Cannabidiol Is a Novel Modulator of Bacterial Membrane Vesicles

Membrane vesicles (MVs) released from bacteria participate in cell communication and host-pathogen interactions. Roles for MVs in antibiotic resistance are gaining increased attention and in this study we investigated if known anti-bacterial effects of cannabidiol (CBD), a phytocannabinoid from Cannabis sativa, could be in part attributed to effects on bacterial MV profile and MV release. We found that CBD is a strong inhibitor of MV release from Gram-negative bacteria (E. coli VCS257), while inhibitory effect on MV release from Gram-positive bacteria (S. aureus subsp. aureus Rosenbach) was negligible. When used in combination with selected antibiotics, CBD significantly increased the bactericidal action of several antibiotics in the Gram-negative bacteria. In addition, CBD increased antibiotic effects of kanamycin in the Gram-positive bacteria, without affecting MV release. CBD furthermore changed protein profiles of MVs released from E. coli after 1 h CBD treatment. Our findings indicate that CBD may pose as a putative adjuvant agent for tailored co-application with selected antibiotics, depending on bacterial species, to increase antibiotic activity, including via MV inhibition, and help reduce antibiotic resistance.


INTRODUCTION
Outer membrane vesicles (OMVs) and membrane vesicles (MVs) are released from Gram-negative and Gram-positive bacteria and participate in inter-bacterial communication, including via transfer of cargo molecules (Dorward and Garon, 1990;Li et al., 1998;Fulsundar et al., 2014;Jan, 2017;Toyofuku et al., 2019). MVs are released in greater abundance from Gram-negative, than Gram-positive bacteria and their production seems crucial for bacterial survival and forms part of the stress response (McBroom and Kuehn, 2007;Macdonald and Kuehn, 2013;Jan, 2017). Gram-negative bacteria generate, in addition to common one-bilayer vesicles (OMV), also double-bilayer vesicles (O-IMVs), and in some stress conditions other types of MVs (Pérez-Cruz et al., 2016) and therefore we will use the umbrella term "membrane vesicles" (MVs) hereafter. MVs are important in biofilm formation and dissemination of toxins in the host (Wang et al., 2015;Cooke et al., 2019).
MVs participate in host-pathogen interactions (Gurung et al., 2011;Koeppen et al., 2016;Bitto et al., 2017Bitto et al., , 2018Codemo et al., 2018;Turner et al., 2018;Cecil et al., 2019) and may also be involved in antibiotic resistance, for instance by protecting biofilms from antibiotics via increased vesiculation (Manning and Kuehn, 2011). Furthermore, MVs from Porphyromonas gingivalis have been linked to metabolic remodeling in the host (Fleetwood et al., 2017), while MVs from Neisseria gonorrhoeae have been shown to target host mitochondria and to induce macrophage death (Deo et al., 2018). Besides roles for cellular and bacterial communication, the use of MVs as nano-carriers for various compounds, including for antibiotic and vaccine delivery, has also raised considerable interest in the research community (Gnopo et al., 2017;Rüter et al., 2018;Tan et al., 2018;Wang et al., 2018).
The regulation of bacterial MV biogenesis and release may therefore be of great importance, both in relation to interbacterial communication, including biofilm formation, their host interactions as commensals, as well as in host-pathogen interactions and in antibiotic resistance.
Cannabidiol (CBD) is a phytocannabinoid from Cannabis sativa with anti-inflammatory (Martin-Moreno et al., 2011), anticancerous (Pisanti et al., 2017;Kosgodage et al., 2018) and anti-bacterial activity (Hernández-Cervantes et al., 2017). While immunoregulatory roles for cannabinoids have been reported in infectious disease (reviewed in Hernández-Cervantes et al., 2017), and C. sativa has been identified as a natural product with a capability of controlling bacterial infections, including a strong anti-bacterial activity against antibiotic resistant strains (Appendino et al., 2008), a link between CBD and bacterial MV release has hitherto not been investigated.
As our recent work identified CBD as a potent inhibitor of extracellular vesicle (EV) release in eukaryotes (Kosgodage et al., 2018;Gavinho et al., 2019), we sought to investigate whether CBD may work via phylogenetically conserved pathways, involving bacterial MV release from bacteria. As we, and other groups, have previously shown that cancer cells can be sensitized to chemotherapeutic agents via various EV-inhibitors (Jorfi et al., 2015;Koch et al., 2016;Muralidharan-Chari et al., 2016;Kosgodage et al., 2017), including CBD (Kosgodage et al., 2018(Kosgodage et al., , 2019, we sought to establish whether in bacteria, similar putative MV modulatory effects could be utilized to sensitize bacteria to antibiotics.

