Chronic Obstructive Pulmonary Disease (COPD) is the third leading cause of death globally. COPD includes two clinical phenotypes, emphysema and chronic bronchitis. Initially, factors that contribute to the onset of COPD include genetic factors, pollution, cigarette smoke, and exposure to diverse chemicals. Subsequently, exacerbation of COPD correlates with bacterial colonization and respiratory viral infections. The primary bacterial pathogens implicated in COPD exacerbation include Pseudomonas aeruginosa, Moraxella catarrhalis, Hemophilus influenzae, and Streptococcus pneumonia.
Pathogenic bacteria can survive in the lungs of COPD patients for several months. This may be attributed to the formation of biofilms, which may also be responsible for recurring episodes of acute exacerbations
[1]
. Biofilms are dense micro-communities, often attached to inert surfaces, that are encapsulated by secreted polymers
[2]
. The development of a biofilm is a dynamic process
[3]
. Initially, a planktonic microbe attaches itself to a surface, and it then subsequently aggregates with other microbes in the formation of a biofilm.
Patients who suffer from exacerbations frequently experience poor quality of life, accelerated decline in lung function, increased healthcare expenses, and increased morbidity and mortality. Studies testing patients’ sputum cultures have determined that approximately 50% of COPD exacerbations are attributed to bacterial lung infections
[4]
.
The Global Initiative for Chronic Obstructive Pulmonary Disease (GOLD) publishes guidelines that are widely accepted by clinicians in the United States and internationally as the standard on which to base therapy. The guidelines offer recommendations on how to initiate therapy in patients newly diagnosed with COPD, as well as recommendations on how to modify therapy in patients who experience frequent exacerbations. The GOLD guidelines offer recommendations regarding how to treat acute exacerbations, including antimicrobials, mucoregulators, and the use of bronchodilators and anti-inflammatory drugs
[5]
.
Natural products have consistently served as a valuable source for antimicrobial drug candidates . In recent years, studies have been performed to evaluate the medicinal applications of cannabinoids, which are found in Cannabis plants. Results of numerous studies highlight the potential antimicrobial applications of multiple substances found in Cannabis plants . Cannabinoids can be segmented as either endogenous or exogenous
[8]
. In the treatment of bacterial infections, results from animal studies demonstrated that exogenous cannabinoids, particularly Tetrahydrocannabinol (THC), can minimize resistance to numerous pathogens, including Listeria monocytogenes, Treponema pallidum, Legionella pneumophila, and Staphylococcus aureus
[9]
. Cannabidiol (CBD), another exogenous cannabinoid, has been studied in numerous in vitro studies. Results of these studies demonstrated that CBD has both bacteriostatic and bactericidal activity against several bacteria, including methicillin-susceptible Staphylococcus aureus (MSSA), Methicillin-Resistant Staphylococcus aureus (MRSA), Streptococcus mutans, and Streptococcus faecalis
[6]
[7]
. In a study conducted by Luz-Veiga et al., CBD displayed antimicrobial effects against Pseudomonas aeruginosa and Escherichia coli
[10]
. Inhibition and eradication of P. aeruginosa biofilms with CBD have been demonstrated on soft contact lenses
[11]
. Promising antibacterial activity with CBD has been reported with Salmonella typhimurium and Salmonella newington
[12]
. Anti-biofilm activity of CBD has also been demonstrated for the fungal pathogen Candida albicans
[13]
. Studies with Cannabigerol (CBG) have shown anti-biofilm activity with S. mutans, and quorum sensing and biofilm formation of Vibrio harveyi were reduced in the presence of CBG
[14]
[15]
. Cannabinoids can also be useful for enhancing the activity of antibiotics when used together with antibiotics
[16]
. Overall, these results highlight the broad applications of cannabinoids as antimicrobial therapeutic alternatives.
The most common bacterium implicated in acute exacerbation of COPD (AECOPD) is Pseudomonas aeruginosa, which is a gram-negative, aerobic species that is responsible for opportunistic infections in immunocompromised patients
[17]
. P. aeruginosa excels in its ability to form biofilms. Due to the difficulty of delivering antibiotics directly to the bacteria, these biofilms pose a significant challenge for treating infections caused by this organism. P. aeruginosa’s biofilm-forming abilities reinforce the need for novel pharmacotherapies that exhibit activity against this pathogen. There are multiple strains of P. aeruginosa that differ from one another in their genome, growth conditions, and mechanisms of biofilm formation. PA14 is one of the most well-studied strains of P. aeruginosa and holds particular importance in research as it is a clinical isolate of this bacterial species. Although PA14 is more virulent, its genome is highly conserved with that of PA01. We therefore chose PA14 for this study to gain insight into the potential clinical utility.
