Uncovering the Hidden Antibiotic Potential of Cannabis

Article abstract of DOI:10.1021/acsinfecdis.9b00419

The spread of antimicrobial resistance continues to be a priority health concern worldwide, necessitating the exploration of alternative therapies. Cannabis sativa has long been known to contain antibacterial cannabinoids, but their potential to address antibiotic resistance has only been superficially investigated. Here, we show that cannabinoids exhibit antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), inhibit its ability to form biofilms, and eradicate preformed biofilms and stationary phase cells persistent to antibiotics. We show that the mechanism of action of cannabigerol is through targeting the cytoplasmic membrane of Gram-positive bacteria and demonstrate in vivo efficacy of cannabigerol in a murine systemic infection model caused by MRSA. We also show that cannabinoids are effective against Gram-negative organisms whose outer membrane is permeabilized, where cannabigerol acts on the inner membrane. Finally, we demonstrate that cannabinoids work in combination with polymyxin B against multidrug resistant Gram-negative pathogens, revealing the broad-spectrum therapeutic potential for cannabinoids.

Full text of DOI:10.1021/acsinfecdis.9b00419

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Letter
Uncovering the hidden antibiotic potential of Cannabis
Maya A. Farha, Omar M El-Halfawy, Robert T. Gale, Craig MacNair, Lindsey
A. Carfrae, Xiong Zhang, Nicholas Jentsch, Jakob Magolan, and Eric D Brown
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00419 • Publication Date (Web): 04 Feb 2020
Downloaded from pubs.acs.org on February 5, 2020
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Uncovering the hidden antibiotic potential of Cannabis
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,2,
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1,2
Maya A. Farha †, Omar M. El-Halfawy
†; Robert T. Gale ; Craig R. MacNair ; Lindsey A.
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Carfrae ; Xiong Zhang , Nicholas G. Jentsch ; Jakob Magolan ; Eric D. Brown *
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1
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8N
3
Z5, Canada
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Michael G. DeGroote Institute of Infectious Disease Research, McMaster University, 1200 Main
Street West, Hamilton, Ontario, L8N 3Z5, Canada
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Microbiology and Immunology Department, Faculty of Pharmacy, Alexandria University, 1 El-
Khartoum square, Azarita, Alexandria, 21521, Egypt
*
†
To whom correspondence should be addressed (ebrown@mcmaster.ca)
These authors contributed equally
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Abstract
The spread of antimicrobial resistance continues to be a priority health concern worldwide,
necessitating exploration of alternative therapies. Cannabis sativa has long been known to contain
antibacterial cannabinoids, but their potential to address antibiotic resistance has only been
superficially investigated. Here, we show that cannabinoids exhibit antibacterial activity against
MRSA, inhibit its ability to form biofilms and eradicate pre-formed biofilms and stationary phase cells
persistent to antibiotics. We show that the mechanism of action of cannabigerol is through targeting the
cytoplasmic membrane of Gram-positive bacteria and demonstrate in vivo efficacy of cannabigerol in a
murine systemic infection model caused by MRSA. We also show that cannabinoids are effective
against Gram-negative organisms whose outer membrane is permeabilized, where cannabigerol acts on
the inner membrane. Finally, we demonstrate that cannabinoids work in combination with polymyxin B
against multi-drug resistant Gram-negative pathogens, revealing the broad-spectrum therapeutic
potential for cannabinoids.
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Keywords
Cannabinoids; antibacterial activity; broad-spectrum; membrane activity; in vivo efficacy; drug
discovery
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Public Health agencies around the globe have identified antimicrobial resistance as one of the
most critical challenges of our time. The rapid and global spread of antimicrobial-resistant organisms in
recent years has been unprecedented. So much so that the world health organization (WHO) published
its first ever list of antibiotic-resistant "priority pathogens", made up of 12 families of bacteria that pose
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the greatest threat to human health . Among them, Staphylococcus aureus is the leading cause of both
healthcare and community-associated infections worldwide and a major cause for morbidity and
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mortality , especially with the emergence and rapid spread of methicillin-resistant S. aureus (MRSA),
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which is resistant to all known β-lactam antibiotics . Worse yet, resistance to vancomycin, linezolid
and daptomycin has already been reported in MRSA clinical strains, compromising the therapeutic
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alternatives for life-threatening MRSA infections . Further, antibiotic-resistant Gram-negative
infections have increasingly become a pressing issue in the clinic. Indeed, of the bacteria highlighted
by the WHO, 75% are Gram-negative organisms. Among the currently approved antibiotics in clinical
use, the latest discovery of a new drug class dates back to more than 30 years ago. The rapid loss of
antibiotic effectiveness and diminishing pipeline beg for the exploration of alternative therapies.
Cannabis plants are important herbaceous species that have been used in folk medicine for
centuries. Increasing scientific evidence is accumulating for the efficacy of its metabolites in the
treatment, for example, of epilepsy, Parkinson disease, analgesia, multiple sclerosis, Tourette’s
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syndrome and other neurological diseases . At a very nascent stage are investigations into the potential
of cannabis metabolites as antibacterial therapies. To date, assessments of their antibacterial activity
have been few and superficial. In vitro studies have shown that cannabinoids inhibit the growth of
Gram-positive bacteria, mostly S. aureus, with no detectable activity against Gram-negative
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organisms , where the clinical need is highest. Further, the mechanism of action has remained elusive
and there has been little validation of antibacterial activity in vivo.
