Potential cannabidiol (CBD) repurposing as antibacterial and promising therapy of CBD plus polymyxin B (PB) against PB-resistant gram-negative bacilli

This study aimed to assess the ultrapure cannabidiol (CBD) antibacterial activity and to investigate the antibacterial activity of the combination CBD + polymyxin B (PB) against Gram-negative (GN) bacteria, including PB-resistant Gram-negative bacilli (GNB). We used the standard broth microdilution method, checkerboard assay, and time-kill assay. CBD exhibited antibacterial activity against Gram-positive bacteria, lipooligosaccharide (LOS)-expressing GN diplococcus (GND) (Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis), and Mycobacterium tuberculosis, but not against GNB. For most of the GNB studied, our results showed that low concentrations of PB (≤ 2 µg/mL) allow CBD (≤ 4 µg/mL) to exert antibacterial activity against GNB (e.g., Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii), including PB-resistant GNB. CBD + PB also showed additive and/or synergistic effect against LOS-expressing GND. Time-kill assays results showed that the combination CBD + PB leads to a greater reduction in the number of colony forming units per milliliter compared to CBD and PB alone, at the same concentration used in combination, and the combination CBD + PB was synergistic for all four PB-resistant K. pneumoniae isolates evaluated. Our results show that CBD has translational potential and should be further explored as a repurposed antibacterial agent in clinical trials. The antibacterial efficacy of the combination CBD + PB against multidrug-resistant and extensively drug-resistant GNB, especially PB-resistant K. pneumoniae, is particularly promising.

CBD in concentrations up to 256 µg/mL was not antibacterial for any of the tested GNB (27 species, 70 strains) (Supplementary Table 1). We also evaluated higher concentrations of CBD for E. coli ATCC 25922, K. pneumoniae ATCC 13883, A. baumannii ATCC 19606, and P. aeruginosa ATCC 27853, but again no antibacterial activity was observed up to 8.192

Antibacterial activity of CBD in combination with PB (CBD + PB) against GN bacteria. Screen-
ing by broth microdilution method with fixed concentration of CDB (256 µg/mL). We observed antibacterial activity of the combination CBD + PB against 8/13 different species (47/52 strains) of GNB, including standard strains (Table 1) and clinical isolates ( Table 2). For the combination CBD (256 µg/mL) + PB (0.01-512 µg/mL), compared to PB alone, we observed a minimal threefold reduction in the PB concentration required for CBD antibacterial activity against PB-susceptible GNB (non-fermenting GNB and Enterobacterales). Also, the combination of CBD + PB against K. pneumoniae led to a twofold reduction in PB concentration compared to PB MIC, while only a onefold reduction was observed for P. aeruginosa (Tables 1 and 2, and Supplementary Fig. 2A).
Regarding intrinsically PB-resistant GNB, the combination of CBD + PB was not antibacterial even in the presence of PAβN. The exception was E. tarda ATCC 15947, for which the combination CBD + PB + PAβN was also antibacterial, again showing the antibacterial activity of CBD with lower PB concentrations (Table 3).
Confirmation by checkerboard assay. For K. pneumoniae (n = 12), E. coli (n = 4), A. baumannii (n = 2), and P. aeruginosa (n = 2), checkerboard assay was performed to confirm the in vitro antibacterial activity and to assess the different proportions of each substance in the combination CBD + PB (Table 4).
For most of the GNB (including PB-resistant K. pneumoniae), 2-4 µg/mL of CBD were enough to inhibit bacterial growth when combined with low concentrations of PB (≤ 2 µg/mL) (Table 4). Particularly, the combination of ≤ 2 µg/mL of CBD plus ≤ 0.5 µg/mL of PB was antibacterial to most PB-resistant clinical isolates of K. pneumoniae ( Supplementary Fig. 3).