MATERIALS AND METHODS
MV Isolation From E. coli VCS257 and S. aureus subsp. aureus Rosenbach E. coli (VCS257, Agilent, La Jolla, CA) and S. aureus subsp. aureus Rosenbach (ATCC 29247, USA) static cultures were grown in Luria-Bertani (LB) broth for 24 h at 37 • C. The growth phase before vesicle isolation was exponential; the volume of the cultures was 20 ml. For MV isolation, ultracentrifugation and nanoparticle tracking analysis (NTA) were used based on previously established methods by other groups (McCaig et al., 2013;Klimentova and Stulik, 2015;Roier et al., 2016). E. coli and S. aureus cultures were maintained by plating on Mueller-Hinton agar plates and weekly sub-culturing was performed according to previously established methods (Iqbal et al., 2013).
Before MV isolation, all bacterial growth medium (LB broth) was pre-treated before use by ultracentrifugation at 100,000 g for 24 h to ensure minimum contamination with extracellular vesicles (EVs) from the medium (Kosgodage et al., 2017).
For MV isolation, bacteria were grown in EV-free medium (as described above) for 24 h at 37 • C, the culture medium was collected and centrifuged once at 400 g for 10 min for removal of cells, followed by centrifugation at 4,000 g for 1 h at 4 • C to remove cell debris. The resultant supernatant was then centrifuged for 1 h at 100,000 g at 4 • C and the isolated MV pellet was resuspended in Dulbecco's phosphate buffered saline (DPBS; ultracentrifuged and sterile filtered using a 0.22 µm filter) and centrifuged again at 100,000 g for 1 h at 4 • C. The resulting MV pellet was sterile filtered (0.45 µm) once and then resuspended in sterile filtered DPBS. The quantitative yield of vesicles was ∼6.5 × 10 9 MVs per liter of culture. The isolated MV pellets were then either used immediately, or stored at −80 • C for further experiments.

Transmission Electron Microscopy (TEM) Imaging of Bacterial MVs
A suspension of isolated MVs (1.4 × 10 8 MVs/ml) was used for TEM imaging. MV samples (10 µL) were applied to mesh copper grids, prepared with glow discharged carbon support films, and incubated for 2 min. The grids were then washed five times with 50 µl of 1 % aqueous uranyl acetate. Grids were left to dry for 5 min before being viewed. Micrographs were taken with a JEOL JEM 1230 transmission electron microscope (JEOL, Japan) operated at 80 kV at a magnification of 80,000 to 100,000. Digital images were recorded using a Morada CCD camera (EMSIS, Germany) and processed via iTEM (EMSIS).

