Potential of Cannabidiol (CBD) to overcome extensively drug-resistant Acinetobacter baumannii

Abstract

Extensively drug-resistant (XDR) Acinetobacter baumannii poses a serious clinical challenge due to its resistance to nearly all available antibiotics, including carbapenems and colistin. Cannabidiol (CBD), a non-psychoactive phytochemical from Cannabis sativa L., has recently shown promising antimicrobial activity. This study evaluates the antibacterial and anti-biofilm effects of CBD against XDR A. baumannii isolates and explores its mechanism of action and potential as an adjunct therapeutic agent. Twenty-six A. baumannii isolates collected from ICU medical devices were identified using MALDI-TOF/MS. Antimicrobial susceptibility was assessed by disk diffusion and broth microdilution to determine MICs and MBCs for CBD and standard antibiotics. Synergistic effects were evaluated via checkerboard assays and FICI values. Biofilm inhibition and eradication were assessed using crystal violet and MTT assays. Time-kill studies, membrane integrity assays (DNA/protein leakage, NPN uptake, membrane depolarization), and scanning electron microscopy (SEM) were employed to investigate bactericidal kinetics and membrane-disruptive mechanisms. CBD exhibited activity against antimicrobial resistance isolates (MIC: 3.9 to > 500 µg/mL). Remarkably, CBD synergized with gentamicin, meropenem, and colistin, reducing their effective concentrations by up to 1,000-fold. Combination therapy significantly inhibited and eradicated biofilms. Time-kill assays demonstrated rapid, concentration-dependent killing, with complete bacterial clearance at 4× MIC within 2 h. Mechanistic assays and SEM confirmed that CBD induces extensive membrane damage. These findings highlight CBD’s potential as an effective adjunct to conventional antibiotics for treating XDR A. baumannii infections, offering a novel strategy to counteract antimicrobial resistance.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12906-025-05056-w.

Introduction

Acinetobacter baumannii is an opportunistic, Gram-negative bacterium that has emerged as a major cause of hospital-associated infections (HAIs) worldwide, with no standard therapeutic recommendation for its management and control [1]. It primarily affects critically ill and immunocompromised patients, leading to severe infections such as ventilator-associated pneumonia, bloodstream infections, urinary tract infections, meningitis, and wound infections [2–4]. A. baumannii’s remarkable ability to survive in hospital environments, resist desiccation, and persist on medical equipment—particularly in intensive care units (ICUs)—makes it a persistent challenge in healthcare settings [4–7].

A major concern with A. baumannii is its exceptional ability to develop multidrug resistance (MDR), defined as resistance to at least one agent in three or more antibiotic classes, including cephalosporins, polymyxins, carbapenems, and aminoglycosides. Additionally, extensively drug-resistant (XDR) A. baumannii is resistant to all available antibiotic classes, including last-resort drugs such as colistin and carbapenems. Pan-drug-resistant (PDR) A. baumannii exhibits resistance to all antimicrobial agents across all antibiotic classes, posing an even greater clinical challenge. Consequently, treatment options for infections caused by XDR and PDR A. baumannii are extremely limited, leaving clinicians with few therapeutic options, such as antibiotic combinations, which may be associated with safety concerns [8, 9].

In addition to its resistance mechanisms, a range of virulence factors contribute to the antimicrobial resistance of A. baumannii, including outer membrane proteins, lipopolysaccharides, capsules, phospholipases, nutrient acquisition systems, efflux pumps, protein secretion systems, quorum sensing, and biofilm formation [10–12]. These factors enhance the pathogen’s ability to survive under stressful conditions, evade immune responses, and increase its resistance to antibiotics.

Highly drug-resistant A. baumannii infections have been associated with high mortality rates, approximately 44%, particularly among critically ill patients, leading to prolonged hospital stays and increased healthcare costs [13]. The convergence of these resistance mechanisms has led to the alarming global spread of antimicrobial resistance A. baumannii, prompting the World Health Organization (WHO) to classify it as a critical-priority pathogen requiring urgent research into novel therapeutic strategies [14]. Addressing this challenge necessitates the development of innovative treatment approaches, including alternative antimicrobial agents, biofilm-disrupting compounds, and synergistic combination therapies. One promising avenue is the repurposing of existing compounds with antimicrobial properties.

Cannabidiol (CBD) is a major non-psychoactive phytocannabinoid derived from Cannabis sativa L., with a molecular weight of 314.5 Da. Its chemical structure consists of a pentyl-substituted bis-phenol (pentylresorcinol) core linked to an alkyl-substituted cyclohexene terpene ring system. Structurally, CBD comprises three key components: (1) aromatic phenol groups, which contribute to its antioxidant and potential antimicrobial properties; (2) a terpenoid cyclohexene ring, a common feature in cannabinoids that influences their bioactivity; and (3) a pentyl side chain, which plays a crucial role in CBD’s interaction with lipid membranes and protein targets [15, 16]. Among the more than 100 cannabinoids identified in C. sativa L., CBD is one of the most biologically active, demonstrating anti-inflammatory, analgesic, and neuroprotective properties [17–19].

Recently, CBD has gained attention for its potential antimicrobial properties against various drug-resistant pathogens, including both Gram-positive and Gram-negative bacteria [20]. CBD exhibits bactericidal activity against Methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pneumoniae, and Clostridioides difficile, primarily through membrane disruption [21]. Additionally, CBD has demonstrated efficacy against Gram-negative pathogens such as A. baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa when used in combination with the conventional antibiotic polymyxin B, suggesting a potential synergistic effect [22, 23]. Among these, CBD has shown potential activity against intracellular Mycobacterium tuberculosis, the causative agent of tuberculosis [23–25].

Although CBD has demonstrated antimicrobial properties, its efficacy against antimicrobial resistance A. baumannii and the precise mechanisms involved remain poorly understood. This study aims to bridge this gap by elucidating the mode of action of CBD, including its impact on bacterial membrane integrity and biofilm formation, providing a foundation for future therapeutic applications.

