Volume 2, Issue 4
This review article from an experienced pharmaceutical microbiologist discusses the risks of microbial contamination for the full range of cannabis-derived products and recommends the most appropriate microbiological quality requirements for each product.
This review article, written from the point of view of an experienced pharmaceutical microbiologist, discusses the risks of microbial contamination for the full range of cannabis-derived products and recommends the most appropriate microbiological quality requirements for each of these products by benchmarking against the microbiological quality recommendations from the American Herbal Products Association (AHPA), the World Health Organization (WHO), European Pharmacopeia (Ph. Eur.), and the U.S. Pharmacopeia (USP) for powdered cannabis, dietary supplements, herbs, botanical products, and nonsterile drug products.
With a rapidly emerging state-regulated industry producing both recreational and medicinal cannabis products, there are many questions surrounding the microbiological attributes of cannabis-derived products. This review article discusses the risk of microbial contamination of these products and the most appropriate microbiological specifications and test methods for the widely different products that range from smoked powdered cannabis buds to chemically defined pharmaceutical drug products. Part of the challenge is that cannabis-derived products based on the consumer (medical and recreational) and mode of use may be viewed as herbal products, recreational drugs, foods, or pharmaceutical drug products. Critical issues to be addressed by state regulators and the cannabis industry alike are the microbiological specifications and test methods for these widely different products.
What are the biggest risks with cannabis-derived products? In medical use, smoking cannabis has been used to control weight loss associated with the human immunodeficiency virus (HIV) and acquired immune deficiency syndrome (AIDS), preventing nausea associated with chemotherapy, and alleviating pain associated with a range of illnesses. These patient populations will be more susceptible to microbial infection than recreational users. With respect to mode of administration, the microbiological quality requirement will be widely different for cannabis that is smoked, vaped, topically applied, or eaten. As many medicinal cannabis users have severely compromised immune systems, medical cannabis distributed by licensed producers in the Netherlands and Canada is irradiated for control of the bioburden but not to sterilize the product.
Does the scientific literature report concerns with the microbial contamination of cannabis? This issue was addressed by the author in an earlier publication that may have escaped the cannabis industry’s attention because of its publication in a pharmaceutical trade journal (1). Groups prone to chronic pulmonary aspergillosis (CPA) associated with medical or recreational use of cannabis (2–12) include immune-compromised individuals receiving chemotherapy and corticosteroids; solid organs and stem cell transplant recipients; and HIV or AIDS patients. In addition to CPA that has a high mortality rate, if not treated early, chronic use of smoked cannabis is associated with allergic broncho-pulmonary aspergillus (ABPA) (13). An authoritative review on the Aspergillus and Aspergillosis website (14) stated that the overall risk of aspergillosis appeared low, given the large number of cannabis smokers. An exception noted would be invasive aspergillosis in highly immune-compromised patients. This suggests that medical cannabis that is smoked should have more stringent fungal specifications than other products.
In addition, cannabis may be contaminated with other human pathogens during its cultivation, processing, and distribution. Outbreaks of hepatitis B (15) hepatitis A (16), and salmonellosis (17) have been reported. The author expects that this type of contamination may be more likely in field-grown cannabis plants using irrigation water, and poor hygiene practices among harvesters and processors, than greater capitalized greenhouse-grown cannabis operations.
Published surveys of the microbial populations of powdered cannabis in peer-reviewed journals are few and of varying technical rigor. Higher mold numbers using the selective Sabouraud Dextrose Agar (SDA) for cannabis compared to tobacco (200 to 300 versus 104 to 107 CFU/g), with the predominant fungi Penicilliumspp., Aspergillus fumigatus, and A. flavus have been reported (18).
This study reported that cigarette smoke was negative for mold but cigarettes are tightly rolled and burn at higher temperatures than joints. Other studies have shown that smoke from both burning tobacco and marijuana cigarettes contain fungal spores (19,20). In an evaluation of the quality of medicinal-grade and coffee shop-purchased cannabis in the Netherlands (21), it was reported that irradiated medicinal-grade cannabis contained less than 10 CFU/g enterobacteria and Gram-negative bacteria (bile-tolerant, Gram-negative bacterial count) and less than 100 CFU/g molds and aerobic bacteria (total aerobic microbial count) while 10 samples of coffee shop cannabis ranged from less than 10 to 80,000 CFU/g bile-tolerant, Gram-negative bacterial counts and 120 to 480,000 CFU/g total aerobic microbial counts.