This study focused on investigating the activity of CBD and CBG on PA14 biofilms. Parallel assays were performed with CBD and CBG to assess inhibition as well as eradication of PA14 biofilms. The study also evaluated CBD and CBG combination formulations on both PA14 inhibition and eradication.
2. Materials and Methods
<xref ref-type="bibr" rid="scirp.145944-"></xref>2.1. Bacterial Strains, Growth Media, Conditions, and Materials
The P. aeruginosa strain PA14 used in this study was a gift from Dr. Wen Shi, Forsyth Institute/Harvard Dental School. Tryptic Soy Agar (TSA) and Tryptic Soy Broth (TSB) media (Becton Dickinson Company, Sparks, MD, USA) were used to culture the planktonic form of PA14. PA14 was incubated with agitation at 37˚C for 24 hours until absorbance at OD490nm was ≥ 1. CBD and CBG at concentrations of 1 mg/mL were purchased from Sigma-Aldrich, St. Louis, MO, USA.
2.2. Biofilm Growth and Staining Conditions
For biofilm assays, a PA14 overnight culture incubated at 37˚C under aerobic conditions in a rich medium (TSB) was diluted 1:100 into M63 minimal medium
[18]
. M63 medium was prepared as a 5X M63 stock solution by dissolving 15 g KH2PO4, 35 g K2HPO4, and 10 g (NH4)2SO4 in 1 L of water, and diluting the 5X stock solution 1:5. M63 was supplemented with magnesium sulfate (1 mM), glucose (0.2%), and casamino acids (0.5%). 100 μL of the diluted culture was transferred into each well of a 96-well Falcon 3911 microtiter plate (Becton Dickinson and Company, Sparks, MD, USA). The microtiter plate was incubated for 24 hours at 37˚C. After incubation, the plate was inverted, and the liquid was removed. The plate was rinsed with water by gently submerging it in a small container of water. Water was removed and the rinsing process was repeated a second time. This removed unattached cells and media components and significantly lowered background staining. To stain, 125 μL of a 0.1% solution of Crystal Violet (CV) (Sigma-Aldrich, St. Louis, MO, USA) in water was added to each well of the microtiter plate. The microtiter plate was incubated at room temperature for 10 - 15 min. After staining, the plate was rinsed 3 - 4 times with water by submerging it in a container of water as outlined above and blotted vigorously on a stack of paper towels to remove excess cells and dye. The microtiter plate was inverted and dried for a few hours.
2.3. The Effect of Cannabinoids in Inhibiting the Growth of PA14 Biofilms
To assess the effects of CBD and CBG in inhibiting the growth of a new biofilm, varying concentrations of each cannabinoid, ranging from 6.25 µG/mL to 200 µG/mL, were mixed with PA14 in M63 media and incubated in a 96-well Falcon 3911 microtiter plate (Becton Dickinson and Company, Sparks, MD, USA) overnight. As a control, M63 and PA14 were combined without the cannabinoid. The biofilms were subsequently incubated for 24 hours, which has been determined as the optimal time for biofilm growth of PA14. To view the results qualitatively, 0.1% crystal violet was used to stain wells and observe ring formation. Absorbance measurements were taken by solubilizing CV in 30% acetic acid to measure results quantitatively. Absorbance readings were measured by pipetting the contents from each well into a new microtiter plate and placing the plate in a Multiskan SkyHigh Microplate Spectrophotometer (Thermo Fisher Scientific) and reading at 550 nanometers. Quadruplicate experiments were performed.
2.4. The Effect of Cannabinoids in Eradicating PA14 Biofilms
To assess the effects of CBD and CBG in eradicating a preexisting biofilm, M63 media was mixed with PA 14 and incubated in a 96-well microtiter plate overnight. The contents of the inoculated wells were then gently aspirated and rinsed three times by carefully aspirating the contents of each well and replacing them with fresh M63. Each cannabinoid at the varying concentrations noted above was then mixed with PA14 in M63 and incubated overnight. Additional wells on the plate served as a control. The rationale for the choice of media and incubation time is outlined above. To view the results qualitatively, 0.1% crystal violet was used to stain wells and observe ring formation. Absorbance measurements were taken by solubilizing CV in 30% acetic acid to measure results quantitatively, as described above. Quadruplicate experiments were performed.
2.5. Effect of Two Cannabinoids on PA14 Biofilms
The effect of using CBD and CBG together was evaluated for both biofilm inhibition and eradication. The above procedures were performed as described above, but the concentrations of each cannabinoid were halved to maintain the overall concentration of cannabinoids in the bacteria and M63 mixture.