Therefore, this study aims to comprehensively evaluate the antibacterial and anti-biofilm activities of CBD against highly drug-resistant A. baumannii, with a particular focus on its potential to disrupt bacterial membrane integrity and enhance the efficacy of conventional antibiotics. By providing insights into the antimicrobial effects of CBD, this study aims to establish its potential as a therapeutic agent against highly drug-resistant A. baumannii, contributing to the development of alternative strategies for managing drug-resistant bacterial infections.

Methods

CBD and bacterial isolates

An analytical-grade CBD solution (≥ 98% purity in methanol, Product No. C-045) was purchased from Supelco (Sigma-Aldrich, St. Louis, MO, USA). A total of 26 Acinetobacter spp. isolates were obtained from medical devices in the Intensive Care Unit (ICU) of Phayao Hospital, Thailand. These devices included endotracheal tubes, infusion sets, urinary catheters, and nasogastric tubes, as described in a previous report [26]. A. baumannii ATCC 17978 was used as a reference strain.

Identification of Acinetobacter baumannii using MALDI-TOF/MS

Acinetobacter spp. isolates were identified using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/MS) with the Bruker Autoflex maX TOF/TOF Biotyper (Bruker Daltonics, Bremen, Germany) at the Synchrotron Research and Applications Division, Synchrotron Light Research Institute (Public Organization), Thailand. A total of 26 bacterial isolates belonging to the Acinetobacter genus were cultured on tryptic soy agar (TSA) plates (Difco Laboratories, Inc., New Jersey, USA) at 37 °C for 24 h. Colonies were prepared for analysis using the formic acid extraction method, as described in previous studies [27, 28]. Briefly, a single purified bacterial colony was transferred into 300 µL of sterile water, thoroughly mixed with 900 µL of absolute ethanol (Sigma Chemical Co., St. Louis, MO, USA), and centrifuged at 12,000 × g for 2 min. The supernatant was discarded, and the pellet was resuspended in 50 µL of 70% formic acid (Sigma Chemical Co., St. Louis, MO, USA) and 50 µL of acetonitrile (Sigma Chemical Co., St. Louis, MO, USA). The mixture was centrifuged again at 12,000 × g for 2 min. One microliter of the resulting supernatant was spotted onto a MALDI target plate (MTP 384 target plate polished steel BC; Bruker Daltonics, Bremen, Germany) and allowed to air-dry at room temperature. The dried spot was overlaid with 1 µL of HCCA matrix solution (α-Cyano-4-hydroxycinnamic acid; Bruker Daltonics) prepared in 50% acetonitrile and 2.5% trifluoroacetic acid (Sigma-Aldrich Chemical Co., Madrid, Spain) and allowed to air-dry again.

The MALDI-TOF Biotyper analysis was conducted by processing the mass spectrum profiles using FlexControl software version 3.4 (Bruker Daltonics). The spectra were recorded in the positive linear mode at the laser frequency of 2,000 Hz and in the mass range of m/z 2,000–20,000 Dalton. Calibration was performed using the E. coli strain DH5α, which presents ribosomal protein mass with RNA and myoglobin at peaks of 4,365.30, 5,096.80, 5,381.40, 6,255.40, 7,274.50, 10,300.10, 13,683.20 and 16,952.30 m/z. Two-thousand shots of the mass spectrum profile data were collected from each sample. The mass spectrum profiles were processed with Flexcontrol V3.4 (Karlsruhe, Germany). Protein mass fingerprints were analyzed with a recalibration tolerance of 300 parts per million (ppm). The resulting protein mass fingerprint data were then transferred to the MALDI Biotyper Realtime Classification (RTC) software version 4.0 for comparison with a reference mass fingerprint database to identify the species.

Antimicrobial susceptibility testing

Firstly, the antibiotic susceptibility of 26 isolates was tested using the disc diffusion (Kirby-Bauer) method, following the Clinical and Laboratory Standards Institute (CLSI) M100 guidelines, 30th edition (2020) [29]. Bacterial suspensions were adjusted to a 0.5 McFarland standard and inoculated onto Mueller-Hinton agar (MHA) from Difco Laboratories, Inc., New Jersey, USA, followed by the placement of 13 antibiotic disks from Oxoid Limited, United Kingdom, including amikacin (AK), ceftazidime (CAZ), ciprofloxacin (CIP), colistin (CO), gentamicin (CN), doxycycline (DO), cefepime (FEP), imipenem (IMP), meropenem (MEM), piperacillin (PRL), trimethoprim-sulfamethoxazole (SXT), tetracycline (TE), and piperacillin/tazobactam (TZP). Plates were incubated at 37 °C for 18 h, and inhibition zones were measured and interpreted using the CLSI breakpoint guidelines, with results reported as susceptible (S), intermediate (I), or resistant (R). E. coli ATCC 25922 was used as an internal control.

Antimicrobial activity assay

The minimum inhibitory concentration (MIC) values of CBD and antibiotics—gentamicin, meropenem, and colistin—against antimicrobial resistance A. baumannii strains were determined using the microdilution method in accordance with CLSI guidelines [29]. CBD, gentamicin, meropenem, and colistin were serially diluted in Mueller-Hinton broth (MHB), starting at concentrations of 500 µg/mL, 10,000 µg/mL, 5,000 µg/mL, and 500 µg/mL, respectively. Each A. baumannii strain was cultured at a density of 5 × 10⁵ CFU/mL and incubated at 37 °C for 24 h. Following incubation, 10 µL of resazurin (Alpha Chemika, Mumbai, Maharashtra, India) was added, and the plates were further incubated for 4 h to observe color changes. A shift from blue to pink indicated bacterial growth, while the MIC was defined as the lowest drug concentration that prevented this change. MHB media solution (without bacteria added) was used as a negative control to check for contamination and confirm the accuracy of the MIC readings. To determine the minimum bactericidal concentration (MBC), 10 µL of samples at concentrations exceeding the MIC were spotted onto MHA. The MBC value was established based on the absence of bacterial colony formation, as described in previous studies [30, 31].