For PB-susceptible P. aeruginosa ATCC 27853 and HC103, and plasmid-mediated colistin-resistant (MCR-1) E. coli 72H strains, the checkerboard assay was also performed in the presence of PAβN. The results showed that the combination of CBD (4 µg/mL) + PB was antibacterial only in the presence of PAβN (Table 4).
The fractional inhibitory concentration index (FICI) of the combination of CBD + PB was not calculated due to the absence of antibacterial activity (MIC) of CBD against GNB.
For GND M. catarrhalis ATCC 25238, N. meningitidis ATCC 13077, and N. gonorrhoeae ATCC 19424, the FICI of the combination CBD + PB was calculated because both CBD and PB alone showed antibacterial activity (MIC). Thereby, CBD + PB showed additive and/or synergistic effect against these GND (Table 5, and Supplementary Fig. 4).
Time-kill assays. Time-kill assays showed that the combination CBD + PB leads to a greater reduction in the number of CFU/mL compared to PB alone (at the same concentration used for the combination) for all four clinical isolates of PB-resistant K. pneumoniae evaluated (Fig. 1). Also, the overall reduction in CFU/mL of the combination CBD + PB relative to PB alone was above 2 log 10 for many time points, further confirming the synergistic effect of CBD and PB (Table 6).  25 . c Andrade et al. 26 . d Fernandes et al. 27 . e Clímaco et al. 30 . f Galetti et al. 29 .

Strain
Antibacterial activity of the combination CBD + PB (µg/mL)

Discussion
CBD antibacterial activity against GP and GN bacteria, and Mycobacterium tuberculosis. We observed antibacterial activity of ultrapure CBD against GP bacteria, M. tuberculosis, and LOS-expressing GND; but not against GNB as recently reported [14][15][16] . Preliminary results regarding ultrapure CBD activity in GP bacteria and its absence in GN bacteria (including MDR and XDR strains) were partially presented by us at ASM Microbe 2018 and published in the abstract book 13 .    31 . For N. gonorrhoeae, we employed the broth microdilution method instead of agar dilution. Blaskovich et al. used a broth culture medium composed of a lower content of lysed horse blood (3%) for S. pneumoniae and S. pyogenes, and a modified broth according to standards from the American Type Culture Collection (ATCC) for Neisseria spp., which does not contain blood 16 . The presence of blood in culture media (e.g., MH-F broth) increases CBD MIC, as observed in our study for S. aureus (fourfold dilution increased CBD MIC) when compared to only CAMHB, a result similar to previous reports 12 . These differences may have contributed to our higher CBD MIC values against fastidious bacteria compared to those of Blaskovich et al. 16 .
For M. tuberculosis, we observed lower CBD MIC values than those of Blaskovich et al. They used a 5-day incubation period , the addition of 12.5uL of 20% Tween 80 into resazurin, and a culture medium supplemented with ADC (albumin, dextrose, catalase) (Difco Laboratories), 0.5% glycerol, and 0.02% tyloxapol 16 . We used a period of incubation of 7 days, no Tween 80 into resazurin, a culture medium supplemented with OADC (oleic acid, albumin, dextrose, catalase), and glycerol 0,4%. The difference between OADC and ADC is the presence of oleic acid at 0.5 g/L.
Our results reinforce that CBD alone is not antibacterial against GNB (MDR/XDR or susceptible to antibiotics), because we evaluated 27 species (70 strains) of GNB species most commonly involved in HAI as well as in community infections, expanding the panel of GNB and human pathogens investigated (Supplementary Table 1).
In an attempt to understand this lack of effect, we used various efflux pump inhibitors to potentially improve CBD activity. However, our data also showed no role of efflux pumps that are commonly involved in antibiotic extrusion from GN cell. Therefore, we hypothesized that CBD was inactive due to low permeability through the cell envelope (outer membrane) of GNB.
The absence of antibacterial activity of CBD against GNB may be related to LPS molecules and outer membrane proteins, from the outer membrane, which would lead to the impermeability of macromolecules and limited diffusion of hydrophobic molecules, such as CBD [14][15][16] . Our results of in vitro CBD antibacterial activity against GP bacteria and M. tuberculosis, and the absence of CBD antibacterial activity against GNB, support a role for LPS in hindering CBD activity.
