Since the discovery of penicillin in 1928 by Sir Alexander Fleming, antibiotics have saved millions of lives from fatal infections world-wide.
However, with time bacteria have developed mechanisms to escape the effects of antibiotics-they have become resistant.
With fewer antibiotics available to treat resistant bacterial infections, the possibility of entering a pre-antibiotic era is looming ahead.
Alternative strategies are being explored and helper compounds are attracting attention. Helper compounds are non-antibiotic compounds with the capability of enhancing the efficacy of antibiotics.
How to boost antibiotics
One such helper compound has been suspected to be cannabidiol (CBD); a cannabinoid from the cannabis plant. Now a research team from University of Southern Denmark, has published a scientific study proving the effect of CBD.
Janne Kudsk Klitgaard is Principal Investigator and corresponding author. First author is Ph.D. student Claes Søndergaard Wassmann. The study is published in the journal Scientific Reports.
When we combined CBD and antibiotics, we saw a more powerful effect than when treating with antibiotics alone. So, in order to kill a certain number of bacteria, we needed less antibiotics, they say.
Bacteria clones spread globally
In the study, CBD was used to enhance the effect of the antibiotic bacitracin against Staphylococcus aureus bacteria; a major human pathogen that frequently causes community- and hospital-acquired disease.
Multidrug-resistant clones of this pathogen have spread globally. In some countries, treatment of bacterial infections with these resistant bacteria are difficult and the problem is projected to be an ever-larger problem in the future.
According to the researchers, the combination of CBD and antibiotics may be a novel treatment of infections with antibiotic resistant bacteria.
How do the bacteria die?
Three things happened with the Staphylococcus aureus bacteria, when the researchers treated them with the combination in their study:
- The bacteria could no longer divide normally.
- The expression of certain key genes (cell division and autolysis genes) in the bacteria was lowered.
- The bacterial membrane became unstable.
Anti-resistance must be stopped
According to the researchers, overuse of antibiotics is the main cause of antibiotic resistance.
If we combine an antibiotic with a helper compound, that enhances the effect of the antibiotic, we need less antibiotic to achieve the same effect. This may contribute to the development of fewer resistant bacteria, says Janne Kudsk Klitgaard.
Since the discovery of penicillin in 1928 by Sir Alexander Fleming, antibiotics have saved millions of lives from fatal infections world-wide. However, with time bacteria have developed mechanisms to escape the effects of antibiotics. The amount of antibiotics used seems to be directly related to development of antibiotic resistance.
Similarly, a growing number of multi drug resistant (MDR) bacteria is a result of inadequate intentions to solve the resistance problem and increasing unmet demands for new antibacterial drugs1.
With fewer antibiotics available to treat MDR bacterial infections, the possibility of entering a pre-antibiotic era is looming ahead. Alternative strategies are being explored and helper compounds, also known as antibiotic potentiators or resistant breakers, are attracting attention2.
Helper compounds are non-antibiotic compounds functioning as adjuvants for antibiotics to operate in synergy through various mechanisms including efflux pump inhibition, inhibition of enzymes, and changes in membrane permeability, all of which may contribute to increasing the efficacy of a specific antibiotic 3,4.
Drugs found to contain helper compound properties are normally used for treatment of non-infectious diseases but may contain some degree of antibacterial activity itself5. Helper compounds are usually associated with side-of-action in the central and peripheral nervous system as local anaesthetics and in psychopharmaceutic practice, where they usually block membrane associated transporter activity5.
Overuse of antibiotics is the main cause of antibiotic resistance. Therefore, by combining an antibiotic with a helper compound less antibiotic is needed in order to achieve bacterial growth inhibition or killing compared to using the antibiotic alone. This strategy may therefore decrease the likelihood of resistance development, and investigations to identify efficient helper compounds are thus important.
Cannabinoids are categorised as either endogenous cannabinoids, which are cannabinoids produced by the human body, or exogenous cannabinoids, which are produced either by plants such as Cannabis sativa or synthetically.
Cannabinoids act on the endocannabinoid system of the human body6 consisting of two G-protein coupled receptors (GPCR). These are named cannabinoid type 1 and 2 (CB1 and CB2) receptors, and, depending on the specific cannabinoid, the binding results in either an agonistic or antagonistic downstream effect7.
Besides endocannabinoids being ligands for the endocannabinoid receptors, exogenous cannabinoids are also ligands for the receptors. One of the best characterised exogenous ligands is tetrahydrocannabinol (THC). It is a partial agonist for both CB1 and CB2 receptor mediating effects such as analgesia, muscle relaxation, and antiemetic effects, but also results in negative effects such as anxiety, psychosis, and sedation.
Another exogenous cannabinoid is cannabidiol (CBD), which has been observed to decrease the adverse negative effects of THC. CBD is an antagonist of both CB1 and CB2 receptor leading to anti-sedative, anti-psychotic, and anxiolytic effects7. However, these are not the only known effects of CBD, as it is able to cause a variety of different effects such as inhibition of cancer cell growth8, neuroprotection in both neuro-degenerative diseases such as Parkinson’s Disease9 and post-ischemia10, and anti-inflammatory effects as in type-1 diabetes11.