Checkerboard assays

The combination of CBD and antibiotics (gentamicin, meropenem, and colistin) was evaluated by checkerboard assays, according to CLSI guidelines [29], to assess the interaction between CBD and antimicrobial agents against A. baumannii isolates A24 and A29. These isolates exhibited high-level resistance to all 13 antibiotics tested, indicating an XDR phenotype. CBD and antibiotics were two-fold diluted in MHB to generate a two-dimensional concentration gradient. CBD concentrations ranged from 1× MIC to 1/128× MIC, while the antibiotics—gentamicin, meropenem, and colistin—were diluted from 1× MIC to 1/1024× MIC. A 96-well microtiter plate was used, with one antibiotic diluted across the rows and the second across the columns, forming a checkerboard layout. Bacterial cells inoculum of 5 × 10⁵ CFU/mL was added to each well, and the plate was incubated at 37 °C for 24 h. The MIC values for each compound, both individually and in combination, were determined by identifying the lowest concentration that inhibited bacterial growth. CBD and antibiotic interactions were assessed using the Fractional Inhibitory Concentration Index (FICI), which classifies interactions as synergy (FICI ≤ 0.5), additive effect (0.5 < FICI ≤ 4), or antagonism (FICI > 4). FICI was calculated as the sum of FIC(a) and FIC(b), where FIC(a) = MIC of CBD in combination/MIC of CBD alone and FIC(b) = MIC of the antibiotic in combination/MIC of the antibiotic alone.

Biofilm formation and eradication assay

To assess the effect of CBD on biofilm formation, we proceeded as described in previous studies with some modifications [21, 32]. A. baumannii isolate A29 was cultured and adjusted to a final concentration of 1 × 10⁸ CFU/mL. A 96-well microtiter plate was inoculated with 200 µL of the bacterial suspension per well. CBD was then added at concentrations of 1×, 2×, and 4× MIC. Additionally, each antibiotic was tested at 1× MIC. The potential synergistic effect of CBD in combination with antibiotics was also evaluated at the appropriate concentration. The plate was incubated at 37 °C for 24 h to allow biofilm formation. After incubation, non-adherent cells were removed by washing with phosphate-buffered saline (PBS), and the wells were heat-dried at 60 °C. Biofilm biomass was quantified using 0.1% crystal violet (LOBA Chemie Pvt. Ltd., Mumbai, India) staining for 15 min, followed by two PBS washes. The bound stain was solubilized in 200 µL of 95% ethanol (CRL Chemicals Co., Ltd., Bangkok, Thailand) for 20 min at room temperature, and absorbance was measured at 595 nm. Biofilm formation was calculated using the formula: (A_t/A_c) ×100, where Aₜ is the OD₅₉₅ of CBD-treated samples, and A_𝚌 is the OD₅₉₅ of the untreated control.

For the eradication assay, biofilms were first allowed to form for 24 h, as described above. After incubation, planktonic cells were removed, and the biofilms were rinsed with PBS. The appropriate treatment was then applied, and the plate was incubated for an additional 24 h at 37 °C. Biofilm biomass was quantified using 0.1% crystal violet staining. To assess the viability of preformed biofilms, the wells were incubated with 5 mg/mL of MTT (3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyl tetrazolium bromide) (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at 37 °C. After staining, the solution was discarded, and DMSO (Merck, Darmstadt, Germany) was added to dissolve the formazan product. The optical density was then measured at 570 nm.

Time-kill kinetics assay

The antibacterial activity of CBD against the representative A. baumannii isolate A29 was evaluated using a time-kill assay, following a previously established method [21, 33]. The tested strain was grown overnight in Tryptic soy broth (TSB) medium (HiMedia Laboratories Pvt. Ltd., Mumbai, India) until reaching the logarithmic phase. The cultures were then adjusted to an initial inoculum of approximately 5 × 10⁵ CFU/mL before being treated with CBD alone at concentrations of 1×, 2×, 4×, and 8× MIC or in combination with antibiotics at the appropriate synergistic dose. Untreated cultures served as growth controls. Bacterial cultures were incubated at 37 °C with constant shaking to ensure proper aeration. At predetermined time points (0, 1, 2, 4, 8, 16, and 24 h), aliquots were taken, serially diluted in PBS and plated onto TSA (HiMedia Laboratories Pvt. Ltd., Mumbai, India) plates. The plates were incubated at 37 °C for 18–24 h, and bacterial colonies were counted to determine CFU/mL at each time point. A bactericidal effect was defined as a reduction of ≥ 3 log₁₀ CFU/mL compared to the initial inoculum, whereas a decrease of < 3 log₁₀ CFU/mL indicated a bacteriostatic effect.

Outer membrane permeabilization analysis

The disruption of the outer membrane in A. baumannii isolate A29 was initially assessed by evaluating DNA and protein leakage, following previously established methods [33]. Bacterial cultures were treated with CBD at concentrations of 1×, 2×, and 4× MIC for 1 h at 37 °C. DNA leakage was quantified by measuring the optical density (OD) at 260 nm using a NANO-400 A Micro Spectrophotometer (Hangzhou Allsheng Instruments Co., Ltd., Hangzhou, China), while protein leakage was analyzed using the Bio-Rad DC Protein Assay Kit (Bio-Rad Laboratories, Inc., USA) and compared against a bovine serum albumin (BSA) (Bio-Rad Laboratories, Hercules, CA, USA) standard curve.

Additionally, outer membrane permeability and membrane potential were assessed by staining the treated bacterial cells with N-phenyl-1-naphthylamine (NPN) (TCI, Tokyo, Japan) and rhodamine 123 (Rh123) fluorescence dyes (Sigma Chemical Co., St. Louis, MO, USA), respectively, as described in a previous report [33]. The fluorescence intensity of NPN (excitation/emission: 350/420 nm) and Rh123 (excitation/emission: 480/530 nm) was measured, and the relative fluorescence intensity (%) was calculated using the formula: (F1/F0) × 100, where F0 represents the fluorescence intensity of untreated cells and F1 corresponds to that of treated cells. A positive control using 0.1% Triton X-100 (Sigma Chemical Co., St. Louis, MO, USA) was included for comparison, while PBS inoculated with the same bacterial suspension served as the negative control.