Here, we show that cannabinoids exhibit antibacterial activity against MRSA, inhibit its ability
to form biofilms, eradicate pre-formed biofilms and stationary phase cells persistent to antibiotics. We
reveal that the mechanism of action of cannabigerol (CBG) is through targeting the cytoplasmic
membrane of Gram-positive bacteria and demonstrate in vivo efficacy of CBG in a murine systemic
infection model caused by MRSA. We also show that cannabinoids are effective against Gram-negative
organisms whose outer membrane is permeabilized, where CBG acts on the inner membrane. Finally,
we demonstrate that cannabinoids work in combination with polymyxin B against multi-drug resistant
Gram-negative pathogens, revealing the broad-spectrum therapeutic potential for cannabinoids. In all,
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our findings position cannabinoids as promising leads for antibacterial development that warrant
further study and optimization.
We began our study investigating the antibacterial, anti-biofilm and anti-persister activity of a
variety of commercially available cannabinoids, including the five major cannabinoids,
cannabichromene (CBC), cannabidiol (CBD), cannabigerol (CBG), cannabinol (CBN), and ∆9-
tetrahydrocannabinol (THC), as well as a selection of their carboxylic precursors (pre-cannabinoids)
and other synthetic isomers (18 unique molecules total, Fig. 1) against methicillin-resistant S. aureus
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(MRSA) (Supplementary Table 1). Susceptibility tests were conducted according to the Clinical and
Laboratory Standards Institute (CLSI) protocol against MRSA USA300, a highly virulent and prevalent
community-associated MRSA. Overall, antibacterial activities for the five major cannabinoids (and
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some of their synthetic derivatives) were in line with previously published work . Seven molecules
were potent antibiotics with minimum inhibitory concentration (MIC) values of 2 µg/mL, including
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CBG, CBD, CBN, cannabichromenic acid (CBCA) and THC along with its Δ - and exo-olefin
regioisomers. We observed moderate loss of potency associated with the benzoic acid moiety (CBG,
CBD, and THC were more potent than CBGA, CBDA, THCA) and when n-pentyl substituent was
replaced with n-propyl (CBD and THC were superior to CBDV and THCV) (Supplementary Table 1).
These two modifications appeared to have an additive detrimental effect on antibacterial activity
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THCVA, CBDVA). The two most common human metabolites of THC, (±) 11-nor-9-carboxy-Δ9-
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THC, and (±) 11-hydroxy-Δ -THC, as well as cannabicylol were inactive at the highest concentrations
screened (MIC > 32 µg/mL) (Supplementary Table 1).
Biofilm formation by MRSA, typically on necrotic tissues and medical devices, is considered
an important virulence factor influencing its persistence in both the environment and the host
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organism . These highly structured surface-associated communities of MRSA are typically associated
with increased resistance to antimicrobial compounds and are generally less susceptible to host immune
factors. We assessed the ability of the various cannabinoids to inhibit the formation of biofilms by
MRSA, using static abiotic solid-surface assays in which MRSA was treated with increasing
concentrations of cannabinoids under conditions favouring biofilm formation (Supplementary Fig. 1).
In all, the degree of inhibition of biofilm formation correlated with antibacterial activity; those
cannabinoids with potent activity against MRSA strongly suppressed biofilm formation and vice versa
(Supplementary Fig. 1, Supplementary Table 1). The five major cannabinoids clearly repressed MRSA
biofilm formation, with CBG (Fig. 2a) exhibiting the most potent anti-biofilm activity. Indeed, as little
as 0.5 μg/mL (1/4 MIC) of CBG inhibited biofilm formation by ∼50% (Fig. 2b). Thus, this experiment
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underlined the strong inhibitory effect of cannabinoids on biofilm formation; this sub-MIC level of
CBG did not affect planktonic growth (Supplementary Fig. 2). Interestingly, we also evaluated the
effect of CBG on pre-formed biofilms by determining its minimal biofilm eradication concentration
(MBEC); CBG could eradicate pre-formed biofilms of MRSA USA300 at 4 µg/mL (Fig. 2c).
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Another challenge in the treatment of MRSA infections is the formation of non-growing,
dormant ‘persister’ subpopulations that exhibit high levels of tolerance to antibiotics11-13. Persister cells
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have a role in chronic and relapsing S. aureus infections such as osteomyelitis , and endocarditis .
Here, we evaluated the killing activity of a series of cannabinoids against persisters derived from
stationary phase cells of MRSA USA300 (Supplementary Fig. 3). These have been previously shown to
be tolerant to conventional antibiotics such as gentamicin, ciprofloxacin and vancomycin11, 17-18. In
general, the anti-persister activity correlated with potency against actively dividing cells as determined
by MIC assays (Supplementary Table 1). Again, CBG was the most potent cannabinoid against
persisters, whereas oxacillin and vancomycin were ineffective at concentrations that otherwise kill
actively dividing cells (Supplementary Fig. 3, Fig. 2d). More specifically, CBG killed persisters in a
concentration-dependent manner starting at 5 µg/ml. Notably, CBG rapidly eradicated a population of
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10 CFU/ml MRSA persisters to below the detection threshold within 30 minutes of treatment (Fig.
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We selected CBG (Fig.2a) for further studies of mechanism and in vivo efficacy. Not only did
CBG potently inhibit MRSA (MIC 2 µg/mL), repress biofilm formation (Fig. 2b), eradicate pre-formed
biofilms (Fig. 2c) and effectively eradicate persister cells (Fig. 2d), but it is non-psychotropic, non-
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sedative and constitutes a component of Cannabis for which there is high therapeutic interest . While
it has many desirable pharmacological properties, CBG also possesses several desirable physico-
chemical properties as a medicinal chemistry lead in terms of molecular weight, number of hydrogen
donors and acceptors, number of rings and rotatable bonds (Table 1). Nevertheless, CBG suffers from
high lipophilicity (high log P) and low aqueous solubility (Table 1). These values were not a bottleneck
to our studies, but in moving CBG as a lead, such properties could be addressed in medicinal chemistry
campaigns. To our advantage, we were able to synthesize CBG efficiently from olivetol and geraniol,
two inexpensive precursors, in one synthetic operation. We were cognisant that such facile synthetic
access would enhance the potential for subsequent medicinal chemistry-based development efforts.