Not much is known regarding antimicrobial effects of cannabinoids and even less on the mechanism of action. Endocannabinoids and exogenous cannabinoids such as CBD have been observed to inhibit growth of bacteria12–14, yet the use of cannabidiol as an antibiotic adjuvant has not been studied so far.
In the present study, we aim to characterise cannabidiol as a potential helper compound against resistant bacteria in combination with the cyclic peptide antibiotic bacitracin (BAC). BAC is a mixture of related cyclic peptides operating as a bactericidal antibiotic by interfering with the cell wall and interrupting the biosynthesis of the peptidoglycan leading to cell lysis15.Go to:
The combination of CBD and BAC is effective against Gram-positive bacteria
Initially, we validated the antimicrobial effect of cannabidiol (CBD) against the Gram-positive bacterium Methicillin-Resistant Staphylococcus aureus (MRSA) as previously published by Appendino and colleagues14 but also for Enterococcus faecalis (E. faecalis), Listeria monocytogenes (L. monocytogenes), and Methicillin-Resistant Staphylococcus epidermidis (MRSE).
We found the Minimum Inhibitory Concentration (MIC) to be 4 µg/mL for S. aureus, L. monocytogenes, and the MRSE strain and 8 µg/mL for E. faecalis, indicating that Gram-positive bacteria are sensitive towards CBD (Table 1).
*FIC index indicates synergy when FIC index ≤ 0.5, indifference when 0.5 < FIC index < 4, and antagonism when FIC index > 437.
|Strain||MIC CBD (µg/mL)||MIC BAC (µg/mL)||Fold reduction in MIC of BAC when combined with ½xMIC CBD||FIC Index*|
|MRSA USA300 FPR3757||4||64||32–64||0.5|
|E. faecalis (13–327129)||8||64||≥64||0.375|
|L. monocytogenes (EGD)||4||512||8||0.625|
|MRSE (933010 3F-16 b4)||4||32||64||0.5|
To determine whether CBD would induce a higher susceptibility of BAC in Gram-positive bacteria, MICs of BAC were determined for the four Gram-positive bacteria in the presence of CBD. Remarkably, the MIC of BAC was decreased by 8 to at least 64-fold when combined with 1/2 x MIC of CBD compared to MIC of BAC alone in the different Gram-positive strains (Table 1). Furthermore, the Fractional Inhibitory Concentration (FIC) index was determined for each Gram-positive bacteria.
The results showed a FIC index at 0.5 for both MRSA USA300 and MRSE and 0.375 for E. faecalis indicating weak synergistic effect between the compounds CBD and BAC (Table 1). After combining CBD with other antibiotics, both similar and different types, we concluded that CBD had the best effect together with BAC (see Supplementary Figure S1).
To assess the potentiating effect of CBD on BAC over time, measurements of bacterial growth over 24 hours in the presence of either CBD alone or in combination with BAC were performed. The assessment concentrations of CBD were at 2 µg/mL and 8, 16, and 32 µg/mL for BAC. As seen in Fig. 1a, growth of S. aureus is inhibited by 2 µg/mL CBD and 16 µg/mL BAC combined compared to monotherapies of the individual compounds.
The results suggest that CBD can potentiate the antimicrobial effects of BAC. Similarly, growth measurements of E. faecalis, MRSE, and L. monocytogenes on monotherapies and combination (Fig. 1b–d), suggests that the combination of CBD and BAC is useful against other Gram-positive bacteria.
To clarify whether CBD and BAC act in synergy, time-kill assays were performed (Fig. 1e). CBD and BAC reduced the viability by 6 log10 CFU/mL compared to CBD alone. The result shows that a clear synergistic effect in fact exists between CBD and BAC, and that the effect is bactericidal. The slight re-initiation of growth after 8 hours is almost certainly caused by degradation or oxidation of the cannabinoid16.
To verify that the decreased CFU upon combination treatment is caused by killing of the bacteria and not due to clustering of the cells, microscopy was performed at the time 1, 2, 4, and 8 hours post treatment (Supplementary Figure S2). Images show no additional clustering of the cells treated with the combination compared to the other treatments.
To further assess the spectrum of use for the combination of CBD and BAC, growth of Gram-negative bacteria upon treatment was measured as well. The Gram-negative bacteria tested were strains of Pseudomonas aeruginosa, Salmonella typhimurium, Klebsiella pneumoniae, and Escherichia coli (Supplementary Figure S3).
Experiments for CBD and BAC against the Gram-negative bacteria revealed MIC values above 128 µg/mL for all tested bacteria, presumably due to the outer membrane. In addition, the experiments did not reveal any synergy between CBD and BAC in the concentrations tested, limiting the use of the combination to Gram-positive bacteria.