Western Blotting
Protein was isolated from MV pellets using Bacterial Protein Extraction Reagent (B-PER, ThermoFisher Scientific, U.K.), pipetting gently and shaking the pellets on ice for 2 h, where after samples were centrifuged at 16,000 g at 4 • C for 20 min and the resulting supernatant collected for protein analysis. Samples were prepared in 2x Laemmli buffer, boiled at 95 • C for 5 min, electrophoresed by SDS-PAGE on 4-20 % TGX gels (BioRad, U.K.), followed by semi-dry Western blotting. Approximately 10 µg of protein was loaded per lane and even protein transfer was assessed by Ponceau S staining (Sigma, U.K.). Blocking of membranes was performed for 1 h at room temperature (RT) in 5 % BSA in TBS-T. The membranes were then incubated with the anti-OmpC (Outer-membrane protein C antibody; orb6940, Biorbyt, U.K.; diluted 1/1000 in TBS-T) overnight at 4 • C, followed by washing in TBS-T and incubation for 1 h in antirabbit-HRP conjugated secondary antibody at RT. Visualization was performed using ECL (Amersham, U.K.) and the UVP BioDoc-ITTM System (U.K.). Nanoparticle Tracking Analysis for Assessment of MV Release From E. coli VCS257 and S. aureus subsp. aureus Rosenbach MVs were isolated from control and CBD-treated bacterial cultures as described above. Nanoparticle tracking analysis (NTA) was performed using the Nanosight LM10 (Malvern, U.K.), equipped with a 405 nm diode laser and a sCMOS camera. MV pellets were resuspended in equal volumes (100 µl) of DPBS before NTA analysis to ensure comparable analysis of quantification. Before application, samples were diluted 1:50 in sterile-filtered EV-free DPBS and applied at a constant flow rate, maintaining the number of particles in the field of view in the range of 20-40 with a minimum concentration of samples at 5 × 10 7 particles/ml. Camera settings were according to the manufacturer's instructions (Malvern), five 60 s videos per sample were recorded and replicate histograms averaged. Each experiment was repeated three times.
CBD-Mediated MV Release Inhibition in E. coli VCS257 and S. aureus subsp. aureus Rosenbach E. coli and S. aureus cultures were cultivated using EV-free Müeller-Hinton broth for 24 h. An inoculate of 0.1 ml of bacteria, in a 20 ml culture volume of bacterial growth medium (Luria-Bertani (LB) broth), were grown at exponential phase overnight, as assessed by OD600. The bacterial cells were then washed using DPBS at 4,000 g for 10 min and seeded in 1.5 ml triplicates in micro centrifuge tubes. For treatment with CBD, CBD (GW research Ltd) was applied at concentrations of 1 or 5 µM and incubated with the bacterial cultures for 1 h at 37 • C. Treatments were performed in triplicates, including DMSO as a control. MV isolation following CBD and control treatment was carried out using step-wise centrifugation and ultracentrifugation as before. Changes in MV release were assessed by quantifying numbers of MVs by NTA analysis as described above, with each experiment repeated three times. Cell viability was assessed before the start of every experiment and after treatment with CBD compared to controls determined by colony forming unit (CFU) measurement.

Disc Diffusion Test for Assessment of CBD-Mediated Enhancement of Antibiotic Treatment
Discs were impregnated with the following antibiotics (all from Sigma-Aldrich): colistin (10 µg/ml), rifampicin (15 µg/ml), erythromycin (50 µg/ml), kanamycin (1,000 µg/ml) and vancomycin (5 µg/ml). Concentration of the antibiotics used was based on previously published and established MIC values (Maclayton et al., 2006;Moskowitz et al., 2010;Kshetry et al., 2016;Rojas et al., 2017;Goldstein et al., 2018). E. coli and S. aureus agar plates were prepared for the disc diffusion test (Iqbal et al., 2013) by soaking a sterile paper disc in 5 µM CBD and placing it in the middle of the agar plate, while the impregnated antibiotic discs were placed equidistant to the CBD disc. Zones of inhibition were assessed after 24 h using the Kirby-Bauer test.

Proteomic Analysis of MVs Released From CBD Treated and Control Untreated E. coli VCS257
To assess differences in E. coli VCS257 MV protein composition in response to CBD treatment, MVs were isolated as before, after 1 h treatment with 1 µM or 5 µM CBD treatment or control untreated, respectively. MVs were assessed by SDS-PAGE (using 4-20 % gradient TGX gels, BioRad, U.K.) and silver staining using the BioRad Silver Stain Plus Kit (1610449, BioRad, U.K.), according to the manufacturer's instructions (BioRad). For assessment of proteomic changes, MVs were subjected to liquid chromatography-mass spectrometry (LC-MS/MS) analysis. MVs from CBD treated, vs. non-treated E. coli were run 1 cm into a SDS-PAGE gel and the whole protein lysate cut out as one band, whereafter it was processed for proteomic analysis (carried out by Cambridge Proteomics, U.K.). Peak list files were submitted to Mascot (in-house, Cambridge Center for Proteomics) using the following database: Uniprot_Escherichia_coli_20180613 (4324 sequences; 1357163 residues).