We determined the MIC of CBG against 96 clinical isolates of MRSA using the CLSI protocol. The
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corresponding frequency distribution of MICs is presented in Fig. 2e. The MICs ranged from 2 – 8
µg/mL with a resulting MIC of 4 µg/mL. The range of MIC values was tight with only one outlier
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strain detected to have a very low MIC of 0.0625. Overall, this activity compares favourably with
conventional antibiotics for these multi-drug resistant strains.
Given its growth inhibitory action on Gram-positive bacteria, we reasoned isolating resistant
mutants to CBG would be a straightforward approach to gather insights into its bacterial target. Indeed,
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resistance mutations can often be mapped to a drug's molecular target . To this end, MRSA was
repeatedly challenged with various lethal concentrations of CBG, ranging from 2-16x MIC, to select
for spontaneous resistance in MRSA (Fig. 3). No spontaneously resistant mutants were obtained,
indicating a frequency of resistance less than 10−10 for MRSA. We also attempted to allow MRSA
bacteria to develop resistance to CBG by sequential subcultures via 15-day serial passage in liquid
culture containing sub-MIC concentrations of CBG and, again, no change in the MIC of CBG was
detected (Fig. 3). While these experiments were unsuccessful probes of mechanism, they suggested
very low rates of resistance for CBG, a highly desirable property for an antibiotic.
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We turned to chemical genomic analysis to generate hypotheses for the target of CBG. Such
studies can reveal patterns of sensitivity among genetic loci that are characteristic of the mechanism of
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action of an antibacterial compound . We confirmed that the model Gram-positive bacterium B.
subtilis was susceptible to CBG (MIC 2 µg/mL), and screened a CRISPR interference knockdown
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library of all essential genes in B. subtilis for further sensitization to CBG. In the absence of
induction, relying on basal repression (which leads to a ~3-fold repression of the knockdown library22),
we were unable to detect any knockdowns sensitized to sub-lethal concentrations of CBG (Fig. 3).
Low-level induction identified some sensitive and some suppressing clones, however follow-on work
with the individual knockdowns in liquid culture via full checkerboard analysis (combining xylose, the
inducer, with CBG) failed to confirm sensitivity or suppression. In all, we were unable to identify bona
fide chemical genetic interactions among essential genes of B. subtilis and CBG. We next aimed to
query the non-essential gene subset, this time using the Nebraska Transposon Mutant Library, a
sequence-defined transposon mutant library consisting of 1,920 strains, each containing a single
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mutation within a nonessential gene of CA-MRSA USA300 , again looking for genetic enhancers or
suppressors to generate target hypotheses (Fig. 3, Supplementary Fig. 4a). While we were unable to
uncover genetic suppressors at supra-lethal concentrations of CBG, we identified 41 transposons as
sensitive across 3 different sub-lethal concentrations of CBG (Supplementary Table 2). Analysis of
these transposons revealed a significant enrichment for genes encoding proteins that are localized at the
cytoplasmic membrane (Supplementary Fig. 4b) and enrichment for genes encoding functions in
processes that take place at the cytoplasmic membrane, such as cellular respiration and electron
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transport chain (Supplementary Fig. 4c). In all, chemical genomic profiling with CBG generally linked
its activity to cytoplasmic membrane function.
The lack of clear targets among the essential gene products, the predominance of chemical
genetic interactions linked to membrane function, and the difficulty generating resistant mutants,
suggested that CBG might act on the cytoplasmic membrane of MRSA. Indeed, the propensity of
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membrane-active compounds to generate resistance is frequently low . Further, the bacterial
membrane is critical for cell function and survival, and is essential irrespective of the metabolic status
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of the cell, including non-growing and persisting cells . The strong action of CBG on persister cells
would be consistent with such a mode of action. Thus, we assessed the ability of CBG to disrupt
membrane function using the membrane potential-sensitive probe, 3,3'-dipropylthiadicarbocyanine
iodide (DiSC (5)). In DiSC -loaded MRSA cells, CBG caused a dose-dependent increase in
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fluorescence that occurred at a concentration consistent with the MIC of CBG (Fig. 3). To probe the
possibility that CBG selectively dissipated membrane potential (Δψ) component of proton motive
force, we tested for synergy with sodium bicarbonate, a known perturbant of ΔpH, that has been shown
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to synergize with molecules that reduce Δψ . A lack of synergy between these compounds suggested
CBG disrupts the integrity of the cytoplasmic membrane (Supplementary Fig. 5). In order to evaluate
the potential membrane activity of CBG on mammalian cells, we evaluated its hemolytic toxicity
across varying concentrations (Supplementary Fig. 6). CBG was hemolytic only at 32 µg/mL, above its
MIC of 2 µg/mL, suggesting some specificity for prokaryotic cells.
Having established strong in vitro potency for CBG against MRSA, we next sought to evaluate
the in vivo efficacy in a murine systemic infection model of MRSA. The effect of CBG on a systemic
infection mediated by the CA-MRSA USA300 strain is shown in Fig. 4. A dose-dependence study
(Supplementary Fig. 7) informed that a dose of 100 mg/kg is most effective while remaining tolerable.
To evaluate tolerability, we treated mice with 100 mg/kg CBG and assessed their change in body
weight over various time points and found no significant changes (Supplementary Fig. 8). Additionally,
no signs of acute toxicity have been reported in a pharmacokinetic study of 120-mg/kg doses of
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CBG .. Overall, CBG displayed a significant reduction in bacterial burden in the spleen by a factor of
.8-log in CFU compared to the bacterial titer seen with the vehicle (pꢀ<ꢀ0.001, Mann–Whitney U-
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test). Further, the in vivo efficacy of CBG was comparable to that of the antibiotic control, vancomycin
administered at a similar dose. In all, CBG displayed promising levels of efficacy in the systemic
infection model.
