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ORIGINAL RESEARCH
published: 22 April 2021
doi: 10.3389/fmicb.2021.656471
Edited by:
Fabian Cieplik,
University Medical Center
Regensburg, Germany
Reviewed by:
Denise Muehler,
University Medical Center
Regensburg, Germany
Shahper Nazeer Khan,
Aligarh Muslim University, India
*Correspondence:
Muna Aqawi
muna.aqawi@mail.huji.ac.il
Specialty section:
This article was submitted to
Antimicrobials, Resistance
and Chemotherapy,
a section of the journal
Frontiers in Microbiology
Received: 20 January 2021
Accepted: 30 March 2021
Published: 22 April 2021
Citation:
Aqawi M, Sionov RV, Gallily R,
Friedman M and Steinberg D (2021)
Anti-Bacterial Properties
of Cannabigerol Toward
Streptococcus mutans.
Front. Microbiol. 12:656471.
doi: 10.3389/fmicb.2021.656471
Anti-Bacterial Properties of
Cannabigerol Toward Streptococcus
mutans
Muna Aqawi1,2*, Ronit Vogt Sionov1, Ruth Gallily3, Michael Friedman2and
Doron Steinberg1
1Biofilm Research Laboratory, Faculty of Dental Medicine, Institute of Dental Sciences, The Hebrew University of Jerusalem,
Jerusalem, Israel, 2School of Pharmacy, Institute for Drug Research, The Hebrew University of Jerusalem, Jerusalem, Israel,
3The Lautenberg Center for General and Tumor Immunology, Hadassah Medical School, The Hebrew University
of Jerusalem, Jerusalem, Israel
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: Streptococcus mutans, phytocannabinoids, Cannabigerol, dental caries, bacteriostasis
INTRODUCTION
Dental caries, also known as tooth decay, is the most common disease of the oral cavity (Krzy´
sciak
et al., 2014) and one of the most prevalent chronic human disease worldwide (Robert et al., 2007).
Dental caries is a transmissible, complex disease that is caused by prolonged periods of low pH
in the mouth, resulting in a net mineral loss from the teeth (Kutsch, 2014). It develops through a
complex interaction over time between acid-producing bacteria, fermentable carbohydrates, teeth,
and saliva (Krzy´
sciak et al., 2014). More than 700 different bacterial species have been detected in
the oral cavity of humans (Larsen and Fiehn, 2017). Among them, is the highly cariogenic bacterium
associated with dental caries, Streptococcus mutans (S. mutans).
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Streptococcus mutans is a gram-positive, facultatively
anaerobic bacterium with acidogenic and aciduric properties (De
Sousa et al., 2015;Chu et al., 2016), whose presence within the
dental biofilm is an important feature for the establishment and
the development of cariogenic dental plaques (Silva et al., 2008).
S. mutans resides in supragingival plaque, a biofilm formed above
the gumline in the oral cavity (Fozo and Quivey, 2004). A major
core feature contributing to the organism’s pathogenicity is its
ability to transport and metabolize a wide range of nutrients
in the hosts’ diet and saliva into organic acids (Lemos et al.,
2019). The production of organic acids lowers consequently the
pH of the surrounding environment, and over time the drop
in pH leads to tooth demineralization and the development of
caries. S. mutans is capable of surviving in low pH environments
(Matsui and Cvitkovitch, 2010) and can metabolize various
sugars including glucose, fructose, and sucrose, and acidify the
environment to a pH as low as 3.5 (Belli and Marquis, 1991).
The potential use of Cannabis in anti-bacterial therapies
has recently emerged. In vitro studies have shown that
cannabinoids inhibit the growth of Gram-positive bacteria,
mostly Staphylococcus aureus, with no detectable activity against
Gram-negative organisms (Turner and Elsohly, 1981;Appendino
et al., 2008). Recently, Blaskovich et al. (2021) observed similar
anti-bacterial effect of Cannabidiol (CBD) toward antibiotic-
sensitive and antibiotic-resistant Staphylococcus aureus with a
MIC around 1–5 µg/ml. CBD also had anti-bacterial effect on
other gram-positive bacteria (e.g., Streptococcus pneumoniae and
Clostridioides difficile), as well as a subset of Gram-negative
bacteria that includes the “urgent threat” pathogen Neisseria
gonorrhoeae. Of note, the bacteria didn’t develop resistance to its
anti-bacterial activity (Blaskovich et al., 2021).