Scanning Electron Microscopy (SEM)

To examine the effects of CBD on A. baumannii isolate A29, bacterial cultures were adjusted to 1 × 10⁸ CFU/mL and treated with 4× MIC of CBD at 37 °C for 2 h. After treatment, bacterial cells were collected by centrifugation at 3000 × g for 5 min, and the pellet was resuspended in 1 mL of 2.5% glutaraldehyde (Sigma Chemical Co., St. Louis, MO, USA) for fixation at 4 °C for 3 h. Following fixation, the cells were washed with PBS and dehydrated using a graded ethanol series (30%, 50%, 70%, and 90%, each for 30 min). The samples were then subjected to critical point drying (CPD) and gold coating before imaging with scanning electron microscopy (SEM) using a TESCAN Vega III (Czech Republic) [33].

Cytotoxicity assay

The cytotoxicity of CBD was assessed in human embryonic kidney (HEK) 293 cells using MTT assay [34]. The cells were maintained in 60 mm dishes in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Waltham, MA, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Waltham, MA, USA) and 1% penicillin/streptomycin (Sigma Chemical Co., St. Louis, MO, USA), and incubated at 37 °C in a humidified atmosphere of 5% CO₂. HEK293 cells were seeded at 1 × 10³ cells/100 µL in 96-well culture plates and treated with various concentrations of CBD (0.5–5 µg/mL) for 24, 48, and 72 h. The cells were then incubated with 20 µL of 5 mg/mL MTT for 4 h, after which the formazan crystals were dissolved in 200 µL of DMSO. Absorbance was measured at 570 nm. Untreated cells cultured in DMEM were used as the negative control to represent 100% cell viability. The percentage of viable cells was calculated, and CBD cytotoxicity was expressed as the IC₅₀ value, determined using a linear equation of the relative viability rate versus CBD concentration.

Statistical analysis

All experiments were performed in at least three independent replicates, and the results are expressed as mean ± standard deviation (SD). Statistical analysis was conducted using one-way ANOVA followed by Dunnett’s multiple comparison test to compare treated groups with the control, or two-way ANOVA followed by Bonferroni post-tests. Analyses were performed using GraphPad Prism 5.01 (GraphPad Software, Inc., La Jolla, CA, USA). A p-value < 0.05 was considered statistically significant, indicating a meaningful difference between the experimental conditions and the control.

Results

Species confirmation of A. baumannii using MALDI-TOF/MS and its antibiotic susceptibility profile

In this study, MALDI-TOF MS identified all 26 isolates as A. baumannii. Of these, 24/26 (92.3%) had a log score between 2.0 and 3.0, and 2/26 (7.7%) had a log score between 1.7 and 1.9 (Table S1). According to the manufacturer, a log score between 2.0 and 3.0 indicates a highly probable species identification and secure genus identification, while a score between 1.7 and 1.999 indicates probable species identification and genus identification. A score value less than 1.7 is considered unreliable for identification. Therefore, all 26 isolates of antibiotic resistant A. baumannii will be treated with CBD and current antibiotics in future studies.

A total of 26 A. baumannii isolates were tested for resistance across 11 profiles using a panel of 13 antibiotics. It is important to note that the 13 antibiotics selected for the disk diffusion assay were recommended by the CLSI for testing A. baumannii. These antibiotics represent multiple antibiotic classes—penicillins (PRL), β-lactam/β-lactamase inhibitor combinations (TZP), parenteral cephems including cephalosporins (CAZ, FEP), carbapenems (IMP, MEM), lipopeptides/polymyxins (CO), aminoglycosides (AK, CN), tetracyclines (DO, TE), fluoroquinolones (CIP), and folate pathway antagonists (SXT). This comprehensive panel was used to accurately assess antimicrobial resistance patterns and to classify the isolates as multidrug-resistant (MDR) or XDR according to standardized clinical criteria. The isolates exhibited diverse resistance patterns, with several showing MDR and XDR. The resistance patterns are summarized in Table 1. Notably, isolates A24 and A29 were identified as XDR, being resistant to all 13 antibiotics tested. This highlights the extreme drug resistance in these strains, including resistance to both first-line and last-resort antibiotics, such as carbapenems and colistin.

Table 1.

Antibiotic resistance profile of A. baumannii isolated from medical devices in the intensive care unit (ICU)

Pattern No.	Resistance patterns	Isolates	Classification
1	CN, MEM	A33	Not MDR
2	CAZ, CN, CO, MEM	A36	MDR
3	CAZ, CN, MEM, PRL	A31, 35	MDR
4	CAZ, CN, CO, MEM, PRL,	A32	MDR
5	CAZ, CN, MEM, PRL, TZP	A34	MDR
6	CAZ, CIP, CN, FEP, IMP, MEM, PRL, TE, TZP	A8	MDR
7	CAZ, CIP, CN, FEP, IMP, MEM, PRL, TE, TZP	A1, A2, A3, A4, A5, A6, A7, A9, A10, A14	MDR
8	AK, CAZ, CIP, CN, CO, DO, FEP, IMP, MEM, PRL, TE, TZP	A13	XDR
9	CAZ, CIP, CN, DO, FEP, IMP, MEM, PRL, SXT, TE, TZP	A20	MDR
10	AK, CAZ, CIP, CN, DO, FEP, IMP, MEM, PRL, SXT, TE, TZP	A25, A26, A27, A28, A30	MDR
11	AK, CAZ, CIP, CN, CO, DO, FEP, IMP, MEM, PRL, SXT, TE, TZP	A24, A29	XDR

Abbreviations: AK Amikacin, CAZ Ceftazidime, CIP Ciprofloxacin, CN Gentamicin, CO Colistin, DO Doxycycline, FEP Cefepime, IMP Imipenem, MEM Meropenem, PRL Piperacillin, SXT Trimethoprim-sulfamethoxazole, TE Tetracycline, TZP Piperacillin/tazobactam, MDR Multidrug-resistant, XDR Extensively drug-resistant

Effectiveness of CBD against antimicrobial resistance A. baumannii

We evaluated the efficacy of CBD in combination with three representative antibiotics—gentamicin, meropenem, and colistin—using the broth microdilution method. These antibiotics were selected as they represent major classes—aminoglycosides, carbapenems, and polymyxins—that are commonly employed as last-resort treatments against highly drug-resistant A. baumannii. The MIC and MBC values of each compound are summarized in Table 2.