To date, antibacterial activity of cannabinoids against Gram-negative organisms has largely
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been ruled out, since reported MICs values fall in the 100-200 µg/mL range . We confirmed this,
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obtaining MICs >128 µg/mL for all of the tested cannabinoids against the model Gram-negative
organism Escherichia coli. Given the observed action of CBG on the cytoplasmic membrane of MRSA,
we reasoned that CBG (and other cannabinoids) might be equally effective on the Gram-negative
counterpart, the inner membrane. Further, just as many antibacterial compounds fail to work against
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Gram-negative pathogens due to a permeability barrier , we reasoned that low permeability across the
outer membrane (OM) may be the reason for the poor efficacy of cannabinoids. Thus, we investigated
the antibacterial profile of the five major cannabinoids against E. coli, where their permeation was
facilitated through the OM by means of chemical perturbation. To this end, we set up checkerboard
assays to assess the interaction of CBG (Fig. 5a) and the four other main cannabinoids (Supplementary
Fig. 9) with the membrane perturbant, polymyxin B against E. coli. Remarkably, all five major
cannabinoids gained potent activity in the presence of sub-lethal concentrations of polymyxin B.
Indeed, all interactions were deemed synergistic (Fig. 5, Supplementary Fig. 9). For example, CBG,
which was inactive against E. coli (>128 µg/mL), was strongly potentiated when combined with a sub-
lethal concentration of polymyxin B (1 µg/mL in the presence of 0.062 µg/mL polymyxin B). A similar
synergistic interaction was observed with polymyxin B nonapeptide, a less toxic derivative of
polymyxin B that strictly perturbs the outer membrane in Gram-negative bacteria (Supplementary Fig.
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0), suggesting that induction of outer membrane permeability is sufficient to allow entry and activity
of CBG. We further assessed whether OM perturbation by genetic means would lead to similar results
by evaluating the activity of CBG against a number of strains where the OM was compromised (Fig.
5
b). In an E. coli ΔbamBΔtolC deletion strain, which renders E. coli hyperpermeable to many small
molecules, due to loss of BamB, a component of the β-barrel assembly machinery for OM proteins and
TolC, the efflux channel in the outer membrane, CBG had a MIC of 4 µg/mL, on par with its Gram-
positive activity. Similarly, in a hyperporinated, 9 strain of E. coli, where a recombinant pore was
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introduced in the OM and all nine known TolC-dependent transporters deleted , CBG activity became
evident with a MIC of 8 µg/mL. Finally, in an Acinetobacter baumannii deficient in
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lipooligosaccharide (LOS-), which effectively alters the permeability of the OM , CBG activity was
enhanced greater than 128-fold, resulting in a MIC value of 0.5 µg/mL. Overall, these results suggest
that cannabinoids face a permeability barrier in Gram-negative bacteria and further imply that
cannabinoids inhibit a bacterial process present in Gram-negative pathogens, and likely common to that
in Gram-positive pathogens.
To this end, we investigated whether CBG acted on the inner membrane (IM) of E. coli as well
as the OM. IM and OM permeability were determined, respectively, from ortho-Nitrophenyl-β-
galactoside (ONPG) and nitrocefin hydrolysis in an E. coli strain constitutively expressing a
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cytoplasmic β-galactosidase and a periplasmic β-lactamase while lacking the lactose permease, as
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described in the literature . As shown in Fig. 5c, CBG specifically acted on the IM, and only in the
presence of polymyxin B at a sub-lethal concentration that had minimal effects on the IM alone. We
observed that CBG (+polymyxin B) induced major permeability changes in the inner membrane,
indicated by a time-dependent marked increase in optical density values due to ONPG hydrolysis as a
result of unmasking the cytoplasmic β-galactosidase, which can only occur with destabilization of IM
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(Fig. 5c). CBG exhibited no action on the OM (Supplementary Fig. 11). Overall, the mechanism of
bacterial killing by CBG in E. coli is likely loss of IM integrity and requires antecedent OM
permeabilization.
Combination antibiotic therapy is becoming an increasingly attractive approach to combat
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resistance . So too is the strategy of using an OM perturbing molecule to facilitate the permeation of
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compounds that are otherwise active only on Gram-positive bacteria . We assessed the therapeutic
potential of the adjuvant polymyxin B in combination with CBG to inhibit the growth of priority Gram-
negative pathogens such as A. baumannii, E. coli, Klebsiella pneumoniae, and Pseudomonas
aeruginosa (Fig. 5d). We employed conventional checkerboard assays to determine the interaction and
potency of CBG and polymyxin B when used concurrently against various multi-drug resistant clinical
isolates. In all cases, synergy was evident, suggesting the potential for combination therapy of the
cannabinoids with polymyxin B against Gram-negative bacteria.
In summary, we have investigated the therapeutic potential of cannabinoids, specifically CBG,
through a comprehensive study of in vitro potency on biofilms and persisters, as well as mechanism of
action studies and in vivo efficacy experiments. Most notably, we have uncovered the hidden broad-
spectrum antibacterial activity of cannabinoids and demonstrated the potential of CBG against Gram-
negative priority pathogens. Taken together, our findings lend credence to the idea that cannabinoids
may be produced by Cannabis sativa as a natural defense against plant pathogens. Notwithstanding,
cannabinoids are well-established as drug compounds that have favourable pharmacological properties
in humans. The work presented here suggests that the cannabinoid chemotype represents an attractive
lead for new antibiotic drugs.
