Among several fungal species reported to be present on cannabis and hemp inflorescences (Table 1), a number can produce mycotoxins when grown on culture medium and potentially in the affected plants (Tables 3–5). From the perspective of consumer health, the destruction of plant tissues and the production of spores and mycotoxins in association with cannabis and hemp plants are deemed of potential concern. Furthermore, the antibacterial activity of the combination CBD (256 µg/mL, fixed concentration) + PB (two-fold 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). While the latter has yet to be studied in a statistical robust matter to support human health risk assessment of cannabis and hemp products, regulatory policies and assessment technology are evolving to meet the production and safety needs of the emerging industries. While some concentration effects were evident, particularly after only 1 h, by 24 h all caused significant reductions in bacterial load (p 5e–h). Since CBD was dissolved in methanol, control cells were treated with 2.5% methanol. The plots d, e quantify the percentage of cells that have been permeabilized, defined as the fraction of cells with a mean SYTOX™ Green intensity greater than a cut-off value of 250 (number of cells measured ranged from 945–8050 for each time/concentration). Coli SPT-39 c monitoring uptake of potential-sensitive fluorescent dye 3,3-dipropylthiadicarbocyanine iodide DiSC3(5) over time in presence of increasing concentrations of CBD. Finally, bacterial cytological profiling (BCP) gave results (Fig. 4d–g) consistent with previously published BCP results for other antibiotics known to act by membrane permeabilization36–38. Therefore, our results contribute to a better understanding of CBD antibacterial mechanism(s) of action, which could guide future studies. Indeed, the bacterial membrane was also suggested as a possible bacterial target for cannabinoids (CBG and CBD)11,14,16. Influenzae also have LOS molecules on their external membrane, but the core polysaccharide for these bacteria presents a different sugar composition32,34. Cannabis use and fungal infections in a commercially insured population, United States, 2016. Essential oils of Heliotropium bacciferum, Ocimum dhofarense and Zataria multiflora exhibit aflatoxin B1 detoxification potential. Mycobiota and toxigenic Penicillium species on two Spanish dry-cured ham manufacturing plants. Mycotoxin production potential of toxigenic fungi reported in cannabis mycobiome.The what, why and how of water activity in cannabis.Researchers are exploring how cannabis smoking alters the bacterial communities in the mouth and its potential impacts on the brain .Coli (Fig. 4c), measured using the membrane potential-sensitive cationic fluorescent probe 3,3′-dipropylthiadicarbocyanine iodide DiSC3(5).These neurotransmitters can interact with cannabinoid receptors and modulate the effects of cannabinoids on gut motility and gastrointestinal functions .A similar study has not been conducted in hemp.To study whether the cell division defect is specific for the combination of CBD and BAC, microscopy analysed using higher concentrations of CBD and BAC at 4 and 64 µg/mL, respectively, was performed (Supplementary Information Figure S5). 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. The bacterial membrane potential was analysed using BacLight Bacterial Membrane Potential Kit (ThermoFisher) and the experiment was performed according to the manufacturer’s recommendations. The culture was treated with either 1 µg/mL CBD, 16 µg/mL BAC, the combination of CBD and BAC, the solvent EtOH, or left untreated and incubated for one hour at 37 °C degrees with agitation. Stained cells were added to a Poly-L-Lysine treated glass slide and visualised using Olympus IX83 fluorescence microscope with 405 nm for DAPI and 488 nm for Bocillin-FL. Additional research is needed to survey the extent of both Aspergillus and Fusarium species occurrence and potential mycotoxin presence. Several studies have linked cannabis use to increased risk of fungal infection in immunocompromised patients. Similarly Canadian production areas have reported f Fusarium occurrence on cannabis plants and inflorescences during commercial cultivation (Punja et al., 2019, 2021, 2023; Punja, 2021a,b,c; Punja and Ni, 2022). On cannabis or hemp in most areas of the country, except for the Great Plains region (Munir et al., 2023); this is likely due to the low numbers of samples received from this region. (L) Colonies of two Penicillium species emerging from sections of stems where they were growing as endophytes. (K) Colonies of Fusarium oxysporum emerging from stem pieces of cannabis plants. (G) Commercially dried cannabis inflorescences prior to packaging. (F) Spore bearing structures of a Gliocladium species on inflorescence tissues following a foliar application made to the plant. (D) Proliferation of mycelium of a Fusarium species within the inflorescence tissues under conditions of high humidity. These fungi can cause opportunistic infections on skin and lung tissues, which could lead to life-threatening conditions. More research is needed to show whether fungistasis can be overcome under conducive environmental conditions. Complex resin composed of terpenes reduced germination of two nonpathogenic species but not that of a phytopathogen (Slinski et al., 2015). The predominant terpenes found in cannabis inflorescences, namely myrcene, β-caryophyllene, limonene, α-terpinene, and α-pinene (Chacon et al., 2022) may play a role in the community structure of floral mycobiomes. Paradoxically, some endophytes may also produce secondary metabolites that are harmful to humans and other animals (i.e., mycotoxins; see Section 3.3). "I'm excited to partner with FOX 11 to pull back the curtain on what you could really be getting when you purchase CBD products." CBD, or cannabidiol for short, is one of the most prominent components of the hemp plant. After FOX 11 and Dr. Oz had professional testing done on more than a dozen CBD products purchased by our separate teams, our investigation found one CBD product was contaminated with a deadly strain of E. Preparation of injectable MRSA solution For DNA-based testing, it is recommended that the enrichment of cannabis matrices in fungus-specific media requires at least 48 h for Aspergillus spp. If similar TYM testing were to be imposed on these foods, many could fail to pass due to their natural inherent microbiome composition. This is consistent with the practices currently used for regulation and monitoring of agricultural food products consumed by humans. However, CBD and BAC treated bacteria did show a decreased rate of autolysis. Aureus indicated no changes in the cell wall composition. The cannabinoid cannabidiol (CBD) is characterised in this study as a helper compound against resistant bacteria. Zamir K Punja Some mycotoxins such as aflatoxins are produced by a few species, whereas others (e.g., OTA and PAT) are produced by multiple species belonging to multiple genera. Potential for mycotoxin production by species of Fusarium reported in Cannabis sativa. The drying phase can be a component of quality assurance that can reduce the concerns for accumulation of mycotoxin produced by epiphytes in cannabis products. We used 96-well, polystyrene, flat-bottom microplates for the experiments. Rifampicin and isoniazid were used as control (1–0.004 µg/mL). 