Cannabigerol (CBG) is a non-psychotropic Cannabis-derived
cannabinoid (CB) (Gaoni and Mechoulam, 1964). Several
studies support analgesic, anti-depressant, anti-cancer, anti-
inflammatory, and anti-hypertensive actions for CBG in
mammals (Borrelli et al., 2014;Nachnani et al., 2020). Farha et al.
(2020) demonstrated an anti-bacterial activity of CBG against
methicillin-resistant S. aureus (MRSA). We have previously
shown that CBG did not affect the growth of the gram-negative
Vibrio harveyi, but rather interfered with the quorum sensing
system (Aqawi et al., 2020). Here we have studied the anti-
bacterial activity of CBG against planktonic growing S. mutans.
MATERIALS AND METHODS
Materials
Cannabigerol (CBG) (hemp isolate, 95% purity) was purchased
from NC Labs (Czechia) and dissolved in ethanol at a
concentration of 10 mg/ml. Respective dilutions of ethanol were
used as control.
Bacterial Growth and Kinetics Studies
Planktonic S. mutans UA159 ATCC 700610, Streptococcus sanguis
10556, Streptococcus sobrinus ATCC 27351, and Streptococcus
salivarius ATCC 25975 were grown overnight at 37◦C in 95%
air/5% CO2in brain heart infusion broth (BHI, Acumedia,
MI, United States) until an OD600nm = 1.2–1.3 was reached
(Steinberg et al., 2008). The bacterial cultures were treated with
various concentrations of CBG in BHI and respective dilutions of
ethanol. Untreated bacteria served as control. For kinetic studies,
S. mutans with different starting OD600nm (0.1, 0.2, or 0.4) were
treated with increasing concentrations of CBG (0, 1.25, 2.5, 5, and
10 µg/ml) and the OD595nm was measured every 30 min for a
period of 20 h in a Tecan M200 microplate reader (Tecan Trading
AG, Switzerland) at 37◦C.
Colony Forming Units (CFU)
Colony forming units assay was performed after different
incubation time (0, 2, 4, 6, 8, and 24 h) with CBG (0, 2.5, 5, and
10 µg/ml). 10-fold serial dilutions of the untreated and treated
samples were prepared by repeatedly transferring 100 µl from
one sample to another tube containing 900 µl PBS. After vigorous
vortex, 100 µl of the bacterial suspensions were spread on BHI
agar plates and incubated overnight at 37◦C in the presence of 5%
CO2. After incubation, the number of colonies was counted using
the ImageJ software (Breed and Dotterrer, 1916). The following
equation was used to calculate the CFU per ml in the original
sample:
CFU per ml = Number of colonies ×dilution
factor ×1/volume seeded on the plates.
High Resolution Scanning Electron
Microscopy (HR-SEM)
Planktonic growing S. mutans of OD600nm = 0.1 was treated
with different concentrations of CBG (0, 2.5, 5, and 10 µg/ml)
for 4 h. At the end of incubation, the bacteria were washed
twice with PBS, fixed in 4% glutaraldehyde for 40 min and
washed in double distilled water. The specimens were then
mounted on glass pieces, sputter coated with iridium and
visualized using a MagellanTM 400L High-Resolution Scanning
Electron Microscope (FEI Company, Holland) (Brandwein
et al., 2016). Images were captured randomly from 4 to 5
different areas. The lengths and width of the bacteria were
measured using the ImageJ software. 200 bacteria were measured
for each treatment group from 8 to 9 independent high
magnification images.