Methods
Strains and reagents. Supplemental Table 3 lists bacteria and plasmids used in this work. Supporting
Information S. aureus Table lists the 96 clinical isolates, along with body site location and year of
isolation. Bacteria were grown in cation-adjusted Mueller Hinton broth (CAMHB) at 37°C, unless
otherwise stated. Antibiotics were obtained from Sigma, Oakville, ON, Canada. Pure cannabinoid
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solutions purchased from Sigma, Oakville, ON, Canada were used for all experiments. Only CBG was
synthesized in larger quantity as described below.
Antimicrobial susceptibility testing. Minimum inhibitory concentration (MIC) determination and
checkerboard assays were conducted following the guidelines of CLSI for MIC testing by broth
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microdilution . Persister killing activity of cannabinoids was evaluated against stationary-phase cells
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of S. aureus as previously described .
B. subtilis CRISPRi essential gene knockdown strain collection screen. Overnight cultures of the
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collection (at a 96-well density, n = 289) were performed using the Singer rotor HDA (Singer
Instruments, United Kingdom) in CAMHB. Subsequently, CAMHB with or without CBG were
inoculated using the singer rotor at 96-well density. These experiments were performed either in the
presence of 0.05% xylose (allowing low level of dcas9 expression) or with no xylose induction
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(
basal dcas9 expression). The plates were incubated at 37 C and OD600 was read after 24 h.
S. aureus Nebraska Transposon Mutant Library (NTML) screen. Overnight cultures of the
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NTML (at a 384-well density) were performed using the Singer rotor HDA (Singer Instruments,
United Kingdom) in CAMHB containing erythromycin (5 µg/mL). Subsequently, CAMHB with or
without CBG were inoculated using the singer rotor at 384-well density. The plates were incubated at
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7 C and OD was read after 24 h. Cellular localization and functional (gene ontology, GO-term)
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enrichment analyses were performed using Pathway Tools software and MetaCyc database .
Selection of suppressor mutants of CBG activity in S. aureus. Spontaneous suppressor mutants were
selected for in liquid culture. Briefly, isolated colonies were resuspended in PBS and diluted to a final
OD600 of 0.05 into 200 μL of CAMHB containing CBG (at 4x and 8x MIC) set up in 96-well microtiter
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plates, 36 wells/concentration. Plates were incubated at 37 C for 4 days. Alternatively, bacteria were
treated with a 2-fold series of CBG concentrations spanning the MIC. Bacteria growing at the
maximum sub-MIC concentration were repeatedly passaged in a similar series of CBG concentrations
by 1000-fold dilution every 24 hours. Five CBG dilution series were performed simultaneously and the
cells were passaged for 15 days.
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General molecular techniques. DNA manipulations were performed as previously described . CaCl
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chemically-competent ML35 cells were transformed with pBR322 encoding a periplasmic β-lactamase.
Biofilm formation assays. Biofilm formation was performed in polystyrene 96-well plates in Tryptic
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Soy Broth (TSB) with 1% glucose and detected by the crystal violet method as previously described .
For MBEC determination, biofilms were allowed to form for 24 hours prior to washing off planktonic
cultures with sterile de-ionized water then treatment with CBG at varying concentrations.
Quantification of biofilm was performed as noted above.
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Membrane integrity assays. DiSC (5) assay was performed in S. aureus as previously described . To
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determine outer membrane and inner membrane activity of CBG against Gram-negative bacteria, we
performed β-lactamase and β-galactosidase assays, respectively. Overnight cultures of ML35 pBR322
in TSB with 50 μg/mL ampicillin were 100-fold diluted in fresh pre-warmed TSB and incubated at
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7 C at 220 rpm. Logarithmic phase cells were collected, washed twice in PBS and then resuspended in
PBS at a final OD600 of 0.01. Nitrocefin (30 μM) or ONPG (1.5 mM) - probes for β-lactamase and β-
galactosidase, respectively (final concentration) - were added to the bacterial suspension and
immediately aliquoted to dilution series of CBG and/or PMB at 100 μL final volume. Plates were
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incubated at 37 C and monitored kinetically for color change at 492 and 405 nm (for nitrocefin and
ONPG hydrolysis, respectively). Adequate no drug, no probe and/or cell-free controls were included.
Hemolysis assay. Hemolysis assay using red blood cells (defibrinated sheep blood, Thermo Fisher
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Scientific) was performed as previously described .
Statistical analyses. Statistical analyses were conducted with GraphPad Prism 5.0 and is indicated for
each assay in the figure caption. All results are shown as mean ±SEM unless otherwise stated. In the
case of MIC and checkerboard assays, the experiments were repeated at least three independent times
and the experiment showing the most conservative effects (if applicable) was shown.
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Synthesis of Cannabigerol.
OH
geraniol
OH
OH
TsOH
CH3Cl
r.t. 12 h
HO
HO
olivetol
28 % yield
CBG
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CBG was synthesized using a reported procedure . To a 25 mL round-bottomed flask containing a
magnetic stir were added olivetol (108 mg, 0.6 mmol), chloroform (5 mL), geraniol (174 µL, 1.0
mmo), p-toluene sulfonic acid monohydrate (19 mg, 0.1 mmol). The flask was covered with aluminum
foil and the reaction was stirred at room temperature in the dark for 12 hours at which point TLC
analysis indicated complete consumption of the olivetol substrate. To the reaction was added aqueous
saturated NaHCO (5 mL). The organic phase was removed and washed with water (5 mL). The
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aqueous layer was extracted with additional chloroform (5 mL) and the combined organic extracts were
dried over MgSO and concentrated en vacuo. The crude residue was purified via flash column
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chromatography on silica gel using gradient elution with hexanes and ethyl acetate. CBG was isolated
as an off white powder in 28 % yield (54 mg, 0.17 mmol). Spectral data can be found in Supplementary
Figs. 12-14.