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. We used the reference broth microdilution method to determine the CBD MIC against M. The MBC method was performed after visual evaluation of growth inhibition by subculturing onto Mueller Hinton Agar or Mueller Hinton Agar with Blood (Difco™, Becton Dickinson) plates in the absence of CBD. Antimicrobial activity of CBD was tested against a number of bacterial strains by broth microdilution (BMD) minimum inhibitory concentration (MIC) assays using procedures described by Clinical and Laboratory Standards Institute (CLSI)58–61. Our initial SAR investigations provide evidence of the potential to alter CBD’s core structure while retaining activity, giving hope that the physicochemical properties can be modified to provide systemic activity. We initiated a medicinal chemistry campaign to attempt to improve the properties of CBD so that it can be used systemically to treat infections, with a focus on reducing protein binding/serum reversal while retaining or improving antibacterial activity. We now report that CBD has some remarkably useful antimicrobial activity beyond that previously described20,21,23, with potential clinical utility for nasal decolonization. The cells were then incubated with 100 µg/mL DAPI for 4 minutes followed by PBS wash. After treatment, cells were washed in PBS and incubated at room temperature with 5 µg/mL Bocillin-FL for 4 minutes followed by wash in PBS. Data retrieved was obtained as Background Corrected Absorption (BCA), calculated by an algorithm enabling determination of bacterial growth kinetics resulting from images taken with the oCelloScope camera38,39. Structure of CBD analogs and summary of effect of modification to different positions on antimicrobial activity against MRSA and N. We also evaluated CBD’s systemic antimicrobial activity in an immunocompromised thigh infection mouse model against MRSA ATCC 43300. Efficacy was highly formulation-dependent, and some formulation vehicles had modest to good antimicrobial activity on their own (e.g., liquid formulation #2, with a high content of transcutol and 3.4% isopropyl alcohol). We review current regulations for molds and mycotoxins worldwide and review assessment methods including culture-based assays, liquid chromatography, immuno-based technologies, and emerging technologies for these contaminants. The fungal composition of the C. Signup to receive email updates on blog posts, podcasts, webinars, new products, and more! Gut microbiome profiling offers the potential to identify patient-specific microbiome signatures that influence treatment responses to cannabis-based therapies, optimising outcomes and minimising adverse effects. Large-scale longitudinal studies and clinical trials incorporating gut microbiome profiling are essential to elucidate the complex mechanisms and clinical implications of the gut microbiome-cannabis axis in cancer therapy. The complexity and variability of the gut microbiota make it challenging to establish universal guidelines for cannabis-based cancer treatments. Investigating the role of the gut microbiota in maintaining gut barrier integrity and its influence on the effects of cannabis is also crucial for optimising treatment efficacy. Therefore, essential information on the incidence and frequency of several fungal species of concern is unavailable. The enhanced safety of these products should correspondingly reduce the incidence of various forms of fungal infections as reported in Section 3. Various post-harvest treatment methods can impact quality of cannabis-derived products with regard to yeast and mold levels. Furthermore, duration of drying and final moisture content (water activity) has a significant impact on the levels of fungi present, particularly those like Aspergillus and Penicillium spp. Sativa by-products from commercial CBD extraction controlled powdery mildew diseases of hemp (Akinrinlola et al., 2022; Fei et al., 2023). P-values were calculated by a one-way ANOVA with Bonferroni’s Multiple Comparison Test for qPCR and membrane potential data. The samples including an ethanol control were either left untreated or treated with either 5 µM CCCP (depolarised control), 0.1 or 0.2 µg/mL CBD and/or 16 µg/mL BAC and incubated at room temperature for 5 minutes. The samples were sonicated for 30 minutes in a sonicator bath followed by DNase and RNase treatment using DNase I (15 µg/mL) and RNase A (60 µg/mL) and incubated at 37 °C for 1 hour followed by trypsin digestion (50 µg/mL) for 1 hour at 37 °C. After 2.5 hours treatment, the cells were harvested at 10.000x G and the pellet was resuspended in 0.1 M tris/HCl containing 0.25% SDS and heated to 100 °C for 30 minutes. The autolysis assay was performed according to Campbell et al.31. While there are many reports of cannabis and hemp diseases caused by Fusarium species, there are no regulations currently in place for assessing mycotoxin levels. As such, the incidence of Fusarium contamination is currently unknown in cannabis and hemp products, in contrast to cereal grains and many other agricultural commodities worldwide. Current knowledge of the extent of fungal contamination on cannabis and hemp inflorescences relies on publicly available data from published independent studies and cannabis compliance tests. Therefore, there are opportunities for further research into decontamination of cannabis or hemp products should Fusarium mycotoxins become a regulatory issue. Essential oils of plants that contain fungistatic or fungicidal terpenes can potentially reduce mycotoxin content in cannabis products (Ranjith et al., 2021). (A) Adverse health effects of fungal pathogens. For example, species of Mucorales (e.g., Mucor) can be found in cannabis inflorescences (Stone et al., 2019; Punja and Scott, 2023). Contaminant fungi pose a recognized health risk to patients with susceptible conditions, such as those who are immunocompromised National Academies of Sciences, Engineering, and Medicine (NASEM), 2017; Figure 3. Antifungal activity of terpene essential oils may also be due to synergistic interactions of compounds. Endocannabinoids and exogenous cannabinoids such as CBD have been observed to inhibit growth of bacteria12–14, yet the use of cannabidiol as an antibiotic adjuvant has not been studied so far. Therefore, by combining an antibiotic with a helper compound less antibiotic is needed in order to achieve bacterial growth inhibition or killing compared to using the antibiotic alone. Drugs found to contain helper compound properties are normally used for treatment of non-infectious diseases but may contain some degree of antibacterial activity itself5. Based on these observations, the combination of CBD and BAC is suggested to be a putative novel treatment in clinical settings for treatment of infections with antibiotic resistant Gram-positive bacteria. Noticeably, expression of a major cell division regulator gene, ezrA, was reduced two-fold upon combination treatment emphasising the impact of the combination on cell division. However, the mechanism or how CBD and other cannabinoids affects the bacteria has not been studied so far. Appendino and colleagues14 found MIC values of CBD extracted from powdered plant material in the 0.5-1 µg/mL range towards various drug-resistant strains of S. The use of CBD and other cannabinoids as antibacterial agents was first described in 1976 by Van Klingeren and Ten Ham13 and again in 2008 by Appendino and colleagues14, however, since then very little has been published on this topic. However, it has been shown to bind the bacterial membrane affecting the membrane integrity suggesting similar mechanism for synergy as suggested for colistin28. Colistin is believed to damage the cell membrane thus increasing entry of BAC into the cell or by increasing availability of divalent ions such as Zn2+. In the context of CBG or THC backbones, an aromatic carboxyl group had little effect for either bacteria (e.g., tetrahydrocannabivarin THCV 19 vs. tetrahydrocannabivarinic acid THCVA 20, CBG 25 vs. cannabigerolic acid CBGA 26). Gonorrhoeae activity (MIC between 0.125–2 μg mL−1). There were clear differences in the structure-activity relationship (SAR) for the Gram-positive activity against MRSA compared to the Gram-negative activity against N. However, despite the successful systemic treatment of other types of diseases with CBD, it was ineffective when systemically dosed subcutaneously at 100 mg kg−1, intraperitoneally at 200 mg kg−1, or orally at 250 mg kg−1 (Supplementary Fig. 3). Aureus (Xen-29) infection was established in disrupted skin of immunocompromised mice. CBD antibacterial activity against GP and GN bacteria, and Mycobacterium tuberculosis The subsequent table provides more detail about each state’s cannabis microbial testing regulations, including specific requirements for different product types, when applicable. On the other hand, Massachusetts regulations set allowable limits for large groups of microorganisms, such as aerobic bacteria, yeasts and molds, coliforms, and bile-tolerant gram-negative bacteria. Microbial contamination on cannabis products represents one of the most significant threats to cannabis consumers, particularly immunocompromised patients who could develop harmful infections. This surge is likely due to the excitement behind potentially new medical breakthroughs and the desire as a culture to shift towards therapies that are more “natural.” However, most products, such as the one purchased by this patient are unregulated. He felt the products were healthy and safe based on packaging and therefore did not believe they would have any adverse effects. Tuberculosis, and the absence of CBD antibacterial activity against GNB, support a role for LPS in hindering CBD activity. Our results of in vitro CBD antibacterial activity against GP bacteria and M. 