Furthermore, we also assessed LOS-expressing bacteria such as N. meningitidis, N. gonorrhoeae, and M. catarrhalis. The LOS molecule lacks the O-antigen of LPS 32,33 , thereby allowing us to evaluate a potential role of O-antigen in preventing CBD antibacterial activity (possibly due to the steric effect, hindering CBD from reaching its molecular target).
Even considering that A. baumannii and H. influenzae also have LOS molecules on their external membrane, but the core polysaccharide for these bacteria presents a different sugar composition 32,34 . This fact could explain the absence of CBD antibacterial activity against these GN bacteria. CBD and cannabigerol (CBG, another cannabinoid) have antibacterial activity against A. baumannii only in the absence of the complete LOS molecule, according to previous studies 14, 16 .
The hydrophobic chemical structure of CBD points towards an interaction with lipid in membranes as described by Guard et al. for eukaryotic cells. This interaction alters the biophysical properties of the membrane and affects lipid and cholesterol metabolism 35,36 . Indeed, the bacterial membrane was also suggested as a possible bacterial target for cannabinoids (CBG and CBD) 11,14,16 . Furthermore, Blaskovich et al. also showed that bactericidal concentrations of CBD against S. aureus inhibits the synthesis of proteins, DNA, RNA, and peptidoglycan 16 . Nevertheless, the specific mechanism(s) for the antibacterial activity of CBD has not yet been fully elucidated. Therefore, our results contribute to a better understanding of CBD antibacterial mechanism(s) of action, which could guide future studies. Table 6. Log 10 (CFU/mL) difference between the combination CBD + PB and PB treatment at MEAC. Combinations were considered synergistic (indicated by bold) when the difference was greater (more negative) than 2 log 10 compared to the most active component of the combination (i.e., PB).  (Tables 1 and 2).
For most GNB, our checkerboard results showed that CBD concentrations lower than 4 µg/mL were sufficient for antibacterial activity in the combination CBD + PB. Also, PB concentrations needed for the combination to be antibacterial were up to eight-fold lower than the MIC for PB.
Furthermore, for PB-resistant GNB (highlighting K. pneumoniae strains), 2-4 µg/mL of CBD were enough to lead to bacterial growth inhibition when combined with clinically optimal PB concentrations ( Table 4).
According to EUCAST/BrCAST breakpoints, bacteria presenting PB MIC ≤ 2 µg/mL are categorized as susceptible, and there is a high likelihood of therapeutic success using a standard dosing regimen of PB 23,38 . On the other hand, according to CLSI breakpoints, bacteria with PB MIC ≤ 2 µg/mL are categorized as intermediate as there is no longer a CLSI 'susceptible' category for polymyxins. CLSI argues that polymyxins monotherapy would have limited clinical efficacy and suggests combination therapy with another antibacterial 24 . In our study, we used the EUCAST/BrCAST breakpoint (PB MIC ≤ 2 µg/mL as susceptible) for our analyses and discussion.
Additionally, time-kill results showed the killing effect of the combination of CBD + PB over time, contributing to future studies and perspectives on dose-exposure response relationships and pharmacokinetic/pharmacodynamic parameters.
Among intrinsically PB-resistant GNB, the combination of CBD + PB showed antibacterial activity only against E. tarda. These results may be related to the different intrinsic resistance mechanisms of these bacteria, involving different molecular pathways from two-component systems 3,39 .
For the GND N. meningitidis, N. gonorrhoeae, and M. catarrhalis, the calculation of FICI revealed an additive or synergistic effects for the combination CBD + PB (Table 5). Nevertheless, PB is not used for the treatment of GND-caused infections due to PB intrinsic resistance. However, the synergistic effect may suggest a new insight for this bactericidal activity of the combination CBD + PB, highlighting that PB also neutralizes the endotoxin Lipid A from LPS/LOS of GN bacteria 40 .