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H NMR (700 MHz, CDCl ) δ 6.25 (s, 2H), 5.28 (tq, J = 7.1, 1.3 Hz, 1H), 5.09 – 5.04 (m, 3H), 3.40 (d,
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J = 7.1 Hz, 2H), 2.49 – 2.43 (m, 2H), 2.14 – 2.04 (m, 4H), 1.82 (s, 3H), 1.68 (s, 3H), 1.60 (s, 3H), 1.58
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–
1.54 (m, 2H), 1.36 – 1.28 (m, 4H), 0.89 (t, J = 7.0 Hz, 3H). C NMR (176 MHz, CDCl3) δ 154.92,
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42.89, 139.13, 132.19, 123.89, 121.84, 110.73, 108.52, 39.83, 35.65, 31.63, 30.93, 26.52, 25.81,
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2.68, 22.40, 17.83, 16.32, 14.16. HRMS (ESI) m/z: 315.2329 calcd for C H O [M-H]; 315.24490
21 31 2
obsd.
Mouse infection models. Animal experiments were conducted according to guidelines set by the
Canadian Council on Animal Care using protocols approved by the Animal Review Ethics Board at
McMaster University under Animal Use Protocol #17-03-10. Before infection, mice were relocated at
random from a housing cage to treatment or control cages. No animals were excluded from analyses,
and blinding was considered unnecessary. Seven- to nine-week old female CD-1 mice (Envigo) were
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infected intraperitoneally with 7.5 x 10 CFU of log-phase MRSA strain USA 300 JE2 with 5% porcine
mucin. Treatment of 100 mg/kg CBG or a vehicle solution (60% PEG300 and 5% DMSO) were
administered intraperitoneally immediately post-infection. Mice were euthanized and tissues collected
into phosphate buffered saline (PBS) at necropsy. Organs were homogenized using a high-throughput
tissue homogenizer, serially diluted in PBS, and plated onto solid LB. Plates were incubated overnight
at 37°C and colonies were quantified to determine organ load.
Supporting Information
Table S1, MIC data for various cannabinoids against MRSA; Table S2, chemical genomic interactions;
Table S3, strains and plasmids used; Figure S1, biofilm activities of cannabinoids; Figure S2, kill curve
with CBG; Figure S3, anti-persister activity of cannabinoids; Figure S4, chemical genomic interaction
analysis; Figure S5, checkerboard analysis with bicarbonate; Figure S6, hemolytic activity of CBG;
Figure S7, in vivo dose-dependence of CBG; Figure S8, body weight analysis with CBG; Figure S9,
checkerboard analysis of cannabinoids with polymyxin B against E. coli; Figure S10, checkerboard
analysis with polymyxin B nonapeptide; Figure S11, OM assay with CBG; Figure S12, H NMR
spectra of CBG; Figure S13, C NMR spectra of CBG; Figure S14, high resolution mass spectrum of
CBG.
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Abbreviations
A. baumannii, Acinetobacter baumannii; CBC. cannabichromene; CBD, cannabidiol; CBG,
cannabigerol; CBN, cannabinol; CLSI, Clinical and Laboratory Standards Institute; DiSC (5), 3,3'-
dipropylthiadicarbocyanine iodide; E. coli, Escherichia coli; K. pneumoniae, Klebsiella pneumoniae;
IM, inner membrane; MBEC, minimal biofilm eradication concentration; MIC, minimum inhibitory
concentration; MRSA, methicillin-resistant Staphylococcus aureus; OD, optical density; ONPG, ortho-
Nitrophenyl-β-galactoside; OM, outer membrane; P. aeruginosa, Pseudomonas aeruginosa; PmB,
polymyxin B; S. aureus, Staphylococcus aureus; THC, ∆ -tetrahydrocannabinol
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Author Information
Corresponding Author
*
E-mail: ebrown@mcmaster.ca
Eric D. Brown ORCID: 0000-0002-7624-8112
Author contributions M.A.F., O.M.E., R.T.G., and E.D.B. conceived and designed the research.
M.A.F. and O.M.E. performed all experiments and analyzed data with the exception of the mouse
infection model and the synthesis of CBG. C.R.M. and L.A.C. performed the mouse infection model.
X.Z. and N.G.J. optimized a scalable synthesis of CBG, supervised by J.M. M.A.F. and E.D.B. wrote
the paper, with large input from O.M.E. All authors approved the final version.
Conflict of Interest E.D.B., J.M., M.A.F., O.M.E., and R.T.G. are inventors on a patent application on
the use of cannabinoids for prevention and/or treatment of infections.
Acknowledgements This work was supported by a salary award to E.D.B from the Canada Research
Chairs program and operating funds to E.D.B from a CIHR Foundation grant (FDN-143215); by
a Michael G. DeGroote Centre for Medicinal Cannabis Research post-doctoral fellowship to O.M.E.
Synthetic chemistry was supported by McMaster’s Faculty of Health Sciences and the Michael G.
DeGroote Institute for Infectious Disease Research. We thank Shawn French for preparing the
graphical abstract.
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Susceptibility to Antibiotics by Targeting the Proton Motive Force. ACS Infect Dis 2018, 4 (3), 382-
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90. DOI: 10.1021/acsinfecdis.7b00194.
6. Deiana, S.; Watanabe, A.; Yamasaki, Y.; Amada, N.; Arthur, M.; Fleming, S.; Woodcock, H.;
Dorward, P.; Pigliacampo, B.; Close, S.; Platt, B.; Riedel, G., Plasma and brain pharmacokinetic profile
of cannabidiol (CBD), cannabidivarine (CBDV), Delta(9)-tetrahydrocannabivarin (THCV) and
cannabigerol (CBG) in rats and mice following oral and intraperitoneal administration and CBD action
on obsessive-compulsive behaviour. Psychopharmacology (Berl) 2012, 219 (3), 859-73. DOI:
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0.1007/s00213-011-2415-0.