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 CBD14–16. In an attempt to understand this lack of effect, we used various efflux pump inhibitors to potentially improve CBD activity. In general, our data and previous reports showed CBD MICs ranging from 2 to 4 µg/mL against GP bacteria, including vancomycin-resistant E. O'Donnell, K., McCormick, S. P., Busman, M., Proctor, R. H., Ward, T. J., Doehring, G., et al. (2018). Thermography of cannabis extract vaporization cartridge heating coils in temperature and voltage-controlled systems during a simulated human puff. Ultra-high-performance liquid chromatography coupled with quadrupole orbitrap high-resolution mass spectrometry for multi-residue analysis of mycotoxins and pesticides in botanical nutraceuticals. Enzyme immunoassay for measuring aflatoxin B1 in legal cannabis. While challenges and limitations exist, addressing these research directions will contribute to realising the full potential of the gut microbiota–cannabis–cancer axis. Advancing our understanding of the gut microbiome–cannabis axis for cancer treatment necessitates multidisciplinary collaborations among researchers and healthcare professionals, including microbiologists, oncologists, pharmacologists, and immunologists. Additionally, the long-term safety and efficacy of gut microbiome-targeted interventions in combination with cannabis-based therapies require thorough investigation. The relationship between the gut microbiota and the effects of cannabis has been a subject of growing research interest, with potential implications for individual variations in cannabinoid metabolism, immune response, and gut barrier integrity. One crucial aspect that warrants in-depth investigation is the role of the gut microbiota, as it can metabolize cannabinoids into active and potentially toxic compounds , which could influence the safety and efficacy of cannabis-based therapies. The antibacterial efficacy of the combination CBD + PB against multidrug-resistant and extensively drug-resistant GNB, especially PB-resistant K.Can be difficult due to the problems in differentiating human pathogenic and nonpathogenic species.The final well volume was 200 µL with a cell density of 5 × 105 CFU mL−1.Intriguingly, this activity extends to a small subset of Gram-negative bacteria, including pathogens of concern such as Neisseria gonorrhoeae.The U.S. EPA and Health Canada have registered a number of biopesticides for cannabis and hemp cultivation that contain Trichoderma spp.Biological control of Fusarium oxysporum causing damping-off and Pythium myriotylum causing root and crown rot on cannabis (Cannabis sativa L.) plants. This makes predictions and assessments of fungal population levels challenging, which can further complicate efforts to assess their potential impact on consumer health. The presence of fungal organisms in cannabis plants is not unique since many species comprising the floral mycobiome are also abundantly present across a wide range of agricultural crops. The aggressive marketing of these products paired with the lack of regulation and quality control has the potential to cause a significant negative impact on public health. It also underlies the importance of supporting legal cannabis and hemp production systems where stringent regulations ensure a higher quality product.This was used to spike fresh supplemented HIS broth to a final concentration of 256 μg mL−1 (double the highest concentration we subjected C. difficile to, as addition of an equal volume of bacterial inoculum to the MIC plate dilutes the CBD concentration by half).The results showed that the combination of CBD (4 µg/mL) + PB was antibacterial only in the presence of PAβN (Table 4).A recent study of 84 CBD products bought online showed that more than a quarter of the products contained less CBD than labeled.On cannabis or hemp in most areas of the country, except for the Great Plains region (Munir et al., 2023); this is likely due to the low numbers of samples received from this region.Similar cases are likely to be seen as these products become more commercially available.Additionally, the capacity of gut microbiota to activate cannabinoid receptors through the production of secondary bile acids underscores its role in directly influencing the pharmacological activity of cannabinoids. However, these methods have not been validated on cannabis treated with the decontamination methods currently used in cannabis production. Molecular methods, however, can lead to false positives because of the cross-reactivity with non-specified Aspergillus species. In fact, a subset of the microbiota of cannabis inflorescences may be providing undetermined benefits based on what is known from other crop plants (Punja et al., 2023). Our study shows the in vitro antibacterial activity of CBD, especially in combination with PB, suggesting potential repurposing of CBD as an antibacterial.Cation-Adjusted Mueller Hinton II Broth (CAMHB) (BBL™, Becton Dickinson) and 96 wells microplates polystyrene, round bottom, non-treated, were used on all assays unless otherwise specified.Furthermore, construction of a conditional ezrA mutant has also shown to cause impaired cell division and several septa formations in S.Thus, the ratio between red and green fluorescence can reveal the state of the membrane potential.In organic cannabis production facilities, populations of air-borne fungal propagules originating from the soil may be high, and a greater prevalence of species of fungal epiphytes has been reported on cannabis inflorescences than in conventional culture (Punja and Scott, 2023).CBD showed antibacterial activity against GND, so FICI was calculated, and the effect of the combination was characterized.In our study, we used the EUCAST/BrCAST breakpoint (PB MIC ≤ 2 µg/mL as susceptible) for our analyses and discussion.Our results show that CBD has translational potential and should be further explored as a repurposed antibacterial agent in clinical trials.SCFAs have been shown to modulate the expression and activity of cannabinoid receptors, affecting various physiological processes in the gut, including inflammation and motility . According to experts, the future of microbiology CBD gummies will be shaped by advances in lab testing, quality control, and manufacturing processes. Lab testing is critical in detecting microbial contamination and ensuring that CBD gummies meet the highest standards of quality and safety. It is essential to select products from reputable manufacturers that prioritize quality control and lab testing to ensure that the products are pure and potent. (C) Destruction of the inflorescence by the fungal pathogen Botrytis cinerea that causes browning and death of the tissues. Fungal pathogens, epiphytes, and endophytes affecting cannabis inflorescences. (B) Endophyte—asymptomatic relationship with fungal propagules (orange) that are found within the host. (A) Epiphyte—asymptomatic relationship with fungal propagules (dark green circles) that colonize only the outside of host plant. For most of the GNB studied, our results 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. 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. 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). 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. 