CBD and BAC causes morphological changes
We have established that CBD can potentiate the effect of BAC in Gram-positive bacteria. The next step is to study the mechanism underlying this synergy. First, we looked at the morphological changes of S. aureus USA300 upon exposure to CBD and/or BAC by treating a culture at start exponential phase for 2.5 hours and then performing transmission electron microscopy (TEM) of the cells.
Results showed that CBD and BAC alone caused no morphological changes, as they resembled untreated control and EtOH control. However, as seen in Fig. 2a, treatment with the combination of CBD and BAC resulted in large undivided cells with several septa formations or several initiations of septum formation indicating severe defects in cell division (red arrows) and irregularities around cell envelope (green arrow) (Supplementary Figure S4).
The result was confirmed by staining the Penicillin Binding Proteins (PBPs) in the membrane using Bocillin-FL, a fluorescence-conjugated penicillin V derivative (Fig. 2b) which showed similar morphology (red arrows). As peptidoglycan synthesis occurs both at the septal and peripheral cell wall, we can observe irregularities concerning the peptidoglycan all over the cell surface17. Upon exposure to either CBD or BAC alone regular septum formations were visualised, however, when treated with the combination, several septa formations appeared for some of the cells as visualised by Bocillin-FL as in the TEM images.
This suggests that the combination of CBD and BAC affects the cell envelope causing irregular cell division visualised by multiple septa formations and irregular cell membrane. To study whether the cell division defect is specific for the combination of CBD and BAC, microscopy analysed using higher concentrations of CBD and BAC at 4 and 64 µg/mL, respectively, was performed (Supplementary Information Figure S5).
Images show cells with multiple septa upon treatment with 64 µg/mL BAC, indicating that the effect visualised is not specific to the combination of CBD and BAC. However, it further emphasises the CBD mediated potentiation of BAC, since this phenotype did not appear at lower BAC concentration. Adding a higher concentration of CBD did not seem to cause any division defects.
he combination of CBD and BAC decreases autolysis in S. aureus
Since treatment with the combination of CBD and BAC shows impaired cell division, probably causing an arrest in cell division and potentially decreased cell wall turnover, one could speculate if this would result in decreased autolysis as well.
Therefore, a Triton X-100 induced autolysis assay was performed. S. aureus USA300 were grown until start exponential phase and stressed for one hour with CBD, BAC, CBD+BAC, EtOH or left untreated. Cells were then washed and incubated with or without triton X-100. As suspected, upon treatment with the combination of 1 µg/mL CBD and 16 µg/mL BAC a significant decreased autolysis was observed (Fig. 3) compared to the untreated control from 90 to 300 minutes except at the 150 minute timepoint, indicating cell division arrest.
CBD causes depolarisation of the cytoplasmatic membrane
Since the analysis of the muropeptide composition did not reveal any changes, we investigated the membrane. To evaluate effects on the bacterial membrane, the membrane potential was measured when exposed to either CBD, BAC, or the combination of the two (Fig. 5).
Accumulation of the fluorescent dye DiOC2(3) in healthy bacteria cells with intact membrane potential results in red fluorescence (high red/green ratio), whereas lower concentrations of the dye, due to membrane potential disruption, exhibit green fluorescence (low red/green ratio), as visualised for the depolarised control using CCCP.
Thus, the ratio between red and green fluorescence can reveal the state of the membrane potential. As shown in Fig. 5, even very low concentrations of CBD at 0.1 and 0.2 µg/mL as well as concentration of BAC at 16 µg/mL resulted in a significant lower red/green fluorescence ratio compared to either the untreated or the EtOH control indicating disruption of the membrane potential. However, combining BAC with CBD at either 0.1 or 0.2 µg/mL did not show any significant further membrane depolarisation compared to either CBD or BAC alone.
In this study, we present a putative novel antimicrobial combination for treatment of Gram-positive bacterial infections using the cannabinoid cannabidiol and the cell wall targeting antibiotic bacitracin.
Through growth experiments, it was interestingly found that CBD was able to potentiate the effects of BAC against S. aureus USA300 and other Gram-positive bacteria. However, it was found to be ineffective against Gram-negative bacteria. Upon treatment with the combination of CBD and BAC, it was revealed by TEM that the morphology of the cells had changed compared to cells treated with either CBD or BAC alone or left untreated. The cells showed several septa formations indicating lack of cell separation during cell division causing reduced autolysis, as well as an irregular membrane.
In addition to this, a very important cell division gene, ezrA, turned out to be transcriptionally down regulated upon combination treatment. Changes observed in morphology was not caused by compositional changes in the cell wall muropeptide composition.
Membrane potential changes for the combination of CBD and BAC compared to either CBD or BAC treatment alone did not reveal the mechanism of action for the combination of CBD and BAC. Future studies are therefore focused on the cell division and cell envelope to identify the mechanism of action.
More information: Claes Søndergaard Wassmann et al, Cannabidiol is an effective helper compound in combination with bacitracin to kill Gram-positive bacteria, Scientific Reports (2020). DOI: 10.1038/s41598-020-60952-0