Biological activity of the combination CBD + PB (+ PAβN). CBD alone shows antibacterial activity
against GP bacteria, M. tuberculosis, and LOS-expressing GND; however, it does not show antibacterial activity against GNB, probably due to the presence of LPS molecules and outer membrane proteins, from the outer membrane, resulting in impermeability of CBD.
PB alone shows antibacterial activity against GN bacteria and the use of this antibiotic in clinical practices depends on in vitro susceptibility breakpoints and on bacterial species identified 23,24,38 . PB promotes the destabilization of LPS or LOS, leading to the disruption of the bacterial cell envelope 3 .
Considering the antibacterial activity of the combination CBD + PB against GNB, our results also point to the existence of a CBD molecular target in GNB and indicate that its activity is dependent on bacterial outer membrane destabilization promoted by PB.
CBD is the antibacterial agent in the combination CBD + PB, considering that the concentrations of PB used in the combination were PB MEAC, which are sublethal (subinhibitory) (Fig. 2). This biological activity is supported by time-kill results showing that PB MEAC alone has the same behavior as the growth control in the killing curve (Fig. 1).
Outstandingly, the combination CBD + PB was effective against PB-resistant K. pneumoniae (PB MIC ranged from 4 to 256 µg/mL), considering PB MIC > 2 µg/mL for PB-resistant strains. This fact further points towards an antibacterial activity for CBD, once PB MEAC (sublethal concentrations) were used in the combination CBD + PB.
Thereby, CBD does not restore PB susceptibility for PB-resistant GN bacteria. PB at MEAC acts exclusively as an outer membrane destabilizing agent and does not lead to bacterial cell disruption and death.
Indeed, CBD does not decrease the PB MIC against bacteria, since 'MIC' is the 'minimal inhibitory concentration' of only one antibacterial agent, so, in combination with another substance, the concept of MIC reduction is mistaken and should not be used. The lower PB concentrations of PB in the combination CBD + PB are the PB MEAC.
Furthermore, as a CBD MIC could not be determined for GNB, the antibacterial activity of the combination CBD + PB could not be categorized as synergistic or additive based on checkerboard assay due to the lack of a mathematical factor (MIC) to calculate the FICI. In addition, the attribution of the maximum concentration evaluated in the experiments as a 'MIC' is also a mistake.
Considering the "lack of antibacterial activity (or lack of MIC) of one substance in the drug combination", the association of checkerboard and time-kill assays contributes to a better characterization of the combined antibacterial activity of the two substances. Checkerboard assays show how much drugs concentrations can be decreased while inhibiting bacterial growth, whereas time-kill assays show how much more effective is the combination when compared to each substance alone. Checkerboard data of combination inhibition are important; however, time-kill data are more suitable to categorize the combination effect as synergistic 41 .
Our results showed that low concentrations of PB (lower than the PB MIC against each evaluated GNB) are sufficient to cause the minimal outer membrane destabilization required to allow antibacterial activity of CBD in GNB (except P. aeruginosa and plasmid-mediated colistin-resistant [MCR-1] E. coli strains) ( www.nature.com/scientificreports/ The combination CBD + PB + PAβN was effective against P. aeruginosa and plasmid-mediated colistin-resistant (MCR-1) E. coli strains, for which only CBD + PB was not active. However, these results could not be related to efflux inhibition by PAβN per se, because CBD antibacterial activity alone was not detected in the presence of PAβN. Thereby, our results suggest PAβN permeabilization of the outer membrane contributing to CBD activity, as similarly described for β-lactams in the presence of PAβN against P. aeruginosa, or sensitization of P. aeruginosa to antibiotics (e.g., vancomycin) that are typically incapable of crossing the outer membrane 42 . Indeed, the combination CBD + PB in the presence of PAβN decreases considerably the PB concentrations necessary to allow CBD activity (Table 4).