Statistical Analysis
Histograms and graphs were prepared and statistical analysis was performed using GraphPad Prism version 8 (GraphPad Software, San Diego, U.S.A.). One-way ANOVA and Student's t-test analysis were performed, followed by Tukey's post-hoc analysis. Histograms represent mean of data, with error bars representing standard error of mean (SEM). Significant differences were considered as p ≤ 0.05.

RESULTS
Characterization of MVs From E. coli VCS257 and S. aureus subsp. aureus Rosenbach Isolated MVs were assessed by morphology using transmission electron microscopy (TEM), revealing a poly-dispersed population in the size range of mainly 20-230 nm in diameter for E. coli, including MVs showing inner and outer membranes ( Figure 1A.1), and characteristic one layer membranes for S. aureus MVs, which were in the 37-300 nm range ( Figure 1A.2). Nanoparticle tracking analysis (NTA) verified that the majority of the vesicle population fell in a similar size range under standard culture conditions (mode 143.3 nm; SD ± 72.3 nm for E. coli (Figure 1A.1) and 141.4 nm; SD ± 7.3 nm for S. aureus (Figure 1A.2). Furthermore, Western blotting showed positive for the MV specific marker OmpC ( Figure 1A).

Effects of CBD on Membrane Vesicle
Release From E. coli VCS257 and S. aureus subsp. aureus Rosenbach CBD changed the MV release profile from E. coli compared to control treatment (Figures 1B-D). Modal size of MVs released from E. coli was significantly increased (p = 0.01) after 1 µM CBD treatment, compared to control treated cells, while 5 µM CBD treatment did not have statistically significant effects on MV size (p = 0.0685). Effects on modal size of vesicles released from E. coli between the two doses of CBD was also not statistically significant (p = 0.0643; Figure 1D).
Effects of CBD on E. coli VCS257 MVs was furthermore assessed by TEM, verifying the presence of fewer vesicles per field and showing some change in vesicle size and morphology after CBD ( Supplementary Figures 2A-C). Contrary to what was observed for the Gram-negative E. coli, CBD treatment (5 µM) had no significant effect on MV release from the Gram-positive bacterium S. aureus subsp. Aureus Rosenbach (p > 0.1; Figure 2B).
Effects of CBD on Bacterial Viability of E. coli VCS257 and S. aureus subsp. aureus Rosenbach CBD had negligible effect on E. coli cell viability after 24 h incubation with the lower 1 µM dose, while an 11 % (p = 0.0161) reduction in cell viability was observed in response to 5 µM CBD, but no significant effect was observed on S. aureus cell viability, as assessed by disk diffusion test (Supplementary Figure 1).

CBD Treatment Affects Antibiotic
Sensitivity in E. coli VCS257 CBD treatment (5 µM), when applied in combination with a range of antibiotics tested, was found to sensitize E. coli VCS257 to selected antibiotics, as assessed by an increase in the radius of zone of inhibition, using the disk diffusion test (Figure 3). Significantly enhanced antibacterial effects were found for erythromycin (35 % increase; p = 0.006), rifampicin (50 % increase; p = 0.0007) and vancomycin (100 % increase; p < 0.0001), when combined with CBD treatment (5 µM), compared to antibiotic treatment alone. Notably, vancomycin alone did not have bactericidal effects on E. coli, but only in the presence of CBD. Antibacterial effects of kanamycin were increased by 18 % but this was not statistically significant compared to antibiotic alone (p = 0.09). Zone of inhibition with CBD treatment only was also observed in the E. coli plates (Figure 3), but this was significantly lower than when CBD was combined with antibiotics, except for vancomycin. The zone of inhibition for E. coli caused by antibiotic treatment only, vs. CBD alone, differed also significantly for erythromycin (p = 0.0010), vancomycin (p = 0.0158), rifampicin (p = 0.0003) and kanamycin (p = 0.0008), but not for colistin (p = 0.224). Therefore, while CBD showed some anti-bacterial activity against E. coli when applied in isolation, this was significantly lower than observed for the antibiotics alone (except for vancomycin which did not show antibacterial activity while CBD did). However, when applied in combination, CBD increased bactericidal effects of all antibiotics tested, except for colistin.