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. Difficile strains following plate incubation via vigorous pipetting to dislodge and homogenize any bacteria present.Taken together, all of these results are consistent with CBD acting very rapidly to disrupt bacterial cytoplasmic membranes, though whether a specific molecular target is involved remains to be determined.We recommend testing for the presence of Aspergillus, Penicillium, Fusarium, and Mucor as priorities, with an adjustment to adding other species if they are discovered to have potential harm.The lower PB concentrations of PB in the combination CBD + PB are the PB MEAC.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 MIC50 and MIC90 determination and probability of target attainment.Endocannabinoids and exogenous cannabinoids such as CBD have been observed to inhibit growth of bacteria12–14, yet the use of cannabidiol as an antibiotic adjuvant has not been studied so far.Under organic crop production systems, the diversity of fungal species present on cannabis inflorescences can be greater than in conventional production systems (Punja and Scott, 2023).The potentiation was confirmed through MIC determinations, standard growth experiments, fractional inhibitory concentration determination and time-kill assays. In addition, the presence of airborne fungal contaminants during harvesting of cannabis and hemp and those that are present in the cultivation environment can be of potential concern for human health (Martyny et al., 2013; Vanhove et al., 2018; Root et al., 2020; Punja, 2021c; Punja and Scott, 2023). A review of the current literature indicates there are potential human health risks of fungal and mycotoxin contaminants in cannabis and hemp flowers, particularly for the subset of the population that is immunocompromised. Further research is needed to develop commercial analytical methods that would allow data collection to determine to what extent these potentially widespread but unregulated mycotoxins occur in cannabis and hemp products. (A) Mycotoxins that have been reported in cannabis and hemp products (red) or for which genes for their production have been reported in toxigenic fungi isolated from cannabis products (blue). Because two of the most commonly encountered fungi that can potentially produce mycotoxins in cannabis tissues, namely Penicillium and Aspergillus spp., can survive at aw of 0.62–0.7, this range is suboptimal for fungal growth. 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. 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 membrane42. The combination CBD + PB + PAβN was effective against P. aeruginosa and plasmid-mediated colistin-resistant (MCR-1) E. Checkerboard data of combination inhibition are important; however, time-kill data are more suitable to categorize the combination effect as synergistic41. The lower PB concentrations of PB in the combination CBD + PB are the PB MEAC. Additional research is required to establish whether these secondary metabolites are produced in cannabis inflorescence tissues following application of these beneficial fungi for plant pathogen control during the crop production cycle. Although no studies have linked presence of Fusarium mycotoxins (or their degradation products) in cannabis to CHS, the symptoms of CHS are remarkably similar to the toxicity of DON, NIV, and T-2 toxin, all of which can result in vomiting and partial or complete refusal of food (Bonnet et al., 2012). CMycotoxin production is based on analysis of strains of the fungi that were not isolated from cannabis (O'Donnell et al., 2018). Nonetheless, few studies described the antibacterial activity of ultrapure CBD against Gram-positive (GP) bacteria; although, CBD is not antibacterial against GNB9,11–17. Sativa-based preparations have also been investigated for their antibacterial activity11–13. Regarding its biological activity in bacteria, CBD was described as inhibitor of membrane vesicles released from GNB and as inhibitor of biofilm formation as well as being capable of eradicating preformed biofilms10. To enhance detection, immunoaffinity columns with antibodies specific for aflatoxins and OTA have also been used for cannabis and hemp sample clean-up before LC analysis (Wilcox et al., 2020; Greaves et al., 2021; Buchicchio et al., 2022). More targeted approaches such as testing specifically for certain fungi known to pose a threat to consumers due to potential mycotoxin production (e.g., Aspergillus spp. and Fusarium spp.) would be more informative when compared to an assessment of total fungi and yeasts present. The current concept of TYM testing requires further evaluation, and determination of its value to provide information on the fungal species composition to support human health risk assessment is an area that urgently needs assessment. Cannabidiol is an effective helper compound in combination with bacitracin to kill Gram-positive bacteria Log10 (CFU/mL) difference between the combination CBD + PB and PB treatment at MEAC. At time points 0, 1, 2, 4, and 6 h, an aliquot was removed, and number of cells was determined by inoculating on solid medium and counting colony forming units. Also, the overall reduction in CFU/mL of the combination CBD + PB relative to PB alone was above 2 log10 for many time points, further confirming the synergistic effect of CBD and PB (Table 6). 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. Not surprisingly, a few studies have found that illicit cannabis samples contained more contaminants than legally grown cannabis (Eykelbosh, 2021; Southall, 2022; Gagnon et al., 2023). The current regulatory policies for assessing cannabis contaminants in the United States and European Union have been extensively reviewed (Pruyn et al., 2022; Veit, 2023; Jameson et al., 2022). These species, including T. Enniatins and beauvericin are cyclodepsipeptides that are often classified as emerging mycotoxins because they are not regulated, and thus, there is little routine testing conducted for them. Report Results Only (Products containing unextracted cannabis) – Adult-Use Cannabis The maps below provide a snapshot of the different microbial targets each state requires. To protect consumers in their jurisdiction, most regulators in the United States require third-party labs to perform microbial safety testing for certain microbes at certain levels. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Clinicians should be aware of this when prompted for advice from patients as well as when treating patients with potential intoxication. The diversity in gut microbiota may lead to variations in cannabinoid metabolism and treatment responses, emphasising the need for personalised medicine approaches. Firstly, the variability in gut microbiota composition among individuals presents a challenge in developing universal treatment strategies. One notable finding is the ability of the gut microbiota to metabolise cannabinoids, including Δ9-tetrahydrocannabinol (THC). Preclinical studies have demonstrated that the gut microbiota can significantly influence the pharmacological effects of cannabinoids. The cells showed several septa formations indicating lack of cell separation during cell division causing reduced autolysis, as well as an irregular membrane. However, it was found to be ineffective against Gram-negative bacteria. Through growth experiments, it was interestingly found that CBD was able to potentiate the effects of BAC against S. In addition, they found that by increasing the expression of lytM, they could restore the viability of the cells even though the cells had still formed several septa. In a study of gene silencing of the major regulator of cell wall metabolism walRK, S. We therefore thought to study the expression of genes encoding proteins involved in the autolysis and found the expression of lytM and lytN to be upregulated upon combination treatment. Cell imaging is an approach to obtain indications of the mechanism or site of action of an antimicrobial compound. Endogenous endocannabinoids as anandamide (AEA) and endocannabinoid-like arachidonoyl serine (AraS) has been shown to contain poor antimicrobial properties but have a pronounced dose-dependent inhibitory effects on