Transmission Electron Microscopy (TEM)
Untreated and CBG (10 µg/ml)-treated S. mutans at an
OD600nm = 0.1 were incubated for 4 h. At the end of incubation,
the bacteria were washed twice with PBS, followed by an
overnight fixation in 2% formaldehyde and 2.5% glutaraldehyde
in 0.1 M sodium cacodylate buffer, pH 7.4. The fixed bacteria
were rinsed four times 10 min in 0.1 M cacodylate buffer
and stained with 1% osmium tetroxide and 1.5% potassium
ferricyanide in 0.1 M cacodylate buffer for 1 h. The bacteria
were then washed four times in cacodylate buffer followed by
dehydration in increasing concentrations of ethanol consisting
of 30%, 50%, 70%, 80%, 90%, 95% for 10 min each step,
followed by three times 20 min in 100% anhydrous ethanol, and
two times 10 min in propylene oxide. Each step was followed
by centrifugation at 15,000 gfor 8 s. Following dehydration,
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the cells were infiltrated with increasing concentrations of
Agar 100 resin in propylene oxide, consisting of 25, 50, 75,
and 100% resin for 16 h each step. The bacteria were then
embedded in fresh resin and let polymerize in an oven at 60◦C
for 48 h. Embedded bacteria in blocks were sectioned with
a diamond knife on a Leica Reichert Ultracut S microtome
and ultrathin sections (80 nm) were collected onto 200 mesh,
thin bar copper grids. The sections on grids were sequentially
stained with uranyl acetate and lead citrate for 10 min each
and viewed with Tecnai 12 TEM 100 kV (Phillips, Eindhoven,
Netherlands) equipped with MegaView II CCD camera and
Analysis R
version 3.0 software (Soft Imaging System GmbH,
Münster, Germany).
Microbial Cell Viability Assay
The luminescent BacTiter-GloTM kit (Promega) was used to
quantify the ATP levels in untreated and CBG-treated samples.
Briefly, 100 µl of each sample (CBG = 0, 2.5, 5, 10, and
20 µg/ml for 2 h) was mixed with 100 µl of the reagent
in 96-flat bottom plates (Greiner Bio-One, µClear white clear
bottom plates), and after mixing for 5 min on an orbital shaker,
the luminescence was recorded using the M200 Tecan plate
reader. ATP level was calculated in comparison to the control
using the following equation: (sample luminescence/control
luminescence) ×100.
Membrane Permeability Assay
Changes in the bacterial cell membrane permeability was assessed
by propidium iodide (PI) (Sigma) uptake and the metabolic
activity by calcein AM (BioLegend) staining essentially as
described (Cho and Lee, 2011;Ohsumi et al., 2015). PI enters only
membrane-compromised cells and fluoresces in the red spectrum
when binding to nucleic acids within the cells (Veerman et al.,
2004). On the other hand, calcein AM diffuses passively into the
cytoplasm, where it is converted into green fluorescent calcein
via native esterases. Calcein fluorescence is retained in live cells
but leaks out when the plasma membrane is compromised.
An overnight culture of S. mutans was resuspended in BHI
to an OD600nm = 0.3. The bacteria were then treated with
different concentrations of CBG (0, 1.25, 2.5, 5, 8, and 10 µg/ml)
for 2 h at 37◦C. At the end of incubation, the bacteria
were stained with 10 µg/ml PI and 10 µg/ml calcein AM
for 20 min at 37◦C, followed by flow cytometry (BD LSR-
Fortessa flow cytometer, BD Biosciences) using the 488 nm
excitation laser and collecting the data using the red and green
filters, respectively.
Laurdan Membrane Fluidity Assay
The membrane fluidity of S. mutans was measured using
laurdan (AnaSpec, Fremont, CA, United States) essentially as
described (Bessa et al., 2019). Laurdan is a fluorescence probe
that intercalates into the membrane bilayer and displays an
emission wavelength shift depending on the amount of water
molecules in the membrane (Wenzel et al., 2018). S. mutans
(OD600 = 0.3 nm) was treated with different concentrations
of CBG (0, 4, 6, 8, 10, and 20 µg/ml) at 37◦C for 2 h
and then incubated with 10 µM laurdan for 10 min at
room temperature in the dark. An unstained sample served
as control. Thereafter, the samples were washed four times
in PBS containing 1% glucose and 1% DMSO (PBSGD) and
resuspended in 1 ml of PBSGD. 200 µl of each sample were
added to each well of a µClear black 96-well plate (Greiner Bio-
One, Frickenhausen, Germany) and the fluorescence analyzed
at 30◦C in the M200 Tecan plate reader with an excitation
at 350 nm and an emission scan from 400 to 600 nm.