3. Results
<xref ref-type="bibr" rid="scirp.145944-"></xref>3.1. Effect of CBD and CBG on the Inhibition of PA14 Biofilm Formation
Planktonic PA14 cells assayed by either a disk diffusion assay or an agar well-based assay did not result in any observable antimicrobial activity when treated with either CBD or CBG (data not shown). The ability of CBD and CBG to inhibit biofilm formation is presented in
Figure 1(A)
and
Figure 1(B)
, respectively. As shown, a greater level of inhibition is observed with CBG as compared to CBD. With CBG, 82.3% inhibition relative to control is observed with a concentration of 25 µG/mL. In contrast, CBD did not show inhibition at 25 µG/mL. With CBD, 84.6% inhibition was demonstrated with 100 µG/mL; with CBG, 85.02% inhibition was observed with 100 µG/mL. An initial increase in biofilm was observed at the lower concentrations of both CBD and CBG. This is consistent with results reported with Streptococcus mutans when CBD was added
[7]
. The enhancement of biofilm at low concentrations was also reported with the addition of antibiotics
[19]
.
(A)<p class="imgGroupCss_v"><img class=" imgMarkCss lazy" data-original="https://html.scirp.org/file/1951218-rId15.jpeg?20250924025948" /></p>(B)<xref ref-type="bibr" rid="scirp.145944-"></xref>Figure 1. Biofilm inhibition assay. PA14 grown in the presence of (A) CBD and (B) CBG increasing in concentration (left to right) from 6.25 µG/mL to 200 µG/mL. Results represent the percent of biofilm measured relative to control (no cannabinoid addition) by a Multiskan SkyHigh Microplate Spectrophotometer (Thermo Fisher Scientific) read at 550 nanometers. Quadruplicate experiments were performed.
3.2. Eradication of Preformed PA14 Biofilms by CBD or CBG
Varying concentrations of CBD or CBG, as described above in Methods, were applied to mature PA14 biofilms 24 hours after inoculation.
Figure 2(A)
and
Figure 2(B)
demonstrate the eradication of mature PA14 biofilms treated with different doses of CBD (
Figure 2(A)
) or CBG (
Figure 2(B)
). Eradication of PA14 biofilm was less efficient with CBD as compared to CBG. A concentration of 50 µG/mL showed no eradication with CBD. In contrast, 51.5% eradication was demonstrated with CBG. At 100 µG/mL, 42% eradication was demonstrated with CBD, whereas CBG resulted in 76.2% eradication. The inhibition and eradication experiments show an apparent differential activity between CBD and CBG, with CBG being more active.
(A)<p class="imgGroupCss_v"><img class=" imgMarkCss lazy" data-original="https://html.scirp.org/file/1951218-rId17.jpeg?20250924025949" /></p>(B)<xref ref-type="bibr" rid="scirp.145944-"></xref>Figure 2. Biofilm eradication assay. PA14 biofilms treated with (A) CBD and (B) CBG increasing in concentration (left to right) from 6.25 to 200 μG/mL. Results represent the percent of biofilm measured relative to control (no cannabinoid addition) by a Multiskan SkyHigh Microplate Spectrophotometer (Thermo Fisher Scientific) read at 550 nanometers. Quadruplicate experiments were performed.
Evaluation of CBD and CBG in combination on PA14 biofilms. Because of the higher level of activity observed with CBG as compared to CBD in both the inhibition and eradication experiments, combinations of CBD and CBG were assayed at the same dose level as tested in the individual inhibition and eradication experiments to investigate the effect of the combinations on PA14 inhibition and eradication. The objective was to determine whether CBG in combination with CBD could result in similar inhibition and eradication levels as observed with CBG alone. The inhibition of PA14 biofilm formation with combined CBD-CBG doses is shown in
Figure 3
. Eradication of a mature PA14 biofilm with combined CBD-CBG doses is represented in
Figure 4
. The results demonstrate that 90.6% inhibition with combined CBD-CBG doses, at 100 µG/mL (
Figure 3
), was superior relative to 85.09% eradication obtained with combined CBD-CBG doses (
Figure 4
). Interestingly, when combined with CBD, CBG drives greater inhibition at 100 µG/mL, 90.6% as compared to 84.6% with CBD alone. Similarly, combined CBD and CBG result in greater eradication, 85.09% as compared to 42% with CBD alone. The results demonstrate that CBG is more efficient in the inhibition and eradication of PA14 biofilms than CBD.
<xref ref-type="bibr" rid="scirp.145944-"></xref>Figure 3. Inhibition assay with combined CBD and CBG doses. PA14 grown in the presence of CBD-CBG doses increasing in concentration (left to right) from 6.25 µg/mL to 200 µg/mL. Results represent the percent of biofilm measured relative to control (no cannabinoid addition) by a Multiskan SkyHigh Microplate Spectrophotometer (Thermo Fisher Scientific) read at 550 nanometers. Quadruplicate experiments were performed.