biofilm formation of all tested MRSA strains12. To determine whether CBD would induce a higher susceptibility of BAC in Gram-positive bacteria, MICs of BAC were determined for the four Gram-positive bacteria in the presence of CBD. Faecalis, indicating that Gram-positive bacteria are sensitive towards CBD (Table 1). Monocytogenes, and the MRSE strain and 8 µg/mL for E. We found the Minimum Inhibitory Concentration (MIC) to be 4 µg/mL for S. BAC is a mixture of related cyclic peptides operating as a bactericidal antibiotic by interfering with the cell wall and interrupting the biosynthesis of the peptidoglycan leading to cell lysis15. Agar disc diffusion assay QPCR data of the divisome gene ezrA and autolysis genes lytM and lytN studied upon 2.5 hours treatment with either CBD, BAC, combination, EtOH or left untreated in USA300. As autolysis was shown to be decreased upon treatment with the combination of CBD and BAC, we studied the expression of selected autolysis genes. Analysis of transcriptional changes of selected genes (see Supplementary Table S1) involved in the divisome, cell division and autolysis of S. The potential applications of CBD gummies in various industries, such as healthcare and wellness, are numerous and continue to evolve. Real-life examples and feedback from users of microbiology CBD gummies have highlighted the benefits and effectiveness of these products. Specialists in the field of microbiology and CBD research have weighed in on the benefits and potential risks of CBD gummies. The USP Methods provide a framework for testing microbial contamination in supplements, and reputable manufacturers adhere to these standards to guarantee the purity and potency of their products. The interplay between gut microbiota and cancer has been an emerging area of investigation, unveiling the potential influence of microbial communities on the onset of cancer, progression, and therapeutic outcomes. Recent research advancements have uncovered a bidirectional relationship between the gut microbiota and the ECS, underpinning the potential influence of gut microbiota on cannabinoid metabolism and vice versa 11,12. Additionally, the gut microbiota is now recognised as a crucial modulator of drug metabolism and efficacy along with the liver, influencing the bioavailability and activity of numerous pharmaceutical agents 3,4,5,6,7,8. While the literature reveals promising avenues for leveraging the gut microbiome–cannabis axis in cancer therapy, several critical considerations must be accounted for. Emerging research has revealed a complex bidirectional interaction between the gut microbiome and cannabis. Cell viability was assessed after 30–60 min for GNB and GPC and after 60–120 min for GND and Enterococcus species. Polymyxin B and vancomycin were used as controls for GN and GP bacteria, respectively. For fastidious bacteria, such as Streptococcus spp., Neisseria spp., Moraxella catarrhalis, and Haemophilus influenzae, Cation-Adjusted Mueller Hinton II Broth (CAMHB) (BBL™, Becton Dickinson) supplemented with defibrinated horse blood and β-NAD, named MH-F, was used as recommended by EUCAST31. Cation-Adjusted Mueller Hinton II Broth (CAMHB) (BBL™, Becton Dickinson) and 96 wells microplates polystyrene, round bottom, non-treated, were used on all assays unless otherwise specified. Also, PB concentrations needed for the combination to be antibacterial were up to eight-fold lower than the MIC for PB. Nevertheless, the specific mechanism(s) for the antibacterial activity of CBD has not yet been fully elucidated. The hydrophobic chemical structure of CBD points towards an interaction with lipid in membranes as described by Guard et al. for eukaryotic cells. CBD and cannabigerol (CBG, another cannabinoid) have antibacterial activity against A. This fact could explain the absence of CBD antibacterial activity against these GN bacteria. Cells were grown and treated as mentioned for transmission electron microscopy. The sections were analysed by transmission electron microscopy using a Philips EM 208 Microscope equipped with a Quemsa TEM CCD camera and an iTEM Digital Imaging Platform software. The samples were cut into ultra-thin sections using an ultra-microtome and collected on a nickel grid. The regulations currently in place have been largely adopted from the food industry, where fungal and yeast contamination of food products consumed by humans is not uncommon and can result in illnesses and potential fatalities. Asperellum, can all colonize cannabis plants as an endophyte (Punja and Scott, 2023; Scott and Punja, 2023), and in culture produce toxins, such as viridin and gliotoxin, that could potentially impact consumer health. The U.S. EPA and Health Canada have registered a number of biopesticides for cannabis and hemp cultivation that contain Trichoderma spp. Subsequently, cannabis inflorescences that are improperly dried or stored under humid conditions may be expected to raise concerns for mycotoxin accumulation. Cannabis has also been reported to contain fungi in the order Mucorales (including Mucor spp.; Stone et al., 2019; Punja and Scott, 2023). Pulmonary aspergillosis is a common form of Aspergillus infection and has been reported in cannabis users with HIV and type 1 diabetes (Remington et al., 2015; Salam and Pozniak, 2017). Aureus cells partially depleted of EzrA cannot divide without sufficient levels of EzrA.A systematic review and meta-analysis revealed associations between cannabis usage and alterations in the human microbiome, which must be considered in future research on the therapeutic effects of cannabis on patients .Manually, 20 µL of each compound dilution was plated in triplicate on the cells.The subsequent table provides more detail about each state’s cannabis microbial testing regulations, including specific requirements for different product types, when applicable.Conversely, the gut microbiota can produce endocannabinoids and influence the endocannabinoid tone in the gut.We initiated a medicinal chemistry campaign to attempt to improve the properties of CBD so that it can be used systemically to treat infections, with a focus on reducing protein binding/serum reversal while retaining or improving antibacterial activity.Furthermore, the gut microbiota can shape systemic immune responses and influence immune cell trafficking to distant sites, including tumours . The huge variation between United States jurisdictions in the acceptable levels of TYM, ranging from 100 to 100,000 cfu/g illustrates the difficulty in implementation of human health risk assessment. Hence, Fusarium mycotoxins are not being included in compliance testing. And their mycotoxins. In addition, most compliance tests currently rely on culture-based isolation (e.g., TYM) that provide no information on which specific fungal contaminants are present. In medicinal cannabis, TYM levels of cannabis were reduced (6–4.5 log) when treated with γ-irradiation and by 5-log when treated with cold plasma treatment or e-beam (Jerushalmi et al., 2020). By selecting products from reputable manufacturers, consumers can ensure that they are getting high-quality CBD gummies that are safe for consumption and effective in providing the desired benefits. Lab testing is essential in detecting these contaminants and ensuring that the products are pure and potent. It is essential to choose lab-tested and vegan CBD gummies, such as those offered by Crescent Canna, to ensure that the products are free from contaminants and align with dietary preferences. Microbiology CBD gummies have been found to have a range of health benefits, including stress relief, improved sleep quality, and enhanced overall wellness. Consequently, the composition and function of gut microbiota may impact the local concentration and activity of cannabinoids in the gastrointestinal tract, where gut cancers often originate. Emerging evidence suggests that the gut microbiota could play a critical role in modulating the anti-cancer effects of cannabis. The gut microbiota also participates in the metabolism of dietary compounds and endogenous molecules, contributing to the production of bioactive metabolites that can affect host physiology . Furthermore, the interplay between the gut microbiota, metabolites, and cancer therapies accentuates their potential role in modulating drug efficacy and patient outcomes 33,34. Our studies demonstrate that CBD has consistent activity against a wide range of Gram-positive bacteria, including a variety of drug-resistant strains. A number of earlier21 and subsequent22 publications have reported on the antimicrobial properties of cannabis extracts, as opposed to purified compounds. This article highlights the potential for cannabidiol in the age of antimicrobial resistance. This colorimetric step was additionally performed to allow better visualization of CBD antibacterial activity25. 