Table 2.

Antimicrobial sensitivity of Cannabidiol (CBD) and antibiotics against antimicrobial resistance A. baumannii

No.	Isolates	CBD (µg/mL)		Gentamicin (µg/mL)		Meropenem (µg/mL)		Colistin (µg/mL)
		MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC
1	A1	125.00	250.00	625.00	1250.00	19.53	39.06	3.90	7.81
2	A2	> 500.00	> 500.00	1250.00	2500.00	19.53	39.06	1.95	3.90
3	A3	125.00	250.00	625.00	1250.00	19.53	39.06	3.90	7.81
4	A4	125.00	250.00	625.00	1250.00	19.53	39.06	3.90	7.81
5	A5	125.00	250.00	625.00	1250.00	9.76	19.53	3.90	7.81
6	A6	31.25	62.50	2500.00	5000.00	19.53	39.06	3.90	7.81
7	A7	125.00	250.00	78.13	156.25	9.76	19.53	0.98	1.95
8	A8	> 500.00	> 500.00	39.06	78.13	9.76	19.53	0.98	1.95
9	A9	3.90	7.81	78.13	156.25	9.76	19.53	1.95	3.90
10	A10	> 500.00	> 500.00	19.53	39.06	9.76	19.53	0.98	1.95
11	A13	250.00	500.00	5000.00	10000.00	1250.00	2500.0	125.00	500.00
12	A14	125.00	250.00	5000.00	10000.00	1250.00	2500.0	1.95	3.90
13	A20	125.00	250.00	312.50	625.00	156.25	312.50	1.95	3.90
14	A24	31.25	62.50	500.00	1000.00	156.25	312.50	7.81	15.63
15	A25	125.0	250.00	5000.00	10000.00	9.76	19.53	1.95	3.90
16	A26	125.0	250.00	312.50	625.00	156.25	312.50	1.95	3.90
17	A27	62.50	125.00	1250.00	2500.00	19.53	39.06	1.95	3.90
18	A28	62.50	125.00	2500.00	5000.00	312.50	625.00	3.90	7.81
19	A29	62.50	125.00	312.50	625.00	19.53	39.06	15.63	31.25
20	A30	125.00	250.00	2500.00	5000.00	2500.00	5000.00	0.98	1.95
21	A31	125.00	250.00	5000.00	10000.00	19.53	39.06	0.98	1.95
22	A32	125.00	250.00	5000.00	10000.00	78.13	156.25	7.81	15.63
23	A33	62.50	125.00	500.00	1000.00	78.13	156.25	3.90	7.81
24	A34	125.00	250.00	2500.00	5000.00	78.13	156.25	1.95	3.90
25	A35	125.00	250.00	5000.00	10000.00	156.25	312.50	0.98	1.95
26	A36	> 500.00	> 500.00	5000.00	10000.00	78.13	156.25	7.81	15.62
27	ATCC 17978	250.00	500.00	9.76	19.5	4.88	9.76	0.24	0.49

Resistance (R) is defined as gentamicin > 16 µg/mL, meropenem > 8 µg/mL, and colistin > 4 µg/mL, as indicated by CLSI

Abbreviations: MIC Minimum Inhibitory Concentration, MBC Minimum Bactericidal Concentration

The MIC values of CBD varied widely across the tested A. baumannii isolates, ranging from 3.9 µg/mL to > 500 µg/mL. The lowest MIC was 3.9 µg/mL, as observed in isolate A9, while several isolates (A2, A8, A10, and A36) exhibited MIC values exceeding 500 µg/mL, indicating resistance. The MBC values followed a similar trend, ranging from 7.8 µg/mL to > 500 µg/mL. The MIC values for gentamicin ranged between 19 µg/mL and 5000 µg/mL. Based on CLSI resistance criteria (R > 16 µg/mL), all antibiotic resistant A. baumannii isolates were classified as resistant to gentamicin, demonstrating the limited efficacy of this antibiotic against the tested strains. Meropenem displayed MIC values between 7.8 µg/mL and 2500 µg/mL, with several isolates (A30, A31, A35) exhibiting high MIC values (≥ 2500 µg/mL), confirming carbapenem resistance. However, most A. baumannii isolates showed resistance (R > 8 µg/mL) as per CLSI guidelines, reaffirming the prevalence of carbapenem-resistant A. baumannii (CRAB) among the tested strains. Colistin, the last antibiotic of choice, exhibited the lowest MIC values among the tested antibiotics, ranging from 0.097 µg/mL to 125 µg/mL. The majority of A. baumannii isolates showed susceptibility to colistin, with MIC values below the resistance threshold (R > 4 µg/mL). However, isolates A13, A24, A29, A32, and A36 displayed notably high MIC values, indicating colistin resistance. The reference strain A. baumannii ATCC 17978, a non-resistant strain, remained susceptible to all tested antibiotics. These results suggest that CBD is effective against antimicrobial resistance A. baumannii, particularly XDR strains, providing a potential alternative or adjunct to traditional antibiotics in combating these resistant strains.

CBD enhances the synergistic effect of antibiotics against XDR A. baumannii

To evaluate the potential of CBD as an adjuvant therapy, we assessed its synergistic effects in combination with commonly used antibiotics—gentamicin, meropenem, and colistin—against XDR A. baumannii isolates A24 and A29, both of which exhibit resistance to up to 13 antibiotics. The Fractional Inhibitory Concentration Index (FICI) values, summarized in Table 3, indicate a strong synergistic interaction between CBD and the tested antibiotics, with FICI values ranging from 0.009 to 0.251.

Table 3.