The laurdan Generalized Polarization (GP) was calculated
using the following equation: GP = (I440−I490 )/(I440 + I490)
where I440 and I490 are fluorescence intensities at 440 and
490 nm, respectively.
Nile Red Membrane Staining
Control bacteria (OD600nm = 0.3) or bacteria that have
been exposed to CBG (0, 1.25, 2.5, 5, and 10 µg/ml)
for 2 h, were stained with 10 µg/ml Nile red (APExBIO,
Boston, MA, United States) and 40,6-Diamidine-20-phenylindole
dihydrochloride (DAPI) (Sigma) for 30 min at 37◦C (Sugimoto
et al., 2017). After washing the cells in PBS, the bacteria were
analyzed on flow cytometry (LSR-Fortessa flow cytometer, BD
Biosciences) using the 561 nm yellow-green laser excitation and
collecting the data using the 635 nm filter.
Membrane Potential (MP)
The membrane potential of S. mutans was measured using
cationic dye 3,30-diethyloxacarbocyanine iodide (DiOC2(3);
Molecular Probes, Eugene, OR, United States) by flow cytometry
according to the manufacturer’s instructions. DiOC2(3)exhibits
green fluorescence in all bacterial cells, but the fluorescence shifts
toward red emission at larger membrane potential. An overnight
culture of S. mutans was resuspended in PBS to an OD600nm = 0.3
and exposed to different concentrations of CBG (0, 2.5, 5, and
10 µg/ml) and 30 µM DiOC2(3)for 30 min at room temperature.
The samples were analyzed by flow cytometry (LSR-Fortessa flow
cytometer) using the 488 nm excitation laser and collecting the
data using the green and red filters.
pH Measurements
Streptococcus mutans of OD600nm = 0.1 was treated with different
concentrations of CBG (0, 2.5, 5, and 10 µg/ml) and incubated
at 37◦C for 24 h. At various time points, the pH of the samples
was measured using pH-indicator strips (MColorpHast, Merck
KGaA, Darmstadt, Germany).
Drop Plate Method
Drop plate method was performed after different incubation time
(0, 2, 4, 6, 8, and 24 h). Here, an agar plate was divided into
sectors. In each sector, one drop of 10 µl from untreated and
CBG- treated bacteria (0, 2.5, 5, and 10 µg/ml) was inoculated on
the surface of the agar, and thereafter the plates were incubated
upside down at 37◦C overnight.
Statistical Analysis
Experiments were performed independently three times in
triplicates and the data were analyzed statistically using Student’s
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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 OD595nm .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 OD600nm = 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.
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ttest in Microsoft Excel, with a pvalue of less than 0.05
considered significant.
RESULTS
CBG Halts the Proliferation of Planktonic
S. mutans
We initially analyzed the effect of CBG on S. mutans viability. For
this purpose, S. mutans was exposed to increasing concentrations
of CBG, and the OD595nm monitored after 24 h incubation
(Figure 1A). We found that CBG inhibited in a dose-
dependent manner the planktonic growth of S. mutans with
a MIC of 2.5 µg/ml (Figure 1A). CBG had also an anti-
bacterial effect toward S. sanguis,S. sobrinus, and S. salivarius
(Table 1). To get a better insight into the CBG effect on
the bacterial growth, kinetic growth studies were performed
using different initial bacterial densities (OD600nm = 0.1–
0.4) in the presence of increasing concentrations of CBG
(Figures 1B–D). When starting with an OD600nm = 0.1 or
0.2, CBG treatment arrested the growth of S. mutans at a
concentration of 2.5, 5, and 10 µg/ml, whereas 1.25 µg/ml
only delayed the onset of the bacterial log growth phase
(Figures 1B,C). When increasing the initial OD600nm to
0.4, CBG at 1.25 µg/ml had no effect, CBG at 2.5 µg/ml
showed a delayed onset of the log-phase, while CBG at 5
and 10 µg/ml still retained their growth inhibition effect
without any sign of recovery even after 24 h (Figure 1D).
The OD of the bacteria treated with 10 µg/ml CBG was
even 25% lower after 24 h than the initial OD (Figure 1D).