CBD-Mediated Effects on Antibiotic Sensitivity in S. aureus subsp. aureus Rosenbach
When added to S. aureus subsp. aureus Rosenbach, 5 µM CBD increased the antibiotic activity of kanamycin (30 %; p = 0.0028), as assessed by increased radius of zone around the diffusion disk (Figure 4). CBD did not enhance anti-bacterial activity for the other antibiotics tested and reduced antibacterial effects of both erythromycin and rifampicin (p = 0.0325 and p = 0.0001, respectively). Importantly, there was no halo observed around the FIGURE 3 | CBD sensitizes Gram-negative bacteria E. coli VCS257 to selected antibiotics. Combinatory treatment of CBD with a range of antibiotics (24 h treatment) showed enhanced CBD-mediated antibacterial effects on E. coli VCS257, as assessed by increased radius of zone around the diffusion disks. CBD was most effective in combination with rifampicin (p = 0.0007), vancomycin (p ≤ 0.0001) and erythromycin (p = 0.006). CBD in isolation also had bactericidal effects on E. coli, while combinatory treatment with the antibiotics was most effective. Exact p-values are shown.
FIGURE 4 | CBD sensitizes Gram-positive bacteria S. aureus subsp. aureus Rosenbach to kanamycin. Combinatory treatment of CBD with a range of antibiotics showed enhanced antibacterial effects of kanamycin only on S. aureus, as assessed by an increased radius of zone around the diffusion disk (p = 0.0028). CBD did not enhance bactericidal activity for the other antibiotics tested and reduced bactericidal effects of both erythromycin (p = 0.0325) and rifampicin (p = 0.0001). CBD application in isolation did not form a halo around the diffusion disk in the S. aureus plates, opposed as to what was observed in E. coli, and CBD treatment in isolation is therefore not included in the histogram. Exact p-values are shown.
diffusion disk containing CBD alone in the S. aureus plates, indicating no bactericidal effects of CBD on this strain of S. aureus.

Effects of CBD Treatment on Protein Profiles of MVs Released From E. coli VCS257
Protein composition of MVs was assessed in MVs isolated from E. coli VCS257 after 1 h treatment with 1 µM and 5 µM CBD, respectively, compared to non-treated E. coli MVs, using SDS-PAGE silver stained gels and LC-MS/MS analysis. Silver stained gels revealed some band differences between the three conditions ( Figure 5A). Proteins were further analyzed by LC-MS/MS and peak list files submitted to Mascot (in-house, Cambridge Center for Proteomics, Uniprot_Escherichia_coli_20180613). Hits are listed in Tables 1-3. Compared to untreated MVs, five protein hits were absent in MVs released from the 1 µM CBD treated E. coli and four protein hits were absent in MVs released from the 5 µM CBD treated E. coli, respectively (Table 1 and Figure 5B). When comparing the two CBD treatments, 26 protein hits were specific to the E. coli MVs following 1 µM CBD treatment (Table 2 and Figure 5B) while 68 protein hits were unique to the MVs released from E. coli treated with 5 µM CBD (Table 3 and Figure 5B).