<xref ref-type="bibr" rid="scirp.145944-"></xref>Figure 4. Eradication assay with combined CBD and CBG doses. PA14 biofilms were treated with CBD-CBG doses increasing in concentration (left to right) from 6.25 µG/mL to 200 µG/mL. Results represent the percent of biofilm measured relative to the control (no cannabinoid addition) by a Multiskan SkyHigh Microplate Spectrophotometer (Thermo Fisher Scientific) read at 550 nanometers. Quadruplicate experiments were performed.
4. Discussion
Pseudomonas aeruginosa strain PA14, a burn wound isolate, is significantly more virulent than the routine laboratory strain PA01
[20]
. Although PA14 is more virulent, its genome is highly conserved with that of PA01. Both PA01 and PA14 have been fully sequenced. The genome of PA01 encodes 5700 genes, and the genome of PA14 encodes 5892 genes
[21]
. PA14 harbors two pathogenicity islands which encode unique virulence-associated genes
[22]
.
Treatment of patients infected with P. aeruginosa has been challenging; the bacterium is highly resistant to antibiotics. Resistance to antibiotics is more pronounced in biofilm-based infections
[23]
. The tendency of P. aeruginosa to cause persistent infections with increased mortality rates is attributed to its ability to form biofilms
[24]
. PA01 attaches quickly to a surface and progresses to biofilm formation in an irreversible process. In contrast, PA14 is slower at initial surface attachment, and biofilm formation is reversible. Because of its resistance to current therapeutics, the World Health Organization (WHO) has classified P. aeruginosa as one of the ESKAPE pathogens—pathogens in need of novel therapeutics
[25]
. As a consequence of the multidrug resistance in P. aeruginosa, the search for and discovery of novel therapeutics have been prioritized.
In this study, we show that CBD and CBG display dose-dependent antimicrobial activity in the inhibition and eradication of PA14 biofilms. For PA14, the antimicrobial activity is specific for biofilms. Planktonic PA14 cells assayed by either a disk diffusion assay or an agar well-based assay did not result in any observable antimicrobial activity when treated with either CBD or CBG (data not shown). Anti-biofilm activity is observed when CBD or CBG is used alone, or when the two cannabinoids are used together. Inhibition experiments evaluating each cannabinoid independently demonstrated greater sensitivity with CBG as compared to CBD. Similarly, experiments with combined CBD-CBG doses demonstrated greater sensitivity with inhibition than eradication. In contrast to most other studies that have evaluated the efficacy of cannabinoids as antimicrobial agents by focusing on either CBD or CBG, this study compared PA14 biofilm inhibition and eradication using both cannabinoids, CBD and CBG. Moreover, an additional uniqueness of this study entailed combinations of CBD and CBG evaluated for inhibition and eradication. Other studies reported in the literature have investigated combining a cannabinoid with either an antibiotic
[16]
or an antiseptic compound
[26]
[27]
to enhance activity. This study showed enhanced activity of CBD in both inhibition and eradication assays when combined with a second cannabinoid, CBG. An initial increase in biofilm was observed at the lower concentrations of both CBD and CBG. This suggests a potential risk at sub-inhibitory concentrations. The increase in biofilm observed in our study is consistent with results reported with Streptococcus mutans when CBD was added
[7]
. The enhancement of biofilm at low concentrations was also reported with the addition of antibiotics
[19]
. The results of this in vitro study suggest that these compounds may be a viable option for individuals suffering from a biofilm infection. Additional validation in more complex systems including in vivo models or co-culture systems will be the focus of future studies.
Zeng et al. proposed that the anti-microbial mechanism of CBD against Gram-positive bacteria entails damage to the bacterial cell wall and cell membrane. Significant changes were observed in the proteomic and metabolic profiles. Processes affected include DNA binding, protein translation and expression, and phosphorylation
[28]
. Similarly, Blaskovich et al. demonstrated that protein, DNA, RNA, and peptidoglycan synthesis were all significantly inhibited when CBD was added to S. aureus
[29]
. Whether the anti-microbial mechanism of cannabinoids on P. aeruginosa, a Gram-negative organism, is comparable to the Gram-positive organisms or perhaps elicits changes in quorum sensing
[30]
will be the focus of future studies.
As resistance to antibiotic therapies continues to increase, the necessity for effective drug therapy becomes even more pronounced. Treatment options for Pseudomonas aeruginosa, a pathogen that manifests virulence by forming biofilms that are unresponsive to conventional antibiotics, remain limited. The findings from this study indicate that CBD and CBG exhibit anti-biofilm activity against this difficult pathogen. Because this study examined the effects of cannabinoids on a clinical isolate, the results are more reflective of what would be observed in actual patients. The implications of these discoveries have immense potential, as these compounds could be the future of treating stubborn biofilm infections in COPD exacerbations as well as other infections.
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