7. Delcour, A. H., Outer membrane permeability and antibiotic resistance. Biochim Biophys Acta
009, 1794 (5), 808-16. DOI: 10.1016/j.bbapap.2008.11.005.
Krishnamoorthy, G.; Wolloscheck, D.; Weeks, J. W.; Croft, C.; Rybenkov, V. V.; Zgurskaya,
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H. I., Breaking the Permeability Barrier of Escherichia coli by Controlled Hyperporination of the Outer
Membrane. Antimicrob Agents Chemother 2016, 60 (12), 7372-7381. DOI: 10.1128/AAC.01882-16.
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baumannii in the absence of endotoxin. Mol Microbiol 2018, 107 (1), 47-56. DOI: 10.1111/mmi.13872.
0. Lehrer, R. I.; Barton, A.; Ganz, T., Concurrent assessment of inner and outer membrane
Powers, M. J.; Trent, M. S., Expanding the paradigm for the outer membrane: Acinetobacter
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permeabilization and bacteriolysis in E. coli by multiple-wavelength spectrophotometry. J Immunol
Methods 1988, 108 (1-2), 153-8.
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bacteria. Trends Biotechnol 2013, 31 (3), 177-84. DOI: 10.1016/j.tibtech.2012.12.006.
2. Stokes, J. M.; MacNair, C. R.; Ilyas, B.; French, S.; Cote, J. P.; Bouwman, C.; Farha, M. A.;
Worthington, R. J.; Melander, C., Combination approaches to combat multidrug-resistant
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Sieron, A. O.; Whitfield, C.; Coombes, B. K.; Brown, E. D., Pentamidine sensitizes Gram-negative
pathogens to antibiotics and overcomes acquired colistin resistance. Nat Microbiol 2017, 2, 17028.
DOI: 10.1038/nmicrobiol.2017.28.
33.
CLSI, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow
Aerobically; Approved Standard—Ninth Edition. CLSI document M07-A9. Clinical and Laboratory
Standards Institute: Wayne, PA, 2012.
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against gram-negative enteric bacteria. Antimicrob. Agents Chemother. 1996, 40 (8), 1801-1805.
5. Kim, W.; Zhu, W.; Hendricks, G. L.; Van Tyne, D.; Steele, A. D.; Keohane, C. E.; Fricke, N.;
Vaara, M.; Porro, M., Group of peptides that act synergistically with hydrophobic antibiotics
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Conery, A. L.; Shen, S.; Pan, W.; Lee, K.; Rajamuthiah, R.; Fuchs, B. B.; Vlahovska, P. M.; Wuest, W.
M.; Gilmore, M. S.; Gao, H.; Ausubel, F. M.; Mylonakis, E., A new class of synthetic retinoid
antibiotics effective against bacterial persisters. Nature 2018, 556 (7699), 103-107. DOI:
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0.1038/nature26157.
6. Caspi, R.; Billington, R.; Fulcher, C. A.; Keseler, I. M.; Kothari, A.; Krummenacker, M.;
Latendresse, M.; Midford, P. E.; Ong, Q.; Ong, W. K.; Paley, S.; Subhraveti, P.; Karp, P. D., The
MetaCyc database of metabolic pathways and enzymes. Nucleic Acids Res 2018, 46 (D1), D633-D639.
DOI: 10.1093/nar/gkx935.
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7.
Sambrook, J.; Fritsch, E. F.; Maniatis, T., Molecular cloning: a laboratory manual. 2nd ed.;
Cold Spring Harbor Laboratory: Cold Spring Harbor, New York, 1990.
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Protoc Microbiol 2005, Chapter 1, Unit 1B 1. DOI: 10.1002/9780471729259.mc01b01s00.
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Farha, M. A.; Verschoor, C. P.; Bowdish, D.; Brown, E. D., Collapsing the proton motive force
to identify synergistic combinations against Staphylococcus aureus. Chem Biol 2013, 20 (9), 1168-78.
DOI: 10.1016/j.chembiol.2013.07.006.
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Barker, W. T.; Chandler, C. E.; Melander, R. J.; Ernst, R. K.; Melander, C., Tryptamine
derivatives disarm colistin resistance in polymyxin-resistant gram-negative bacteria. Bioorg Med Chem
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019, 27 (9), 1776-1788. DOI: 10.1016/j.bmc.2019.03.019.
1. Taura, F.; Morimoto, S.; Shoyama, Y., Purification and characterization of cannabidiolic-acid
synthase from Cannabis sativa L.. Biochemical analysis of a novel enzyme that catalyzes the
oxidocyclization of cannabigerolic acid to cannabidiolic acid. J Biol Chem 1996, 271 (29), 17411-6.
DOI: 10.1074/jbc.271.29.17411.
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Table 1. Physiochemical properties of CBG as calculated by ACD/Percepta software..
OH
CBG HO
Phys Chem Profile
Lead-like?
MW
316.48
good
Log P
6.74
very lipophilic
0
1
2
3
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9
0
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0
H-Donors
2
2
9
1
good
good
good
good
H-Acceptors
Rotatable Bonds
Rings
Predicted solubility 0.0003 mg/mL highly insoluble
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OH
H
OH
OH
OH
OH
H
R1
R2
R1
R
R
H
H
R2
R2
O
O
HO
O
HO





R1
R2

CBD
R1
R1

CBC
R1
6
CBN
9-THC
9-THCA
H
n-pentyl
n-pentyl
n-propyl
H
n-pentyl
CBG
H
H
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CO2H
H
CBDA CO2H n-pentyl
CBDV
n-propyl
CBDVA CO2H n-propyl
CBGA CO2H
CBCA CO2H
9-THCV
9-THCVA
H
OH
O
O
OH

CO2H n-propyl
OH
OH
OH
0
1
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0
OH
OH
H
H
H
O
H
O
O
O
CBL
(±) 11-hydroxy-9-THC
(±) 11-nor-9-carboxy-9-THC

8-THC
exo-THC
Fig. 1. Structures of the cannabinoids surveyed in this study.