Altogether, these data suggest that the growth-inhibitory effect
of CBG is affected by the initial cell density, and higher CBG
concentrations are needed to achieve full growth inhibitory effect
at higher densities.
To examine whether CBG is bacteriostatic or bactericidal,
10 µl from each sample was applied on BHI agar plates
at different time points during the 24 h incubation period.
Bacterial growth was observed at all tested CBG concentrations
(Supplementary Figure 1), suggesting that CBG is bacteriostatic,
and the bacterial growth can be recovered when CBG is removed.
To quantify the growth inhibitory effect of CBG, we counted
the colony-forming units (CFUs) at different time points during
the 24 h incubation (Figure 1E). Again, the growth arrest
phenomenon was observed with the CBG-treated cells exhibiting
a multitude lower cell counts/ml than their control counterparts
(Figure 1E). As expected, the control bacteria continued to grow,
reaching a maximum number of live bacteria after 6 h followed
by a decline during the next 18 h (Figure 1E). Bacteria exposed
to CBG at 2.5 µg/ml showed an initial growth arrest, that was
followed by a slow growth rate after 6 h (Figure 1E). At 5 and
10 µg/ml, CBG resulted in a gradual drop in the number of viable
bacteria during the first 8 h, followed by a recovery of surviving
bacteria (Figure 1E). 5 and 10 µg/ml CBG led to a respective
98.5% and 99.9% reduction of viable bacteria at 8 h (Figure 1E).
This finding indicates that CBG at the higher concentrations have
a bactericidal effect, in addition to having a bacteriostatic effect.
TABLE 1 | The minimal inhibitory concentration (MIC) of CBG toward four
different oral bacteria.
Type of bacteria MIC (CBG)
Streptococcus mutans 2.5 µg/ml
Streptococcus sanguis 1µg/ml
Streptococcus sobrinus 5µg/ml
Streptococcus salivarius 5µg/ml
HR-SEM Images Show Altered
Membrane Structures of CBG-Treated
Bacteria
To understand in more depth the effects of CBG, control
and CBG-treated bacteria (2.5, 5, and 10 µg/ml for 4 h)
were visualized under a high-resolution scanning electron
microscope (HR-SEM) (Figure 2A). The CBG-treated
bacteria appear longer at the average in comparison to
control bacteria and folded membrane structures could
often be seen (Figure 2A). At 10 µg/ml CBG, many of the
bacteria seem to be swollen (Figure 2A). When the length
(Figure 2B) and the width (Figure 2C) were measured
using the ImageJ software, the control sample showed an
average length of 0.7 µm and an average width of 0.37 µm,
while bacteria treated with CBG appear larger reaching an
average length of 1 µm and an average width of 0.46 µm at
10 µg/ml.
CBG Leads to Intracellular Accumulation
of Membrane Structures
To further examine the effect of CBG on S. mutans, the
morphology of untreated and CBG (10 µg/ml for 4 h)-
treated bacteria was studied by transmission electron
microscopy (TEM) (Figures 3,4). In the panoramic
low magnification images (Figure 3A), we can clearly
see that most of the control bacteria show structured
nucleoids symmetrically distributed eccentrically in the
dividing bacteria. The cytoplasm of the control bacteria
appears homogenously with electron-dense material and
there are several dividing bacteria with initial membrane
invagination for septum formation (Figure 3A). The
nucleoids of CBG-treated bacteria showed either a distorted
structure or could not been observed (Figure 3B). The
CBG-treated bacteria barely showed any sign of septum
invagination (Figure 3B). Strikingly, the cytoplasm
of the CBG-treated bacteria showed accumulation of
electron-lucent, bright material (Figure 3B). At higher
magnification of control bacteria, we could clearly
distinguish between the intact and well-defined cell wall
(CW), plasma membrane (PM), and cytoplasmic space
with a central nucleoid (N) containing the circular DNA
(Figures 4A,C,E,G). In Figures 4A,C, control bacteria
with initial invagination of the plasma membrane can be
observed. When treated with CBG, there is a disturbance
of the bacterial plasma membrane and mesosome-like
structures of bacterial membranes are observed within
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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.
the bacteria (Figures 4B,D,F,H). In Figure 4B, the CBG-
treated bacteria appeared longer than usual with no sign of
septum invagination.