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. 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. CBD exhibited antibacterial activity against Gram-positive bacteria, M. 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 infections53. The area was then inoculated with 5 × 107 CFU of bacteria in a 10 μL droplet and the droplet spread around the area. On Day 0, an area of the back was shaved, and the skin surface disrupted to facilitate bacterial colonization. On Days −4 and −1, mice were administered with 150 then 100 mg kg−1 cyclophosphamide, respectively. Aureus Xen-29 bioluminescent bacteria was cultured under standard conditions. After incubation, the plates were centrifuged at 1000 × g for 10 min to pellet cells and debris, 25 µL of the supernatant was then transferred to a flat bottom polystyrene 384-well assay plate (Corning, Cat. No. 3680). In rodent models and human cells, ZEA is immunotoxic, induces toxicity in the liver and kidneys, and is hemotoxic (Choi et al., 2012; Ji et al., 2019). It was isolated from 60% of CBD capsules (Narváez et al., 2020), but at levels below limits established for food products (Ji et al., 2019). In Colorado, most cannabis-related emergency department (ED) visits were for CHS (Wang et al., 2021). Gonorrhoeae targeting agent that is not only selective over other Gram-negative bacteria, but also over Gram-positive bacteria.Coli strains, the assay was also performed in the presence of 50 µg/mL of PAβN.The growing dilemma of legalized cannabis and heart transplantation.Clearly, more work is required to decipher the subtle variations in outer membrane composition that affect CBD antibacterial activity, as well as further studies to ascertain whether there are specific pathways targeted by CBD.Additionally, exploring the interplay between the gut microbiota, the immune system, and cannabis can unravel how these interactions impact the immune response against cancer.According to experts, the future of microbiology CBD gummies will be shaped by advances in lab testing, quality control, and manufacturing processes.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. 1. Phytopathogens of Cannabis sativa The current regulatory requirement for enumerating TYM present in cannabis and hemp tissues using plating on culture media does not allow for a direct interpretation as to potential health risks to consumers. Current regulations have addressed the potential for harm from Aspergillus species, yet few reports have confirmed whether mycotoxin production is widespread or is rare in occurrence in cannabis products. It is important to note that fungi listed in Table 1 are commonly found on a wide range of plant species and, hence, are not unique to cannabis and hemp. By sharing their experiences and feedback, users can help manufacturers refine their products and ensure that they are meeting the highest standards of quality and safety. "Microbiology is critical in detecting and preventing microbial contamination, which can compromise the quality and safety of CBD supplements," he notes. Other experts, such as Dr. John Doe, have emphasized the importance of microbiology in ensuring the quality and safety of CBD supplements. Four products had significantly less CBD than advertised, and one product had more CBD than advertised. Five of the products had no detectable levels of CBD in them, despite their labeling. FOX 11 dropped the products off at Nascient, a premium Cannabis testing lab in Chatsworth that was also in the process of testing the products Dr. Oz's team had bought. We purchased a mix of CBD infused products, ranging from edibles, to liquids and creams. While Dr. Oz's team purchased various CBD products in New Jersey for testing, FOX 11 went undercover to several different locations across Los Angeles to purchase our own CBD products for testing. Coli bacteria, two samples contained potentially dangerous levels of ethanol, five samples had no detectable traces of CBD, and only one of the products actually contained what was claimed in its labeling. Two microliter of compound or DMSO were transferred into 96-well black walled polystyrene plates (Corning, Cat. No. 3603), to which was added 148 µL well−1 of the dye-saturated bacterial cells to initiate the reaction. The bacterial growth in those wells was resuspended by pipetting, then plates were read for OD600, which was used to calculate the dilution of cells to a density of 106 CFU mL−1. Standard antibiotic comparators ranged from 0.03–64 µg mL−1 for bacteria, and 0.06–128 µg mL−1 for fungi, and from 0.03–64 µg mL−1 for CBD. The colorimetric step using an aqueous solution of resazurin sodium salt (Sigma-Aldrich) was also performed to allow better visualization of CBD antibacterial activity. 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 assays57,58. MBC values were determined as the lowest concentration of CBD that prevents the growth of the bacterial colony-forming unit in solid culture medium. Cannabis Microbial Testing Regulations by State The cultures were either left untreated or treated with 1 µg/mL CBD and/or 16 µg/mL BAC or ethanol. Bacterial growth was determined by turbidity measurements at OD600 nm. Using an oCelloScope (BioSense Solutions ApS), the bacterial density was measured over a period of 24 hours using the UniExplorer software. MICA and MICB in combination indicate the MIC value of compound A or B, when combined with the other compound (A or B). For the FIC index determination, ¼ volume of each compound or liquid media was added to the wells in the 96-well plate and final ½ volume with same bacterial inoculum was added to the wells afterwards. Consequently, alterations in the gut microbiome composition have been linked to various immune-related diseases, including cancer . Findings suggest that cannabinoids, whether natural or synthetic, hold promise in modulating gut microbiota, influencing inflammation, and improving conditions related to metabolism and the nervous system. Conversely, blocking the CB1 receptor with the anti-obesity drug Rimonabant led to reductions in obesity, inflammatory cytokines, and certain gut bacterial strains . For instance, the CB2R agonist JWH133 demonstrated benefits in cirrhotic rats by reducing bacterial overgrowth and promoting intestinal integrity . This combination therapy reduced inflammation markers and improved intestinal permeability, suggesting a potential avenue for managing gastrointestinal diseases. Treating the bacteria with both CBD and BAC alone or in combination did not change the pattern of the HPLC chromatogram of the muropeptides (Fig. 4) indicating no change in the muropeptide composition. This suggests that the combination of CBD and BAC affects the cell envelope causing irregular cell division visualised by multiple septa formations and irregular cell membrane. Upon exposure to either CBD or BAC alone regular septum formations were visualised, however, when treated with the combination, several septa formations appeared for some of the cells as visualised by Bocillin-FL as in the TEM images. Aureus USA300 upon exposure to CBD and/or BAC by treating a culture at start exponential phase for 2.5 hours and then performing transmission electron microscopy (TEM) of the cells. For a more immediate application of CBD’s antimicrobial activity, clinical trials are being initiated to test CBD as a topical therapy for nasal decolonization of S. In summary, CBD represents the prototypical member of an exciting structural class of antibiotics, with potential to develop new analogs that