Synergistic effect of CBD in combination with antibiotics against XDR A. baumannii

Isolates	Drugs	MIC (µg/mL) of CBD [a]		FIC [a]	Reduction Fold of CBD	MIC (µg/mL) of Drug [b]		FIC [b]	Reduction Fold of Drug	FICI	Outcome
		Alone	Combination			Alone	Combination
A24	Gentamicin	31.25	0.24	0.008	130.21	500.00	31.25	0.063	16.00	0.071	Synergy
	Meropenem	31.25	31.25	1.000	1.00	156.25	0.15	0.001	1041.67	1.001	Additive
	Colistin	31.25	0.49	0.015	63.78	7.81	0.06	0.008	130.17	0.023	Synergy
A29	Gentamicin	62.50	15.63	0.250	4.00	312.50	0.31	0.001	1006.45	0.251	Synergy
	Meropenem	62.50	3.90	0.062	16.03	19.53	0.04	0.001	488.25	0.063	Synergy
	Colistin	62.50	0.49	0.008	127.55	15.63	0.02	0.001	781.50	0.009	Synergy

Abbreviations: CBD Cannabidiol, MIC Minimum inhibitory concentration, FIC Fractional inhibitory concentration, FICI Fractional inhibitory concentration index

Notably, the combination of CBD with gentamicin in isolate A29 resulted in a dramatic 1006.45-fold reduction in the required antibiotic concentration (from 312.5 to 0.31 µg/mL), followed by a 781.50-fold reduction with colistin (from 15.63 to 0.02 µg/mL) and a 488.25-fold reduction with meropenem (from 19.53 to 0.04 µg/mL). These findings highlight the potential of CBD to significantly enhance antibiotic efficacy, suggesting its role as a promising adjunct therapy to combat XDR A. baumannii infections.

Combination treatment with CBD and antibiotics overcomes biofilm recalcitrance of XDR A. baumannii

Since biofilm formation is a major contributor to antibiotic resistance in A. baumannii, complicating treatment strategies. Our investigation of CBD against biofilm recalcitrance in XDR A. baumannii isolate A29 revealed that CBD alone reduced biofilm formation in a dose-dependent manner (Fig. 1A). Notably, combining CBD with commonly used antibiotics—gentamicin, meropenem, and colistin—significantly enhanced biofilm inhibition. Specifically, gentamicin (16.8 µg/mL) with CBD reduced biofilm biomass to 18.4% (81.6% inhibition), meropenem (3.9 µg/mL) with CBD reduced it to 13.1% (86.9% inhibition), and colistin (0.49 µg/mL) with CBD reduced it to 13.9% (86.1% inhibition). Similarly, treatment with CBD inhibited preformed biofilm in a dose-dependent manner, reducing both biofilm biomass and biofilm viability (Fig. 1B and C). These findings highlight the potential of CBD as an adjuvant therapy to enhance antibiotic efficacy and combat biofilm-associated resistance in XDR A. baumannii.

Fig. 1.

Effect of cannabidiol (CBD) and its combination with gentamicin (CN), meropenem (MEM), and colistin (CO) on biofilm formation. The antimicrobial resistance A. baumannii isolate A29 was exposed to CBD alone (at 1×, 2×, and 4× MIC), gentamicin, meropenem, and colistin alone, or the CBD–antibiotic combinations for biofilm formation assays, as well as for assessing preformed biofilm biomass and viability. Biofilm formation (A) and preformed biofilm (B) were evaluated using crystal violet staining, while the viability of preformed biofilm (C) was assessed using the MTT assay. *p < 0.05, ***p < 0.001

Bactericidal kinetics of CBD against XDR A. baumannii

To investigate the antibacterial activity of CBD, we next analyzed its bactericidal kinetics using A. baumannii isolate A29. The time-kill kinetic assay demonstrated a dose-dependent bactericidal effect, as shown in Fig. 2A. At 1× MIC (62.5 µg/mL) and 2× MIC (125 µg/mL), bacterial growth was initially inhibited compared to the untreated control, but complete eradication was not achieved over time (24 h). However, at 4× MIC (250 µg/mL), a significant reduction in bacterial count was observed, with complete eradication occurring within 2 h. The highest concentration, 8× MIC (500 µg/mL), resulted in total bacterial clearance within just 1 h, highlighting a rapid bactericidal effect. Moreover, treatment with CBD in combination with an appropriate synergistic dose of meropenem and colistin had a bactericidal effect within 1 h, similar to treatment with CBD at 500 µg/mL alone (Fig. 2C and D).

Fig. 2.

The effect of CBD on the XDR A. baumannii isolate A29 was assessed using time-kill kinetics. Isolate A29 was exposed to CBD alone at 1×, 2×, 4×, and 8× MIC for 1, 2, 4, 8, 16, and 24 h (A) or in combination with gentamicin (B), meropenem (C), and colistin (D). Viable cells were counted and expressed on a log scale. Dashed bars indicate the bactericidal threshold

These results indicate that CBD exhibits both time- and concentration-dependent killing activity against A. baumannii, with higher doses leading to faster and more effective bacterial eradication.

CBD disrupts outer membrane integrity in XDR A. baumannii

To elucidate the mechanism by which CBD compromises the bacterial outer membrane—a critical barrier for survival—we evaluated DNA and protein leakage in A. baumannii isolate A29 following CBD treatment. As shown in Fig. 3A, exposure to CBD at 62.5, 125, and 250 µg/mL resulted in a dose-dependent increase in DNA leakage, indicating significant membrane disruption. A similar trend was observed in protein leakage (Fig. 3B), where higher CBD concentrations led to greater extracellular protein release, further supporting its membrane-damaging effects.

Fig. 3.