Even though PAβN is a substance commonly used for bacterial efflux pump inhibition in in vitro assays, it is not currently used as a drug in clinical practice. However, a potential use of PAβN as an antibiotic adjuvant that can reduce the effective doses of drugs that require increased outer membrane permeability has been considered 42 . According to the Biopharmaceutics Drug Disposition Classification System, CBD is a class 2 drug, showing low water solubility and high permeability/metabolism depending on CYP3A4, CYP2C19, UGT1A9, and UGT2B7 enzymes 46 .

Pharmacokinetic perspectives of cannabidiol repurposing as antibacterial.
Clinical studies of CBD pharmacokinetics after oral administration were evaluated in different formulations such as capsules, solutions, and oromucosal preparations 47 .
Intravenous (IV) administration is the most usual administration route of antimicrobial therapy in hospitalized and critically ill patients, and CBD pharmacokinetics studies following IV administration have been studied in clinical trials 47 . CBD pharmacokinetics after a 20 mg IV dose was evaluated by a previous study, which showed clearance values of approximately 80 L/h and volume of distribution of 52 L in 70 kg individuals 48 . Thus, as an exercise of translational pharmacokinetics could be done using a classic equation and considering linear pharmacokinetics: www.nature.com/scientificreports/ where τ is the dosing interval, CL is the total clearance, and Css is the steady-state mean plasma concentration. CBD is antibacterial against most Gram-positive cocci (GPC) (e.g., Enterococcus spp. and Staphylococcus spp.) and shows an in vitro MIC of 2 µg/mL or 4 µg/mL. However, CBD is not antibacterial against GNB. Nevertheless, the combination of CBD + polymyxin B (PB) is antibacterial against GNB, including MDR and XDR standard strains and clinical isolates. For most GNB, low concentrations of PB (MEAC, that are lower than PB MIC; and ≤ 2 µg/mL) allow CBD (≤ 4 µg/mL) to exert antibacterial activity against GNB (e.g., Escherichia coli, Acinetobacter baumannii, Enterobacter cloacae), highlighting PB-resistant GNB (e.g., Klebsiella pneumoniae).
Considering data from the literature, to achieve these plasmatic concentrations, an IV administration of a CBD dose of approximately 2 g/12 h or 4 g/12 h would result in plasma exposure higher than MIC (Css) against GPC (alone) and GNB (in the combination CBD + PB). Although these concentrations are target plasma concentrations and considering clearance values previously described, it could be possible to administer CBD doses to reach levels higher than the MIC, since single doses of approximately 6 g are described as well tolerated 46,49 .
To date, the pharmacokinetic data for CBD in the literature refer to another therapeutic context (pharmacoresistant epilepsy or refractory epilepsy treatment), which uses oral administration and different doses, determined by clinical studies specific for the therapeutic purpose described.
Our study shows the in vitro antibacterial activity of CBD, especially in combination with PB, suggesting potential repurposing of CBD as an antibacterial. To achieve this goal, research and development of new CBD formulations are needed to optimize the CBD pharmacokinetics to achieve higher serum concentrations from safe administration of the dosages required for antibacterial therapy. Our results, along with novel CBD formulations, present translational potential to be validated by future clinical studies for the purpose of treating bacterial infections.
In this context, optimization of CBD IV administration and pharmacokinetic parameters could be achieved using nanomaterial-based strategies, such as nanocarriers for increased solubility, stability, and efficacy for antibacterial therapy 50,51 . Thereby, future studies are needed regarding pharmacokinetics and pharmacodynamics, and safety and tolerability of CBD, alone or in the combination CBD + PB, in addition to MIC 50 and MIC 90 determination and probability of target attainment.
We highlight the promising translational potential of CBD repurposing as an antibacterial agent, mainly in the combination CBD + PB against GNB, for rescue treatment for life-threatening infections, highlighting against PB-resistant K. pneumoniae.