The Effect of CBG on ATP Levels in
S. mutans
The microbial cell viability assay was performed to analyze the
effect of CBG on the ATP levels in S. mutans. For that purpose,
S. mutans was exposed to different concentrations of CBG and
the amount of ATP was measured after 2 h. The percentage
reduction in ATP level was calculated in comparison to untreated
and ethanol-treated samples. According to these data, CBG at
1.25 µg/ml reduced the ATP level to 75.0 ±2.2% (Figure 5A;
p<0.05). When increasing the CBG concentrations to 5, 10,
and 20 µg/ml, the ATP levels were reduced to 16.4 ±0.3%,
12.1 ±3.1%, and 12.0 ±1.5%, respectively. Ethanol barely had
any effect. When the ATP level of each sample was divided by
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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.
its own OD595nm to normalize the ATP level to cell density
(Figure 5B), there was only a slight reduction in the relative
ATP level of around 20–30%. Thus, the major reduction in the
ATP levels in Figure 5A was due to the reduced number of
bacteria and the net ATP level per cell is only slightly lower
following CBG treatment.
CBG Alters the Membrane Properties of
S. mutans
We used the hydrophobic fluorescent probe Nile Red to stain
the bacterial membrane of control and bacteria treated with
various concentrations of CBG for 2 h. Despite more membrane-
like structures within the CBG-treated bacteria observed with
TEM (Figures 4D,F,H), there was a dose-dependent decrease
in Nile Red staining (Figures 6A,B). These data suggest that
CBG leads to a reduction in the membrane mass or it
causes a less hydrophobic membrane. Simultaneous staining
of the DNA of the bacteria by DAPI shows only a slight
reduction in DNA content with increasing concentrations of
CBG (Figures 6C,D).
CBG Increases the Membrane
Permeability of S. mutans
To study the effect of CBG on membrane permeability,
untreated and bacteria treated with various concentrations of
CBG for 2 h were incubated with propidium iodide (PI) and
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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.
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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/OD595nm 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.
calcein AM for 20 min before analyzing the cells by flow
cytometry. CBG treatment led to a dose-dependent increase
in PI uptake (Figures 6E,F), while calcein AM staining was
concomitantly reduced (Figures 6G,H). The increase in PI
uptake after CBG treatment indicates an increase in membrane
permeability, while the reduced calcein AM staining is a
combined effect of reduced metabolic activity and leakage of
calcein out of the cells.
CBG Causes Immediate Membrane
Hyperpolarization in S. mutans
To test the direct effect of CBG on the membrane potential
of S. mutans, bacteria were loaded with the membrane-
potential-sensitive cyanine dye DiOC2(3), and then treated
with increasing concentrations of CBG and the green/red
fluorescence intensity immediately monitored by flow
cytometry. As the concentration of the CBG increases,
the red fluorescence intensities were increased by 150%
(Figures 7A,C and Supplementary Figure 2), while the green
fluorescence intensities were decreased by 25% (Figures 7B,C
and Supplementary Figure 2), indicating that CBG causes
hyperpolarization of the membranes. It is noteworthy to
mention that the changes in membrane potential is an
immediate effect of CBG, suggesting that CBG acts on the
plasma membrane.
CBG Reduces the Membrane Fluidity of
S. mutans
The effect of CBG on the bacterial membrane fluidity was
studied by staining the untreated and CBG-treated bacteria
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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
2h.(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.
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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).
with laurdan, which is a fluorescent probe that intercalates into
the membrane bilayer and displays an emission wavelength
shift from 440 to 490 nm depending on the amount of
water molecules in the membrane. An inverse relationship
exists between the laurdan generalized polarization (GP) values
and the degree of membrane fluidity (Colalto, 2018). Also,
the higher the laurdan staining, the higher is the fluidity.
CBG treatment caused an increase in laurdan GP values
(Figure 8A), suggesting a more rigid membrane. The reduced
membrane fluidity is further supported by the observation
that CBG caused a dose-dependent reduction in laurdan
incorporation (Figure 8B).