DISCUSSION
To our knowledge this is the first study to evaluate the putative effects of CBD on the release of membrane vesicles (MVs) from bacteria and effects of CBD on MV profile, including protein composition. In eukaryotic cells, CBD was recently identified as an effective inhibitor of extracellular vesicle (EV) release both in human cancer cells (Kosgodage et al., 2018(Kosgodage et al., , 2019 as well as in the intestinal parasite Giardia intestinalis (Gavinho et al., 2019). Therefore, our present findings may indicate phylogenetically conserved pathways of membrane vesicle release from bacteria to mammals that can be modulated via CBD. Moreover, CBD could enhance the anti-bacterial effect of certain antibiotics in some bacterial types, but also inhibit it in others. This indicates that inhibition of MV release and antibacterial action are likely linked, as previously suggested (Tashiro et al., 2010). Indeed, a recent study using indole derivatives has revealed a role for MVs in antibiotic resistance/persistence, in particular in Gram-negative bacteria tested (Agarwal et al., 2019).
Here we report that CBD significantly reduced MV release in E. coli VCS257, a Gram-negative bacterium, but had negligible effects on membrane vesicle release in S. aureus subsp. aureus Rosenbach, a Gram-positive bacterium, as assessed here by in vitro analysis. In addition, we also found that lower doses of CBD had a stronger MV inhibitory effect in E. coli VCS257 than a higher 5 µM dose (p = 0.0063), and such an effect has also previously been observed for EVs in certain cancer cell types (Kosgodage et al., 2018). Biphasic effects of CBD are indeed recognized (Bergamaschi et al., 2011) and may be reminiscent of "hormesis, " an effect we have suggested could explain its more general medical benefits as well as effects on mitochondrial dynamics (Nunn et al., 2013). Interestingly, at the lower 1 µM concentration, CBD significantly increased the release of a 500 nm peak of MVs, as observed by NTA analysis, while this peak was negligible both in the control treated bacteria and those treated with 5 µM CBD. Such an effect of CBD on MV profile, and protein MV profile as observed by proteomic analysis here, may be relevant in the light of recent recognition of the importance of MV size for cellular entry and uptake (Turner et al., 2018)    2019). The approximately 6.5-fold and 2.5-fold decreases in MV release observed after CBD (1 and 5 µm, respectively) treatment from E. coli, compared to non-treated controls, also correlated with a trend in shift toward proportionally larger vesicles released according to NTA analysis and change in protein profile. The exact mechanism for packaging proteins and other reagents in MVs is not fully understood and given the plethora of targets for CBD (Ibeas Bih et  MVs have also been tested as delivery vehicles for targeted gene silencing using siRNA-packaged MVs (Alves et al., 2016). Whether CBD may be utilized for combinatory application with such approaches may also be of putative interest, in addition to its observed effects in this study, in effectively reducing MV release. In relation to antibiotic activity, cannabinoids including CBD, have been widely studied for their anti-bacterial activity (Wasim et al., 1995;Bass et al., 1996;Appendino et al., 2008;Hernández-Cervantes et al., 2017). For example, C. sativa extracts have previously been shown to have microbicidal activity on various Gram-positive bacteria, including several strains of S. aureus, as well as some Gram-negative bacteria (Wasim et al., 1995;Elphick, 2007;Nissen et al., 2010), with the minimum inhibitory concentrations (MIC) for the main phytocannabinoids, such as CBD, being in the 0.5-5 µM range, which is similar to many modern antibiotics (Van Klingeren and Ten Ham, 1976;Appendino et al., 2008). How precisely CBD may be working as an anti-bacterial agent is still not entirely clear (Appendino et al., 2008), particularly in the light of a plethora of targets for CBD (Ibeas Bih et al., 2015;Hernández-Cervantes et al., 2017), while structure-activity studies indicate that the ability of plant-derived phenolic compounds to interact with membranes and the existence of electrophilic functional groups are important (Miklasinska-Majdanik et al., 2018). Hitherto though, no association has been made into a putative regulatory effect of cannabinoids on bacterial membrane vesicle release. Furthermore, as the current study has revealed changes in proteomic profile of MVs released from E. coli VCS257 following CBD treatment, such findings may inform antibacterial effects of CBD. Using LC-MS/MS analysis to assess