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Fig. 2. Cannabigerol (CBG) is a potent antibacterial, anti-biofilm and anti-persister cannabinoid. a, Chemical
structure of CBG b, Concentration dependence for inhibition of MRSA biofilm formation by CBG. Shown is the
average A600nm measurements of crystal violet stained biofilms and normalized by the OD600 of planktonic cells
with error bars representing one standard error of the mean, S.E.M. (nꢁ=ꢁ4). c, Minimum biofilm eradication
concentration of CBG. CBG is capable of eradicating pre-formed biofilms at a concentration of 4µg/mL (n=8).d,
Time-kill curve of S. aureus USA300 persisters by CBG compared to oxacillin shown as mean ±S.E.M (n=4).
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CBG rapidly eradicated a population of ~10 CFU/ml MRSA persisters to below the detection threshold within
30 minutes of treatment. On the other hand, the β-lactam oxacillin at 160 µg/mL (5x MIC) did not show any
activity against the same population of persisters. e, MIC distribution of CBG against clinical isolates of
9
0
MRSA (n=96). The MIC is 4 µg/mL.
9
0
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Spontaneous
Resistance
Agar plate with
CBG
No CBG
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0
Serial Passages
(Adaptive
Resistance)
CBG concentration
200
150
Knockdown
collection of
Essential genes
CBG 0.5
CBG 1
100
50
0
0
100
200
300
Clone
CBG 1

g/ml
1
.50
.25
1
1.00
Tn mutant
collection of Non-
essential genes
0.75
0.50
0.25
0.00
0
500
1000
1500
2000
NTML clones
70000
6
5
0000
0000
40000
0000
3
Membrane
Integrity Assay
20000
10000
0
0
0.25 0.5
1
2
4
CBG (µg/mL)
Fig. 3. CBG is active on the cytoplasmic membrane of MRSA. Overview of strategies for mechanism of action
determination, culminating in the finding that CBG is active on the cytoplasmic membrane, as determined by
dose-dependent increases in DiSC (5) fluorescence.
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Fig. 4. CBG is efficacious in a systemic mouse model of S. aureus infection. A single-dose treatment is
−
1
administered immediately post-infection: vehicle control (n=7, i.p.), CBG (n=7, 100 mgꢀkg , i.p.) or
−
1
vancomycin control (n=5, 100 mgꢀkg , i.p.). Colony-forming units (CFU) within spleen tissue were enumerated
at 7 h post-infection. CFUs within spleen tissue were also enumerated at an early time point of 30 minutes post-
infection following vehicle control treatment (n=5, i.p.). Horizontal lines represent the geometric mean of the
bacterial load for each treatment group. Administration of CBG resulted in a 2.8-log reduction (pꢀ<ꢀ0.001,
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Mann–Whitney U-test) in CFU when compared to the vehicle control.
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.5
0.3
0.1
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.25
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1
Polymyxin B (µg/mL)
8
4
2
1
8
8
4
2
1
ii
Polymyxin B (µg/mL)
4
2
b
1
MIC (µg/mL)
0
.5
0.5
0.3
0.1
0
0
.5
0.3
Wild-type E. coli
>128
4
0.3
0
.1
0
0
.1
0
E. coli ΔbamBΔtolC
hyperporinated, 9 E. coli
A. baumannii
0
0.005 0.01 0.03 0.06 0.1 0.3 0.5
0
0.005 0.01 0.03 0.06 0.1 0.3 0.5
0.005
0.01 0.03 0.06 0.1 0.3 0.5
0
8
>64
8
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4
8
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2
1
iii
iv
4
2
1
A. baumannii LOS-
0.5
2
1
c
0.5
0.5
0.3
0.1
0
0.5
0.3
0.1
0
0
0
.3
.1
0
1.00
0.75
0
0.01 0.03 0.06 0.1 0.3 0.5
1
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0.01 0.03 0.06 0.1 0.3 0.5
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0
0.005 0.01 0.03 0.06 0.1 0.3 0.5
0
.50
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2
1
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0
.25
2
1
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0
.00
0.5
0.5
0.3
0.1
0
0.5
0.3
0.1
0
2
4
6
8
0
0
.3
.1
0
Time, hr
0
0.005 0.01 0.03 0.06 0.1 0.3 0.5
0
0.01 0.03 0.06 0.1 0.3 0.5
1
0
0.005 0.01 0.03 0.06 0.1 0.3 0.5
Fig. 5. CBG is active against Gram-negative bacteria whose outer membrane is permeabilized, where it acts on
the inner membrane. a, Checkerboard analysis of CBG in combination with polymyxin B against E. coli. The
extent of inhibition is shown as a heat plot, such that the darkest blue color represents full bacterial growth. b,
CBG becomes active against Gram-negative bacteria in various genetic backgrounds where the outer membrane
is compromised. c, CBG acts on the IM of E. coli but only in the presence of sub-lethal concentration of
polymyxin B (PmB), unmasking cytoplasmic β-galactosidase leading to hydrolysis of ONPG as detected via
absorbance reads at 405 nm over time. Conditions were as follows: control (circles), CBG 2 µg/mL (squares),
PmB 0.125 µg/mL (triangles) and PmB 0.125 µg/mL + CBG 2 µg/mL (inverted triangles). d, CBG in
combination with polymyxin B against multi-drug resistant clinical isolates of i, A. baumannii, ii, E. coli, iii, K.
pneumoniae, iv, P. aeruginosa. The extent of inhibition is shown as a heat plot, such that the darkest blue color
represents full bacterial growth.
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