Anti-Bacterial Properties of Cannabigerol Toward Streptococcus mutans

Abstract

Streptococcus mutans (S. mutans) is a gram-positive facultatively anaerobic bacterium and the most common pathogen associated with tooth caries. The organism is acid tolerant and can undergo physiological adaptation to function effectively in acid environments such as carious dental plaque. Some cannabinoids have been found to have potent anti-microbial activity against gram-positive bacteria. One of these is the non-psychoactive, minor phytocannabinoid Cannabigerol (CBG). Here we show that CBG exhibits anti-bacterial activities against S. mutans. CBG halts the proliferation of planktonic growing S. mutans, which is affected by the initial cell density. High-resolution scanning electron microscopy showed that the CBG-treated bacteria become swollen with altered membrane structures. Transmission electron microscopy provided data showing that CBG treatment leads to intracellular accumulation of membrane structures. Nile red, DiOC2(3) and laurdan staining demonstrated that CBG alters the membrane properties, induces membrane hyperpolarization, and decreases the membrane fluidity. CBG-treated bacteria showed increased propidium iodide uptake and reduced calcein AM staining, suggesting that CBG increases the membrane permeability and reduces the metabolic activity. Furthermore, CBG prevented the drop in pH caused by the bacteria. In summary, we present here data showing the mechanisms by which CBG exerts its anti-bacterial effect against S. mutans.

Keywords: Cannabigerol; Streptococcus mutans; bacteriostasis; dental caries; phytocannabinoids.

FIGURE 1

CBG halts the proliferation of planktonic growing S. mutans. (A) The viability of S. mutans after a 24 h incubation with increasing doses of CBG (0–10 μg/ml) as measured by OD_595nm. n = 3; *p < 0.05. (B–D) A kinetic study of the planktonic growth of S. mutans in the presence of increasing concentrations of CBG (0–10 μg/ml) at starting OD_600nm = 0.1 (B), 0.2 (C), and 0.4 (D). (E) The colony forming units of untreated and CBG (0–10 μg/ml)-treated bacteria at various time points. n = 3.

FIGURE 2

CBG alters the membrane structure and the size of S. mutans. (A) HR-SEM images (x50000) of control bacteria or bacteria treated with different concentrations of CBG (0–10 μg/ml) for 4 h. (B) The average length of untreated and CBG (0–10 μg/ml)-treated bacteria. n = 200; *p < 0.05. (C) The average width of untreated and CBG (0–10 μg/ml)-treated bacteria. n = 200; *p < 0.05.

FIGURE 3

CBG alters the morphology of S. mutans. (A,B) Panoramic TEM images (x9700) of control (A) and 4 h CBG (10 μg/ml)-treated (B) bacteria. The arrows point to the nucleoids, the electron-dense and electron-lucent areas, and the invagination septum in control and CBG-treated S. mutans.

FIGURE 4

CBG alters the morphology of S. mutans. Higher magnification TEM images (x59000) of control (A,C,E,G) and 4 h CBG (10 μg/ml)-treated (B,D,F,H) S. mutans. The arrows show the cell wall (CW), cell membrane (PM), nucleus (N), the invagination septum (BF-Binary fission), and the mesosome-like structures.

FIGURE 5

The effect of CBG on the ATP levels in S. mutans. (A) The percentage ATP levels in S. mutans treated with various concentrations of CBG (0–20 μg/ml) for 2 h in comparison to control. n = 3; *P < 0.05. (B) The percentage of the ATP/OD_595nm normalized levels in S. mutans treated with various concentrations of CBG (0–20 μg/ml) for 2 h in comparison to control. n = 3; *p < 0.05.

FIGURE 6

CBG alters the membrane properties of S. mutans. (A,B) The fluorescence intensity of Nile Red membrane staining of control and 2 h CBG (0–10 μg/ml)-treated bacteria as determined by flow cytometry. (C,D) The fluorescence intensity of DAPI staining of control and 2 h CBG (0–10 μg/ml)-treated bacteria as determined by flow cytometry. (E,F) Flow cytometry of PI-stained S. mutans that have been treated with different CBG concentrations (0–10 μg/ml) for 2 h. (G,H) Flow cytometry of Calcein AM-stained S. mutans that have been treated with different CBG concentrations (0–10 μg/ml) for 2 h. (A,C,E,G) are the histograms of flow cytometry. (B,D,F,H) present the geometric mean of the flow cytometry data as a function of CBG concentration.

FIGURE 7

CBG causes membrane hyperpolarization in S. mutans. (A) The red fluorescence of DiOC2(3)-stained S. mutans that have been exposed to different CBG concentrations (0–10 μl/ml) for 30 min. (B) The green fluorescence of DiOC2(3)-stained S. mutans that have been exposed to different CBG concentrations (0–10 μl/ml) for 30 min. (C) The relative fluorescence intensity (RFI) of the red (red lines) and green (green lines) fluorescence of the samples presented in (A,B).

FIGURE 8

CBG reduces S. mutans membrane fluidity. (A) Laurdan generalized polarization (GP) values in S. mutans treated with various concentrations of CBG (0–20 μg/ml) for 2 h. n = 3; *p < 0.05. (B) Fluorescence intensity scan of laurdan stained S. mutans that have been treated with different CBG concentrations (0–20 μg/ml) or EtOH for 2 h.

FIGURE 9

CBG prevents the decrease in pH caused by S. mutans. A kinetic change in the pH values of the medium of untreated and CBG (0–10 μg/ml)-treated S. mutans.