changes in protein profile of MVs from CBD treated and untreated E. coli, respectively, five proteins were found to be absent in the 1 µM CBD treated MVs and 4 proteins were absent in the 5 µM CBD treated MVs, compared to control untreated E. coli MVs. Out of these, 2 proteins overlapped between the two CBD treatments. In addition, comparing 1 and 5 µM CBD treated E. coli MVs, 26 protein hits were unique to MVs released following the 1 µM CBD treatment and 68 protein hits to MVs released following the 5 µM CBD treatment. Using STRING analysis, PPI enrichment p-value was found to be p = 0.0204 for proteins identified as unique to MVs from the 1 µM CBD treatment and p = 1.56 × 10 −6 for proteins identified as unique to MVs from the 5 µM CBD. This indicates that for both treatments these proteins have significantly more interactions among themselves, than what would be expected for a random set of proteins of similar size, drawn from the genome. Such enrichment indicates that the proteins are at least partially biologically connected, as a group. Protein networks are represented showing biological GO pathways and KEGG pathways, respectively, in Supplementary Figures 3, 4 for proteins specific to EVs from E. coli after 1 and 5 µM CBD treatment, respectively. Proteins identified are related to metabolic processes, cellular respiration and antibiotic functions (Supplementary Figures 3A,B, 4A,B).
When assessing the effectivity of CBD to enhance susceptibility of Gram-positive and Gram-negative bacterial species to a range of antibiotics, CBD-mediated MV inhibition rendered E. coli VCS257 significantly more sensitive to erythromycin, vancomycin and rifampicin and somewhat to kanamycin, but did not augment the bactericidal effects observed for colistin. This was somewhat unexpected, given a previous study showing that MVs isolated from the E. coli strain MG1655 could protect bacteria against membrane-active antibiotics such as colistin (Kulkarni et al., 2015). Our finding, that CBD did not sensitize E. coli further to colistin, when applied in combination with this antibiotic, may arise from the fact that a different strain of E. coli (VCS257) was used in the current study, compared to in the study by Kulkarni et al. (2015). It has also been previously shown that the presence of calcium decreases the bactericidal effect of colistin on Paenibacillus polymyxa, suggesting a role for Ca 2+ in generating a protective barrier against colistin (Yu et al., 2015). As CBD is known to modulate calcium (Rimmerman et al., 2013) it can be postulated that this may interfere with the mode of action of colistin. Our findings also indicate that combinatory application of CBD is not effective for all antibiotics, which may possibly be explained by their different modes of action. Importantly, zones of inhibition were observed in the plates which were only treated with the CBD discs in the presence  of E. coli, and this clearly revealed the antibacterial property of CBD. Interestingly, CBD did increase antibacterial effects of vancomycin on E. coli, in spite of vancomycin's limited effectiveness on Gram-negative species, also seen here by the fact that vancomycin alone did not result in a halo around the diffusion disk for E. coli. Therefore, CBD seems to overcome previously established resistance of E. coli to vancomycin, which has reported to partly be due to its inability to significantly penetrate the outer membrane (Zhou et al., 2015). It may also be important to note that erythromycin, rifampicin and kanamycin inhibit protein synthesis, whereas vancomycin is a glycopeptide that inhibits cell biosynthesis in Gram-positive bacteria, while colistin binds to the outer membrane of Gram-negative bacteria, disrupting it. Thus, these antibiotics display very different modes of action.
In the Gram-positive bacterium S. aureus subsp. aureus Rosenbach, CBD increased bactericidal activity of kanamycin only. The reduced ability of CBD to sensitize this Gram-positive bacterium to antibiotics, compared to the significantly higher effects in the Gram-negative bacterium, tallied in with CBD's ability to regulate MV-release, indicating a relevant contribution of MVs to antibiotic resistance. Roles for MVs in protecting biofilms via adsorption of antimicrobial agents have indeed been previously recognized (Schooling and Beveridge, 2006;Manning and Kuehn, 2011;Toyofuku et al., 2019). This also indicates that MV-inhibitors that target membrane vesicles from specific bacteria species, such as CBD here, could be applied in combination with selected antibiotics for tailored antibiotic treatment to tackle antibiotic resistance.