CBG Treatment Prevents the Drop in pH
Caused by S. mutans
A kinetic study of the pH level in the S. mutans culture media
showed that CBG was able to maintain the pH at 7 for at
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Aqawi et al. Activity of Cannabigerol Against S. mutans
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.
least 8 h at all tested concentrations (Figure 9). After 24 h,
the pH in the 2.5 and 5 µg/ml CBG samples had reached
five similarly to the control samples, while 10 µg/ml CBG still
managed to prevent the acidification (pH = 6.5) (Figure 9). The
latter correlates with the prevention of bacterial growth with
this concentration.
DISCUSSION
Recently, the anti-bacterial properties of several medicinal
plants have been explored (Colalto, 2018). Cannabis sativa
is an herbaceous plant that has been used for millennia for
medicinal and recreational purposes. Cannabis is undoubtedly
one of the most widely used illicit drugs (Bewley-Taylor,
2002). Natural products derived from Cannabis and their
analogs have been screened for anti-microbial properties,
in the quest to discover new anti-infective agents. Several
cannabinoids have been found to have potent anti-microbial
activity against Gram-positive pathogens such as MRSA
isolates (Karas et al., 2020). Cannabigerol (CBG) is one of
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.
the phytocannabinoids present in Cannabis sativa L. that
has attracted pharmacological interest because it is non-
psychotropic and is abundant in some industrial hemp varieties
(Navarro et al., 2018).
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Aqawi et al. Activity of Cannabigerol Against S. mutans
Bacteria belonging to the genus Streptococcus are the first
inhabitants of the oral cavity, which can be acquired right
after birth and thus play an important role in the formation
of the oral microbiota (Abranches et al., 2019). In the oral
cavity, many microorganisms have been found to be associated
with dental caries, among them S. mutans is considered
the most cariogenic bacteria (Loesche, 1986). Therefore, the
inhibition of S. mutans is a key objective in the prevention
of dental caries.
In the present study, we aimed to investigate the anti-bacterial
properties of CBG against planktonic growing S. mutans.
We demonstrated that CBG exerts a bacteriostatic effect
at a concentration of 2.5 µg/ml and the growth-inhibitory
effect of CBG is affected by the initial cell density. At the
higher concentrations of 5–10 µg/ml, CBG had a bactericidal
effect as shown by 98.5–99.9% reduction of viable bacteria
after 8 h. This is further manifested by the increased
uptake of PI, that only penetrates bacteria with perforated
membrane. The latter observation suggests that CBG increases
membrane permeability. The dose-dependent reduction
in Calcein AM staining with increasing concentrations
of CBG points to enhanced membrane leakage. These
findings go along with the observation by Blaskovich et al.
(2021) that the primary anti-bacterial action of CBD is
destruction of the membrane. Also, Farha et al. (2020)
provided evidence that CBG acts by perturbing the plasma
membrane and gram-negative bacteria can be sensitized
to CBG after permeabilization of the outer membrane by
polymyxin B. The reduced Calcein AM staining might
also indicate reduced metabolic activity, but the ATP
levels per bacteria were only slightly reduced by CBG after
a 2 h incubation.
Kinetic growth studies showed that CBG at 1.25 µg/ml delayed
the initiation of the bacterial log growth phase, while CBG at
higher concentrations retained their growth inhibition effect
with a sign of recovery after 24 h. This observation indicates
that CBG impedes cell division. This is further confirmed by
TEM, where the number of septal invaginations is strongly
reduced in CBG-treated bacteria in comparison to control
bacteria. The TEM images also showed that CBG leads to a
disturbance of the plasma membrane with an accumulation
of mesosome-like membrane structures within the bacteria.
A similar appearance of mesosome-like structures was observed
by Xiao et al. (2020), when MRSA was treated with an
α/βchimeric polypeptide molecular brush. HR-SEM images
confirmed altered bacteria membrane structures following CBG
treatment. The bacteria appeared swollen, became longer and
wider at the average after a short period of 4 h incubation
with CBG. Moreover, the CBG-treated bacteria showed irregular
folded membrane structures.