Effect of CBD on outer membrane integrity. The A. baumannii isolate A29 was exposed to CBD at concentrations of 1×, 2×, and 4× MIC for 1 h at 37 °C. DNA (A) and protein (B) leakage levels were measured. The relative fluorescence intensity (RFI) of NPN (C) and Rh123 (D) was also assessed. Triton X-100 (0.1%) served as the positive control (TX). ***p < 0.001

To further investigate CBD-induced membrane disruption, we assessed outer membrane permeability and membrane potential using NPN and Rh123 fluorescence dyes, respectively. The NPN uptake assay (Fig. 3C) revealed a significant increase in fluorescence intensity—approximately 25% higher than the untreated control—following CBD treatment. NPN is a hydrophobic fluorescent dye that is typically excluded by an intact bacterial outer membrane. However, when membrane integrity is compromised, NPN can penetrate and interact with the inner membrane, leading to increased fluorescence. This suggests that CBD enhances outer membrane permeability, ultimately weakening bacterial cell integrity.

To determine the effect of CBD on membrane potential, we performed Rh123 fluorescence staining (Fig. 3D). CBD treatment led to a dose-dependent decrease in Rh123 relative fluorescence intensity (RFI), indicating membrane depolarization. Rh123 is a cationic fluorescent dye that accumulates in bacterial cells based on their membrane potential, with a healthy, energized membrane exhibiting strong Rh123 uptake. When membrane integrity is disrupted, depolarization occurs, reducing dye accumulation and leading to lower fluorescence intensity.

Taken together, these findings provide compelling evidence that CBD disrupts bacterial membrane integrity and function, contributing to its potent antibacterial activity against XDR A. baumannii.

Effect of CBD on morphological alterations in XDR A. baumannii

To elucidate the mechanism of action of CBD, scanning electron microscopy (SEM) was used to compare morphological changes in bacterial cells with and without CBD exposure. Figure 4 presents SEM images of bacterial cells at ×15,000 and ×25,000 magnifications. The untreated control bacteria exhibited a smooth, compact surface with an intact cell membrane and no visible surface ruptures (Fig. 4A).

Fig. 4.

Effect of CBD on bacterial cell morphology. A. baumannii isolate A29 was treated with 250 µg/mL of CBD for 2 h at 37 °C. Scanning electron microscopy (SEM) images at ×15,000 and ×25,000 magnifications are shown: (a) represents the untreated control, while (b) represents the CBD-treated cells. Cell damage is indicated by arrows

In contrast, after 2 h of exposure to 250 µg/mL CBD, the bacterial cells displayed severe structural disruption (Fig. 4B). The cell membrane appeared corrugated and wrinkled, with evident damage and surface collapse, as indicated by the arrow. These findings suggest that CBD treatment compromises bacterial cell membrane integrity, leading to intracellular content leakage, membrane shrinkage, and ultimately, cell death.

Cytotoxic effects of CBD in HEK293 cells

To evaluate the cytotoxic effects of CBD in vitro, we conducted an MTT assay using the HEK293 cell line, which is commonly used in biopharmaceutical research, basic medical studies, and therapeutic applications. The results, presented in Fig. 5, showed no significant difference in cytotoxicity across different treatment durations (24, 48, and 72 h). However, at a concentration of 5 µg/mL, CBD exhibited significant cytotoxic effects compared to the control group. The cell viability at 24, 48, and 72 h was 66.4%, 61.2%, and 71.7%, respectively.

Fig. 5.

Cytotoxic effects of CBD in normal human epithelial (HEK293) cells. HEK293 cells were treated with CBD at concentrations ranging from 0.5 to 5 µg/mL for 24, 48, and 72 h. Cell viability was assessed using the MTT assay. ***p < 0.001

The IC₅₀ values of CBD at 24, 48, and 72 h were 11.8, 12.1, and 17.1 µg/mL, respectively, indicating a moderate level of cytotoxicity in normal epithelial cells. These findings suggest that while CBD demonstrates some cytotoxic effects at higher concentrations, its impact on normal cells remains limited at lower concentrations. Therefore, using CBD at lower concentrations in combination treatments of conventional antibiotics may offer a safer and more sustainable approach for future clinical applications.

Discussion

This study evaluated CBD as a therapeutic agent against antimicrobial resistance A. baumannii isolated from medical devices in the ICU. MALDI-TOF MS provided highly probable species identification for all 26 isolates, confirming its reliability as a rapid tool for bacterial identification in clinical microbiology [35]. The ICU is a source of antibiotic resistant pathogens, particularly MDR and XDR A. baumannii, which can be transmitted via contaminated medical equipment, the hands of healthcare workers, the hospital environment, and patient-to-patient, contributing to nosocomial infections [36, 37]. Notably, this study found antimicrobial resistance A. baumannii in used endotracheal tubes, infusion sets, urinary catheters, and nasogastric tubes in the ICU. A retrospective study reveals high rates of MDR in the ICU, with A. baumannii (97.77%), P. aeruginosa (65%), K. pneumoniae (50%), E. faecalis (47.61%), and MRSA (46.55%) being the most prevalent [38]. The antibiotic resistance patterns revealed concerning levels of MDR, XDR and potentially PDR A. baumannii. This study found that 84.6% of the isolates were classified as MDR, followed by XDR at 11.5%. Notably, isolates A24 and A29 exhibited resistance to all 13 antibiotics tested, highlighting the critical issue of XDR. This finding is consistent with global reports of A. baumannii strains exhibiting extreme resistance, which complicates treatment regimens in hospitalized patients and contributes to increased morbidity and mortality [1, 4]. However, we acknowledge that our antibiotic panel did not include certain agents—such as tigecycline and other newer antibiotics—required for a complete assessment of PDR. Therefore, further studies using a broader range of antimicrobial agents are necessary to confirm PDR status.

Since CBD has been recognized as a potential alternative antimicrobial agent, this study confirms its effectiveness against antimicrobial resistance A. baumannii. CBD exhibited antimicrobial activity with a MIC range of 3.9 to > 500 µg/mL. Moreover, CBD showed a potentially more favorable profile compared to gentamicin, a widely used aminoglycoside, which was ineffective, as all antimicrobial resistance A. baumannii isolates were resistant. Similarly, meropenem, a carbapenem antibiotic, displayed limited activity, consistent with the high rates of carbapenem resistance previously reported in A. baumannii [39, 40]. Colistin, a last-resort polymyxin, demonstrated better efficacy, with most isolates remaining susceptible. However, resistance was observed in five strains (19.2%), reinforcing the growing concern regarding colistin resistance in A. baumannii, which aligns with previous findings [39].