Clinical perspectives of cannabidiol repurposing as antibacterial. Drug repurposing may represent a faster approach to identify new antimicrobials since preclinical and clinical parameters of these drugs are already established 52 . Thereby, CBD antibacterial activity might be considered for drug repurposing and should be evaluated in clinical studies (e.g., expanded access), initially against immediately life-threatening condition or serious infections 53  showed that the addition of low concentrations of PB (≤ 2 µg/mL) allow CBD (≤ 4 µg/mL) to exert antibacterial activity against GNB (e.g., K. pneumoniae, E. coli, A. baumannii), including PB-resistant GNB. CBD + PB also showed additive and/or synergistic effect against LOS-expressing GND. Our results show promising translational potential and suggest that CBD might be considered for drug repurposing, especially in the combination CBD + PB against GNB, highlighting PB-resistant K. pneumoniae. Polymyxin B (PB) (United States Pharmacopeia) and ultrapure CBD (99.6%; BSPG-Pharm, Sandwich, UK) were used. As solvents, we used water for PB and methanol (Sigma-Aldrich) for CBD. Our previous standardization showed that methanol at concentrations ranging from 0.006 to 327.68 µL/mL on CAMHB is not antibacterial.

Methods
An aqueous solution of resazurin sodium salt (Sigma-Aldrich) was used to assess the metabolic activity and proliferation of bacterial cells, which were visually determined after bioreduction of the dye (blue) to resorufin (pink) by viable bacteria 25 . Investigation of CBD antibacterial activity against GP and GN bacteria. Antibacterial activity of CBD was investigated against a broad panel of different bacterial species, comprehending GP bacteria (13 different species; 21 strains), GN bacteria (30 different species; 73 strains), including type-strains, quality control strains, and clinical isolates (MDR and XDR strains, international high-risk clones, and also susceptible strains) (Supplementary Table 1) 27,29,30,54,55 .
Microdilution method was performed according to EUCAST recommendations for MIC determination, in agreement with the recommendations from the International Standards Organisation (ISO 20776-1 and ISO 20776-2) 26 . All MIC determination were performed in technical and experimental duplicates and, when the results were disparate, the MIC determination was repeated to confirm the results, considering the highest MIC value detected.
Two-fold serial dilution (256-0.5 µg/mL) of CBD were initially evaluated and MIC values were determined as the lowest concentrations of CBD that inhibit visible bacterial growth in broth culture medium. Polymyxin B and vancomycin were used as controls for GN and GP bacteria, respectively. In addition, ciprofloxacin was used as control for GND (Neisseria spp., M. catarrhalis), and ampicillin for S. pneumoniae and H. influenzae 23 .
Beyond visual evaluation of growth inhibition, 30 µL of a 0.01-0.02% aqueous solution of resazurin sodium salt (Sigma-Aldrich) were added to each well of the microplate. Cell viability was assessed after 30-60 min for GNB and GPC and after 60-120 min for GND and Enterococcus species. This colorimetric step was additionally performed to allow better visualization of CBD antibacterial activity 25  Investigation of CBD antibacterial activity against M. tuberculosis. We used the reference broth microdilution method to determine the CBD MIC against M. tuberculosis H37Rv (ATCC 27294) and also against rifampicin-and isoniazid-resistant M. tuberculosis CF86 (MDR clinical isolate), according to standard procedures 26 . Middlebrook 7H9 broth (Sigma-Aldrich) supplemented with 10% OADC (oleic acid, albumin, dextrose, catalase) and glycerol 0.4% was used and two-fold serial dilution (256-1 µg/mL) of CBD were evaluated.
Rifampicin and isoniazid were used as control (1-0.004 µg/mL). We used 96-well, polystyrene, flat-bottom microplates for the experiments. Each plate was incubated for seven days at 37 °C and 5% CO 2 . After incubation, 30 µL of 0.01% aqueous solution of resazurin sodium salt (Sigma-Aldrich) were added to each well of the microplate, and 24 h later the MIC was determined by fluorescence reading (excitation/emission 530/590 nm) 56 . Efflux  Broth microdilution method to determine CBD MIC was performed in the presence or absence of either PAβN (Sigma-Aldrich) (50 µg/mL), reserpine (Sigma-Aldrich) (50 µg/mL), or curcumin (256 µg/mL), in different assays 57,58 . We considered that a minimal threefold reduction in the MIC values in the presence of efflux pump inhibitors would be indicative of efflux-mediated resistance.