Since the TEM images showed prominent changes in the
membrane structures of S. mutans following CBG treatment,
we performed a series of experiments to test the effect of
CBG on the cell membrane. We initially used Nile Red to
stain the membranes. Our data showed a dose-dependent
decrease in Nile Red staining, suggesting that CBG reduces
the total membrane mass. An alternative explanation for the
reduced Nile Red staining could be CBG-induced alterations in
membrane polarity which affects the fluorescence intensity of
this dye (Sackett and Wolff, 1987). Nile red in polar membranes
fluoresces dark red, while the color shifts to yellow-gold emission
in neutral lipids. In a parallel assay using the membrane
dye laurdan, CBG treatment reduced the incorporation of
this compound in a dose-dependent manner indicative for a
more rigid membrane. On top of these findings, we observed
that CBG caused immediate membrane hyperpolarization in
S. mutans suggesting an effect on ion channels. Altogether, our
data suggest that CBG alters the cell membrane properties of
S. mutans.
Membrane fluidity is a key parameter of bacterial membranes
that undergoes quick adaptation in response to environmental
challenges and has recently emerged as an important factor in
the anti-bacterial mechanism of membrane-targeting antibiotics.
Assessing changes in the overall membrane fluidity and
formation of membrane microdomains is therefore pivotal to
understand both the functional organization of the bacterial
cell membrane as well as the antibiotic mechanisms (Wenzel
et al., 2018). To withstand the acidification of its environment,
S. mutans shifts its lipid profile from saturated fatty acid
(rigid) at pH = 7 to a more unsaturated (fluid) fatty acid at
pH=5(Quivey et al., 2000). Thus, the effect of CBG on
S. mutans membrane properties might contribute to its anti-
bacterial effect by preventing the necessary adaptation to an
acid environment. We observed that CBG prevented the drop
in pH caused by S. mutans. CBG was able to maintain the
pH at 7 for at least 8 h at all tested concentrations. The
maintenance of pH could be related to the reduced proliferation
of the bacteria.
In summary, the present study demonstrates an anti-
bacterial effects of the Cannabis component CBG toward
the cariogenic bacteria S. mutans. CBG acts at several
levels: (1) It exerts a bacteriostatic effect that is affected
by the initial bacterial cell density. (2) It affects the
membrane structure and causes intracellular accumulation
of mesosome-like structures. (3) It causes immediate membrane
hyperpolarization. (4) It reduces the membrane fluidity.
(5) It increases the membrane permeability. (6) It prevents
the drop in pH caused by S. mutans, thereby preventing its
cariogenic property. The interference of CBG with the caries
causative S. mutans may provide a novel innovative way to
combat dental caries.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation.
AUTHOR CONTRIBUTIONS
MA, RS, RG, MF, and DS conceived the idea. MA designed and
performed the experiments, analyzed the data, and wrote the
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Aqawi et al. Activity of Cannabigerol Against S. mutans
manuscript with RS and DS. All authors contributed to the article
and approved the submitted version.
FUNDING
This study was partially supported by the STEP-GTP sisters
fellowship (2019–2021).
ACKNOWLEDGMENTS
We would like to thank Dr. Vitaly Gutkin at The Harvey M.
Krueger Family Center for Nanoscience and Nanotechnology
at the Edmond J. Safra Campus of The Hebrew University of
Jerusalem for his valuable assistance with the SEM analysis.
Our greatest appreciation goes also to Dr. Yael Friedmann
at the Bio-Imaging Unit at the Edmond J. Safra Campus
of The Hebrew University of Jerusalem for performing
the TEM imaging.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fmicb.
2021.656471/full#supplementary-material
Supplementary Figure 1 | Drop method to detect bacterial growth of S. mutans
on BHI-agar plates after treatment with different concentrations of CBG
(0–10 µg/ml) at various time points. At the end of incubation, 10 µl of each sample
was applied in triplicates on BHI agar plates and incubated overnight at 37◦C.
Supplementary Figure 2 | Flow cytometry dot plots of DiOC2(3)-stained
S. mutans that have been exposed to different CBG concentrations (0–10 µl/ml)
for 30 min. The Y-axis shows the red fluorescence, while the X-axis shows the
green fluorescence.
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Copyright © 2021 Aqawi, Sionov, Gallily, Friedman and Steinberg. This is an open-
access article distributed under the terms of the Creative Commons Attribution
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