptococcus mutans UA159 (ATCC 700610), a clinical isolate from a child with active caries, was cultivated in brain-heart infusion (BHI) broth (HiMedia Laboratories Pvt. Ltd., Maharashtra, India) for planktonic growth and in BHI supplemented with 2% sucrose (BHIS) for biofilm formation ( Wolfson et al, 2023 ). The day before the experiment, 100 μl of a frozen bacterial stock was inoculated in 10 ml BHI and cultured in a humidified incubator supplemented with 5% CO 2 .…”

“…For kinetic studies, the OD 600 nm was measured every 30 min at 37°C in a M200 infinite plate reader for 20 h. For checkerboard assay, the bacteria were incubated in various combinations of the two compounds or equal amount of ethanol. Colony forming units (CFUs) were determined at different time points by taking 10 μl of each sample for 10-fold serial dilutions in BHI after vigorous vortexing, and seeding 100 μl of the dilutions onto BHI agar plates, which were incubated for 24 h ( Wolfson et al, 2023 ). The CFU per ml was calculated by multiplying the number of colonies with the dilution factor multiplied with 10 (as 100 μl was seeded out of 1 ml).…”

“…For biofilm formation, S. mutans at an initial OD 600nm of 0.1 was incubated in 200 μl BHI with 2% sucrose (BHIS) in the presence of various concentrations of AA for 24 h in a 96 flat-bottomed tissue culture grade plate at 37°C in a humidified incubator supplemented with 5% CO 2 . At the end of incubation, the biofilms were washed twice with phosphate buffered saline (PBS), and either stained with 0.25% crystal violet in ddw (diluted 1:4 from 1% Gram crystal violet (CV) solution, Merck KGaH, Darmstadt, Germany) for 20 min at room temperature, or the metabolic activity was measured by adding 50 μl of a 0.5 mg/ml MTT solution in PBS followed by a 1 h incubation at 37°C as described ( Wolfson et al, 2023 ). The CV stained biofilms were washed twice with DDW, and the absorbance read at 595 nm in a M200 infinite plate reader.…”

“…For analyzing alterations in membrane potential, control and AA-treated bacteria were resuspended in 1 ml of PBS containing 30 μM of the potentiometric dye DiOC2(3) (BacLight Membrane Potential Kit, Molecular Probes, Life Technologies, Eugene, OR, USA) ( Wolfson et al, 2023 ). The fluorescence intensities were measured in a Fortezza flow cytometer using the excitation/emission of 488 nm/530 nm for green fluorescence and 488 nm/620 nm for red fluorescence.…”

“…Biofilms formed in the presence or absence of AA were stained with 3.3 μM SYTO 9 and 10 μg/ml PI in PBS for 20 min at room temperature, washed in PBS and fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) for 30 min at room temperature and then mounted with 50% glycerol before visualization by a spinning disk confocal microscope (Nikon Corporation, Tokyo, Japan) using the 50 μm pinhole, and the 488 nm filter for SYTO 9 and the 561 nm filter for PI ( Wolfson et al, 2023 ). Images were taken at intervals of 2.5 μm from the bottom to the top of the biofilm.…”