Investigation of CBD antibacterial activity against GNB in the presence of efflux pump inhibitors.
The colorimetric step using an aqueous solution of resazurin sodium salt (Sigma-Aldrich) was also performed to allow better visualization of CBD antibacterial activity.  (Tables 1 and 2).

Investigation of the antibacterial activity of CBD in combination with
To investigate the antibacterial activity of the combination CBD + PB, an initial screening was performed using the reference broth microdilution method with adaptations 26 : Two-fold serial dilutions (512-0.02 µg/mL) www.nature.com/scientificreports/ of PB were evaluated in the presence of 256 µg/mL of CBD (fixed concentration) in each well, including the bacterial growth control wells. Furthermore, the antibacterial activity of the combination CBD (256 µg/mL, fixed concentration) + PB (twofold dilution, 256-0.005 µg/mL) was also evaluated in the presence of PAβN (50 µg/mL), also including the bacterial growth control wells, for 7 species (13 strains). Cell viability assessment with resazurin was also performed as described above.
Confirmation by checkerboard assay. Checkerboard assays were performed to confirm the in vitro antibacterial activity and to assess the different proportions of each substance in the combination CBD + PB 59 . Final PB concentrations ranged from 0.01 to 512 µg/mL and CBD concentrations ranged from 2 to 256 µg/mL. For P. aeruginosa and plasmid-mediated colistin-resistant (MCR-1) E. coli strains, the assay was also performed in the presence of 50 µg/mL of PAβN. Confirmation of the antibacterial activity of the combination CBD + PB was performed against 22 selected strains (Tables 4 and 5).
The antibacterial activity of the combination CBD + PB considered the best well(s) for which concentrations of PB (closest to 2 µg/mL or lower) combined to CBD (lowest concentrations) inhibited bacterial growth. Cell viability assessment with resazurin was also performed as described above.
Time-kill assay. Time-kill assays were used to evaluate the synergistic effect of the combination CBD + PB and were performed as previously described 60 with slight modifications. The assays comprised 4 PB-resistant K. pneumoniae isolates, namely C9, L8, L28, and L29.
A bacterial suspension was prepared on McFarland's 0.5 scale (1.5 × 10 6 colony forming units [CFU] per mL), added to MHB, and incubated at 37 °C under shaking until the logarithmic scale of bacterial growth (McFarland's 1.0 scale) is reached (around 4 h). This suspension was added to different MHB tubes containing the combination CBD (4 µg/mL) + PB MEAC; the combination CBD (2 µg/mL) + PB MEAC; PB MIC; PB MEAC; MHB without drugs or antibiotics as to bacterial growth control. After adding the suspension to each of the tubes (time zero), an aliquot was collected, diluted, and plated onto Mueller Hinton plates for subsequent CFU counting. Tubes were then incubated at 37 °C under shaking and the aliquot collection, dilution, and plating process was repeated after 1, 2, 4, and 6 h 60 . Three independent experiments were performed.
Results of the time-kill assays were analyzed by two different methods: (1) a statistical analysis comparing all treatments with a growth control; and (2) a more straightforward, traditional, and non-statistical method comparing CFU counts between the combination CBD + PB and PB alone 60 . For statistical testing of the former method, means were compared via ANOVA followed by Tukey's test with statistical significance set to 0.05. All computations and graph plotting were performed with Prism 8 software (GraphPad Software, San Diego, CA, USA). For the latter method, combinations were considered synergistic when CBD + PB reduced CFU/mL by at least 2 log 10 compared to PB alone 28 .