have narrow spectrum selective Gram-negative activity against the dangerous pathogen N. Clearly, more work is required to decipher the subtle variations in outer membrane composition that affect CBD antibacterial activity, as well as further studies to ascertain whether there are specific pathways targeted by CBD. Our experiments demonstrate that the lack of CBD activity against most Gram-negative bacteria is related to the presence of the outer membrane and lipopolysaccharide, as membrane-disrupting drugs or LPS-deficient bacteria increase Gram-negative susceptibility to CBD. (I) Swabs taken from dried cannabis samples and streaked onto potato dextrose agar shows the diversity of Penicillium species growing on the medium and producing a range of pigments.Because two of the most commonly encountered fungi that can potentially produce mycotoxins in cannabis tissues, namely Penicillium and Aspergillus spp., can survive at aw of 0.62–0.7, this range is suboptimal for fungal growth.In addition, formation of several septa has been visualised by others by creation of gene knockouts or by performing gene silencing of genes important for cell cycle regulation.The benefits of microbiology CBD gummies are numerous, and they can be attributed to the combination of CBD and the rigorous quality control measures that are in place during production.DiSC3(5) was added to the cells at final concentration of 100 nM, wrapped with aluminum foil, and incubated at 35 ± 2 °C, 220 rpm for 30 min until a stable reduction of fluorescence was observed. Founded Linneas (conducted the bacterial cytological profiling). Conducted biofilm microscopy experiments, D.Q., M.D.S., and J.P. A.M.K. conducted most UQ-based microbiological experiments, with assistance from A.G.E., S.R., M.A., G.J.L., A.O.H., D.M.T.P., and J.Z. ATCC-labeled strains and human cell lines HepG2 (ATCC HB-8065) and HEK-293 (ATCC CRL-1573), were acquired from American Type Culture Collection (ATCC). Twenty-six hours after MRSA infection, mice were euthanised. Additionally, exploring the interplay between the gut microbiota, the immune system, and cannabis can unravel how these interactions impact the immune response against cancer. In conclusion, the emerging field of research on the gut microbiome–cannabis axis presents a promising avenue for advancing cancer treatment strategies. This information could guide the selection of appropriate cannabis-based formulations, dosages, and treatment regimens tailored to everyone’s unique gut microbiota composition and characteristics. These products are not intended to diagnose, treat, therapy, or stop any disease. Some products recommend taking the CBC gummy with food, so you could consume a gummy at each meal or within minutes of a meal. The best CBC gummies are tested by an independent laboratory, so you are assured the products do not contain toxins like heavy metals and pesticides. CBC (cannabichromene) may be a lesser-known cannabinoid, but it is fast gaining popularity as a phytocannabinoid that is non-intoxicating, mood enhancing and a contributor to good health. MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. When cannabis or hemp is smoked in a water pipe, DON contamination may be reduced because DON leaches into water at high temperatures; the water acts as filteration for the smoke (Liu et al., 2019; Feizollahi and Roopesh, 2022). There is only one reported study of DON production in hemp (Bergstrom et al., 2019, 2020). The degradation of these mycotoxins by heat does not always result in reduced toxicity since some degradation products are as toxic as the parent molecule (Kabak, 2009). This study suggests that legalization of cannabis production may be having an intended consequence of improving quality with lower mold contamination, but additional studies are needed to confirm this. A comprehensive toxicology screen including cannabis was negative (Table 4). Bag of CBD gummies consumed by patient marketed as a healthy solution for pain relief. CBD has surged in popularity recently, being marketed in oils, capsules, and candies as a health supplement, claiming to treat a wide variety of medical conditions such as glaucoma, pain, and even having beneficial effects on cancer prevention. CBD is one of many cannabinoids found in marijuana and marijuana-derived products. Considering health, social and economic implications of growing antimicrobial resistance, the World Health Organization (WHO) calls attention to research, discovery, and development of new antibiotics against MDR/XDR ESKAPE pathogens6. Interaction with PB destabilizes LPS or LOS, leading to disruption of the bacterial cell envelop3. The antibacterial activity of polymyxins is due to an electrostatic interaction between the positively charged polymyxin and the phosphate groups of the negatively charged lipid A, on lipopolysaccharide (LPS) or lipooligosaccharide (LOS), in the outer membrane of GNB. The antibacterial efficacy of the combination CBD + PB against multidrug-resistant and extensively drug-resistant GNB, especially PB-resistant K. Our results show that CBD has translational potential and should be further explored as a repurposed antibacterial agent in clinical trials. Furthermore, we also assessed LOS-expressing bacteria such as N. Therefore, we hypothesized that CBD was inactive due to low permeability through the cell envelope (outer membrane) of GNB. However, our data also showed no role of efflux pumps that are commonly involved in antibiotic extrusion from GN cell. These differences may have contributed to our higher CBD MIC values against fastidious bacteria compared to those of Blaskovich et al.16. Gonorrhoeae, we employed the broth microdilution method instead of agar dilution. Gonorrhoeae ATCC 19424, the FICI of the combination CBD + PB was calculated because both CBD and PB alone showed antibacterial activity (MIC). 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. The results showed that the combination of CBD (4 µg/mL) + PB was antibacterial only in the presence of PAβN (Table 4). Thereby, CBD does not restore PB susceptibility for PB-resistant GN bacteria. Outstandingly, the combination CBD + PB was effective against PB-resistant K. Catarrhalis, the calculation of FICI revealed an additive or synergistic effects for the combination CBD + PB (Table 5). These results may be related to the different intrinsic resistance mechanisms of these bacteria, involving different molecular pathways from two-component systems3,39. In our study, we used the EUCAST/BrCAST breakpoint (PB MIC ≤ 2 µg/mL as susceptible) for our analyses and discussion. Evaluation of antimicrobial activity and ethnobotanical study of Cannabis sativa L. Numerous fungicides suppress hemp powdery mildew in the greenhouse in Tennessee, 2021. Biopesticide potential in controlling pathogens and pests of hemp awarded to Kimberly Gwinn. The products for medical use should have a unified national standard that includes mandatory irradiation to eliminate fungal contaminants and protect immunocompromised patients. We recommend testing for the presence of Aspergillus, Penicillium, Fusarium, and Mucor as priorities, with an adjustment to adding other species if they are discovered to have potential harm. In a murine colitis model, activating cannabinoid receptors by THC improved the integrity of the colonic barrier and enhanced the production of protective molecules. Moreover, with cannabinoid treatment, pathogenic bacteria were mitigated, and the presence of beneficial bacteria was enhanced . For instance, recent studies have demonstrated that endocannabinoid anandamide (AEA) can counteract harmful microbiota disruptions caused by severe acute respiratory distress syndrome (ARDS) in mice . In clinical trials exploring cannabis-based therapies for cancer treatment, understanding how gut microbiota composition influences cannabinoid metabolism and toxicity is paramount . Therefore, combining cannabis-based treatments with gut microbiome-targeted interventions may provide synergistic effects, improving the therapeutic properties of cannabinoids. However, much remains to be elucidated regarding the specific mechanisms and clinical implications of the interactions between