CONCLUSIONS
CBD effectively inhibited MV release from the Gram-negative bacterium E. coli VCS257, exhibiting a stronger MV-inhibiting effect at lower dose. In addition, CBD modulated MV protein profiles of E. coli following 1 h treatment. CBD did not have significant effects on MV release in the Gram-positive bacterium S. aureus subsp. aureus Rosenbach. When applied in combination with a range of antibiotics, CBD increased antibacterial effects of selected antibiotics, depending on bacteria type. CBD, in combination with specific antibiotics, may thus possibly be used as an adjuvant to selectively target bacteria to sensitize them to antibiotic treatment and reduce antibiotic resistance.

DATA AVAILABILITY
All datasets generated for this study are included in the manuscript and/or the Supplementary Files.

AUTHOR CONTRIBUTIONS
UK, PM, BA, IK, PW, and SL performed the experiments. UK, JB, AN, JI, and SL analyzed the data. PM, GM, GG, IK, SL, and JI provided resources. UK, SL, and JI designed the study. SL, UK, and AN wrote the manuscript. All authors critically reviewed the manuscript.

ACKNOWLEDGMENTS
This work was supported in parts by a University of Westminster Start-up Grant to SL and an unrestricted grant from GW Pharmaceuticals. Thanks are due to the Guy Foundation for funding the purchase of equipment utilized in this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

SUPPLEMENTARY MATERIAL
The Supplementary Figure 3 | Protein-protein interaction networks of protein hits identified in MVs from 1 µM CBD treated E. coli VCS257. Reconstruction of protein-protein interactions based on known and predicted interactions using STRING analysis. Colored nodes represent query proteins and first shell of interactors; white nodes are second shell of interactors. (A) Biological GO processes are highlighted as follows: red, citrate metabolic process; green, antibiotic metabolic process; yellow, regulation of cellular amide metabolic process; purple, carboxylic acid metabolic process; dark green, regulation of phosphate metabolic process; light blue, cellular respiration; orange, small molecule metabolic process; dark red, negative regulation of translational elongation; dark blue, generation of precursor metabolites and energy. (B) KEGG pathways are highlighted as follows: dark green, oxidative phosphorylation; dark red, citrate cycle (TCA cycle); red, biosynthesis of antibiotics; purple, butanoate metabolism; dark blue, biosynthesis of secondary metabolites; light blue, carbon metabolism; orange, phenylalanine, tyrosine and tryptophan biosynthesis; light green, Microbial metabolism in diverse environments; yellow, Metabolic pathways; violet, glycine, serine, and threonine metabolism. Colored lines indicate whether protein interactions are identified via known interactions (curated databases, experimentally determined), predicted interactions (gene neighborhood, gene fusion, gene co-occurrence) or via text mining, co-expression or protein homology (see color key for connective lines).
Supplementary Figure 4 | Protein-protein interaction networks of protein hits identified in MVs from 5 µM CBD treated E. coli VCS257. Reconstruction of protein-protein interactions based on known and predicted interactions using STRING analysis. Colored nodes represent query proteins and first shell of interactors; white nodes are second shell of interactors. (A) Biological GO processes are highlighted as follows: red, cellular respiration; green, purine-containing compound metabolic process; yellow, electron transport chain; purple, ribose phosphate metabolic process; dark green, purine ribonucleotide metabolic process; light blue, generation of precursor metabolites and energy; orange, nucleobase-containing small molecule metabolic process; dark red, purine ribonucleoside metabolic process; dark blue, organophosphate metabolic process. (B) KEGG pathways are highlighted as follows: red, bacterial secretion system; light green, metabolic pathways; yellow, oxidative phosphorylation; purple, butanoate metabolism; dark green, quorum sensing; light blue, amino sugar and nucelotide sugar metabolism; dark blue, protein export; violet, purine metabolism. Colored lines indicate whether protein interactions are identified via known interactions (curated databases, experimentally determined), predicted interactions (gene neighborhood, gene fusion, gene co-occurrence) or via text mining, co-expression or protein homology (see color key for connective lines).