It is important to note that the combination treatment of CBD with conventional antibiotics—gentamicin, meropenem, and colistin—restored their efficacy by significantly reducing the required doses, up to 1000-fold for gentamicin, 500-fold for meropenem, and 800-fold for colistin, against XDR A. baumannii. This synergistic activity of CBD enabled the antibiotics to be effective at remarkably low concentrations: 310 ng/mL for gentamicin, 40 ng/mL for meropenem, and 20 ng/mL for colistin. Highlighting that the combination of CBD with colistin exhibits greater effectiveness than Polymyxin B, a last-resort antibiotic similar to colistin. A previous study reported that the combination of Polymyxin B at a concentration of 125 ng/mL with CBD (256 µg/mL) reduced the required dose of Polymyxin B when used alone from 1 µg/mL to 125 ng/mL, an 8-fold reduction against clinical A. baumannii [22]. Therefore, considering the potential to overcome XDR A. baumannii, the treatment combining CBD with colistin might be more cost-effective than using Polymyxin B, as it could reduce the required dosage of expensive last-resort antibiotics.

CBD has been reported to inhibit biofilm formation, a major factor in bacterial persistence and antibiotic resistance [21, 41]. Our findings strongly indicate that CBD could serve as a candidate antimicrobial agent against XDR A. baumannii by inhibiting and eradicating biofilms, particularly when used in combination with conventional antibiotics. Biofilms enable bacterial communities to adhere to various surfaces, including medical devices such as catheters, ventilators, and prosthetic implants, allowing pathogens to persist in hospital environments, particularly in ICUs.

The mode of action of CBD in A. baumannii has not been well elucidated; therefore, this study provides a deeper understanding. Firstly, we found that CBD exhibited a rapid bactericidal action against XDR A. baumannii, with higher doses leading to faster and more effective bacterial eradication, suggesting a dose-dependent killing effect. The antibacterial synergy of CBD with meropenem or colistin at low concentrations resulted in bacterial eradication within 1 h, which is faster than the previously observed 4–6 h at 4 µg/mL CBD in clinical isolates of polymyxin B-resistant K. pneumoniae [22]. Further investigations revealed that CBD disrupted bacterial membrane integrity, leading to the leakage of DNA and proteins involved in essential cellular processes, as evidenced by SEM. Ultimately, this leads to bacterial cell death, consistent with the rapid action of CBD in disrupting the bacterial cytoplasmic membrane [21, 22].

Implications for clinical applications

The findings of this study suggest that CBD holds considerable promise as an alternative therapeutic agent for XDR A. baumannii infections. Given its relatively low toxicity profile and its potential to synergize with existing antibiotics, CBD could be integrated into clinical treatment regimens to enhance the efficacy of antimicrobial therapies against highly drug- resistant A. baumannii. However, several critical considerations must be addressed before the clinical application of CBD in this context. First, the pharmacokinetics, bioavailability, and stability of CBD need to be fully characterized to determine optimal dosage and delivery methods [42]. Additionally, while the antibacterial and biofilm-disrupting effects of CBD were observed in vitro, clinical studies are necessary to validate these findings in real-world settings, where factors such as host immune responses and the complexity of human infections may influence therapeutic outcomes.

Building upon these findings, our study highlights the potential of CBD for incorporation into medical device materials to prevent bacterial adhesion and biofilm formation, especially on high-risk devices such as catheters and wound dressings. CBD coatings may disrupt bacterial membranes and reduce the risk of drug-resistant infections. Emerging technologies, such as nanocoatings and controlled-release systems, enhance the feasibility of localized, sustained antimicrobial delivery [43]. Development should align with the “One Health” framework, considering environmental safety, regulatory compliance, and risk assessment to ensure responsible clinical use.

This study has certain limitations. The relatively small number of XDR A. baumannii strains tested constrains the generalizability of our findings regarding the full spectrum of CBD’s antibacterial activity. Further research involving clinical isolates is essential to confirm these results. Future studies should also investigate CBD’s interactions with a broader range of antibiotics and assess its pharmacokinetics and pharmacodynamics in vivo. Moreover, the molecular mechanisms underlying CBD’s antimicrobial effects remain poorly understood, warranting further research to elucidate these pathways and to determine whether CBD exhibits true synergism with antibiotics.

Ultimately, additional research and well-designed clinical trials are required to establish the clinical applicability of CBD. Future investigations should focus on optimizing CBD formulations, evaluating its pharmacokinetic and pharmacodynamic interactions with other drugs, and exploring its synergistic potential with additional antimicrobial agents to enhance therapeutic efficacy.

Conclusion

Our study demonstrates that CBD exhibits potent antibacterial and anti-biofilm properties against XDR A. baumannii, particularly when used in combination with conventional antibiotics such as gentamicin, meropenem, and colistin. Notably, its ability to disrupt membrane integrity represents a key mechanism in overcoming drug tolerance. These findings provide a strong foundation for further investigation of CBD as a novel therapeutic strategy to combat antimicrobial resistance in clinical settings.

Abbreviations

CBD

 Cannabidiol

FICI

Fractional Inhibitory Concentration Index

MBC

Minimum Bactericidal Concentration

MDR

Multidrug resistance

MIC

Minimum Inhibitory Concentration

PDR

Pan-drug-resistant

XDR

Extensively drug-resistant

Authors’ contributions

Conceptualization, A.R., and A.Y.; methodology, A.R., A.Y., A.K, A.S., G.P., C.S., A.D., R.P., N.R., and S.N; writing—original draft preparation, A.R. and A.Y; writing—review and editing, A.R.; project administration, A.R.; funding acquisition, A.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the University of Phayao and the Thailand Science Research and Innovation Fund (Fundamental Fund 2025, Grant No. 5031/2567), as well as the School of Medical Sciences, University of Phayao (Grant No. MS 241001), Thailand.

Data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.