cannabis, the gut microbiota, and cancer. In turn, the gut microbiota can metabolize cannabinoids, affect their pharmacological activity, and modulate the immune response to cannabinoids in the gastrointestinal tract and systemically. The interaction between cannabis, the gut microbiome, and cancer is likely bidirectional, with each component influencing the others in a complex and dynamic manner (Figure 4). Animal studies have demonstrated changes in the abundance of specific bacterial taxa following cannabis exposure 13,84. For instance, commensal bacteria have been found to produce endocannabinoid-like compounds, such as 2-arachidonoyl glycerol (2-AG), which can activate cannabinoid receptors and modulate gut functions . Conversely, the gut microbiota can produce endocannabinoids and influence the endocannabinoid tone in the gut. Studies have also suggested that the ECS might play a role in modulating gut barrier integrity and immune responses, which can influence the gut microbiota 77,79. For example, activation of CB1 receptors has been shown to affect the gut microbiota by altering the gut transit time and promoting changes in gut motility . Pesimquinolones produced by Penicillium simplicissimum and their inhibitory activity on nitric oxide production. Spatio-temporal and cultivar-dependent variations in the cannabis microbiome. The what, why and how of water activity in cannabis. Chemistry and biology of mycotoxins and related fungal metabolites. Evaluating the microbiome of hemp. At present, there is a scarcity of analytical studies on Fusarium mycotoxin accumulation in cannabis and hemp inflorescences. That have been reported to infect cannabis inflorescences can produce an abundance of mycelial growth and mycotoxins under laboratory conditions (Gwinn et al., 2022; Munir et al., 2023). Reports of toxigenic fungi in cannabis and cannabis products sold for consumer consumption or consumed by patients with mycoses. Furthermore, the interaction between the ECS and the gut microbiota is bidirectional, as the ECS can influence the gut microbiota composition and function. Interestingly, germ-free mice, which lack gut microbiota, displayed alterations in the expression of ECS components in the gastrointestinal tract compared to conventionally raised mice . A diagrammatic breakdown of animal and human studies examining cannabis-related use and the impact on gut physiological function. On the other hand, CB2 receptor activation in immune cells within the gut mucosa can influence the inflammatory response and contribute to gut immune homeostasis . As inflammation and gut dysmotility are implicated in cancer development and progression, the ability of gut microbiota to produce SCFAs may play a role in modulating the anticancer effects of cannabinoids. Endophytes can produce antimicrobial compounds that may render them beneficial in the plant (Taghinasab and Jabaji, 2020; Mishra et al., 2022). A number of endophytes that are latent pathogens can be present as pre-and post-harvest contaminants of cannabis inflorescences (Table 1). The origins of these fungi can be traced back to the growing medium used, source of plant propagation materials, or are present externally in the ambient environment. In addition, fungi considered to be epiphytes and endophytes which cause no obvious symptoms in plants have been characterized and are common in stems and roots (Figures 1, 2). A similar study has not been conducted in hemp. The washed cells were then added to the 384-well compound-containing plates for a final volume of 50 µL. Human whole blood (ARCBS; 00150) was washed three times in 0.9% NaCl (Baxter; AHF7124) and resuspended to a concentration of 0.5 × 108 cells mL−1, as determined by manual cell count in a Neubauer haemocytometer. CC50 (concentration at 50% cell viability) values were calculated using nonlinear regression analysis of log(concentration) vs. normalized cell viability, using variable fitting. The cell plates were incubated for 20 h at 37 °C, 5% CO2. The gut microbiome profoundly impacts the development and function of the immune system, including immune cell development, shaping the composition of immune cell populations, and modulating the production of inflammatory mediators . The complex interactions between cannabinoids and gut microbes have the potential to impact the therapeutic efficacy and safety of cannabis-based treatments for various medical conditions, including cancer. Moreover, clinical studies have shown associations between cannabis use and shifts in the gut microbial community, particularly in relation to the abundance of certain bacterial communities 13,85. Understanding the interactions between the ECS and gut microbiota is of growing interest due to their potential implications for human health and disease. Although no studies have compared mycotoxin levels in legalized and illicit cannabis products, the literature suggests that OTA presence can be of concern in illicit cannabis. The most frequently recovered toxigenic fungi from cannabis tissues are Penicillium spp. There is a paucity of published studies on mycotoxin presence in cannabis-derived products (Table 6). The synthesis of mycotoxins involves several metabolic pathways and corresponding enzymes, such as polyketide synthases, nonribosomal peptide synthetase, the mevalonate pathway, or a combination of these. Cannabis hyperemesis syndrome (CHS) is a condition of recurring vomiting that some individuals develop after prolonged cannabis use (Perisetti et al., 2020). However, even though the smoking process can reduce the level of Fusarium mycotoxins, these mycotoxins are absorbed into the bloodstream efficiently and rapidly through inhalation. We are unaware of studies that have discussed the fate of these mycotoxins in combustion. Following the 48 h incubation, the biofilm-containing plates were carefully washed three times with 200 µL per well of saline solution (0.9% NaCl, Baxter Healthcare; Cat. No. AHF7124) using a manual pipette to remove the planktonic cells but leaving the biofilm uninterrupted. Standard antibiotics and CBD were serially diluted in TSB with 3% glucose two-fold across the wells of 96-well polystyrene plates (Corning; Cat. No. 3370), to a concentration range of 0.06–128 µg mL−1, with 100 μL final volume (antibiotic plate). Standard antibiotic comparators and CBD were tested as a 12-point dose response from 0.03–64 µg mL−1. Aureus ATCC (MRSA) and compound concentration range of 0.03–64 µg mL−1 incubated at 37 °C. Several reports have shown that pulmonary aspergillosis developed in patients with malignancies or other immunocompromised states following smoking of cannabis National Academies of Sciences, Engineering, and Medicine (NASEM), 2017. For example, after inhalation of spores, these fungi can enter nasal passages and the lungs where they may cause lung infections, particularly in immunocompromised patients National Academies of Sciences, Engineering, and Medicine (NASEM), 2017; Benedict et al., 2020. Cannabis and hemp can be used in a variety of ways, including as dried flowers that are smoked, inhaled, or vaped; flowers that have undergone an extraction process for cannabinoids; and formulated concentrates that include capsules, gels, creams, suppositories, and tinctures. No use, distribution or reproduction is permitted which does not comply with these terms. The gut microbiota is a dynamic and diverse community of microorganisms residing within the gastrointestinal tract, consisting of bacteria, archaea, viruses, and fungi. The growing interest in understanding how the gut microbiome and cannabis may impact cancer has created a demand for up-to-date, comprehensive reviews to inform researchers and healthcare practitioners. Additionally, the capacity of gut microbiota to activate cannabinoid receptors through the production of secondary bile acids underscores its role in directly influencing the pharmacological activity of cannabinoids. A bacterial suspension was prepared on McFarland's 0.5 scale (1.5 × 106 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). Confirmation of the antibacterial activity of the combination CBD + PB was performed against 22 selected strains (Tables 4 and 5).