The contaminants from one of the 10 samples were identified as the fecal bacterium E. coli and molds of the common genera Penicillium, Cladosporium, and Aspergillus.
This section of my review discusses the definition of cannabis-based products as pharmaceuticals per U.S. Pharmacopeia (USP) <1151> Pharmaceutical Dosage Forms, food classifications, and state cannabis regulations. The hierarchy of risk of microbial infection based solely on the invasiveness of the route of administration would be vaping > smoking > topical application > oral consumption. It is expected that the risk to the cannabis users will be largely determined by their underlying medical status and frequency of usage.
Cannabis Buds Smoked
As far as the author knows, no pharmaceutical drug product is smoked, so how to set a microbiological specification for cannabis buds is more challenging. The literature suggests that microorganisms, especially fungal spores, may survive the burning process, be inhaled into the lungs, and result in infection especially in immunosuppressed users. The microbiological requirements for inhalation products as found in USP <1111> Microbiological Examination of Non-Sterile Products: Acceptance Criteria for Pharmaceutical Preparations and Substances for Pharmaceutical Use are more stringent and less achievable with a plant-derived material than recommended by standard-setting organizations for botanical products (Table I). (See upper right for Table I, click to enlarge.)
What are the microbial specifications for herbal products? Table II compares the microbiological quality recommendations (total aerobic microbial count, total yeast and mold count, bile-tolerant, Gram-negative bacterial count, and absence of specified microorganisms) from the the American Herbal Products Association (AHPA), the World Health Organization (WHO), European Pharmacopeia (Ph. Eur.), and USP for dietary supplements, herbs, and botanical products. (See upper right for Table II, click to enlarge.)
In 2014, the American Herbal Pharmacopoeia (AHP) issued a revised monograph entitled Cannabis Inflorescence-Standards of Identity, Analysis, and Quality Control that provided microbial and fungal limits that were claimed to be consistent with applicable state, federal, and international regulations that were based on botanical products. This monograph is highly influential in the cannabis industry (see Table III). (See upper right for Table III, click to enlarge.)
The USP published a stimulus article entitled “The Advisability and Feasibility of Developing USP Standards for Medical Cannabis” that discussed the issues around setting standards for medical cannabis (22).
The monograph cited the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (23) as a source of appropriate methods. Another good source of microbial methods would be USP <2021> Microbial Enumeration Tests-Nutritional and Dietary Supplements and USP <2022> Microbiological Procedures For Absence Of Specified Microorganisms-Nutritional And Dietary Supplements. The author believes that the limits recommended in the AHP monograph were qualified in terms that medical cannabis may need more restrictive limits addressing the presence of members of the genus Aspergillus, and that the limits recommended will not be suitable for extracted and highly purified cannabinoids where microorganisms would not survive the manufacturing process.
What is missing is a comprehensive survey of the microbial content of cannabis-derived products to serve as a baseline for the development of risk-based microbial specifications, an understanding of the different levels of risk in user populations, and an understanding of the microbiological attributes of products derived by different manufacturing processes. As more U.S. states legalize medical and recreational cannabis products, they are independently setting microbial specifications without this understanding. For example, Table IV contains the microbial limits required by the Colorado Department of Public Health. (See upper right for Table IV, click to enlarge.)
What may be wrong with this approach? These limits emphasize the exclusion of foodborne bacterial pathogens E. coli and Salmonella and the numbers of molds in the products. In food microbiology, it is generally viewed as impractical to screen for pathogenic E. coli in routine food surveillance programs, and this type of testing would be reserved for outbreak investigations. Setting a limit for the number of E. coli per gram of product as indicative of recent fecal contamination would be recommended by the author over specific pathogenic E. coli screening.
Salmonellae in powdered cannabis may survive despite the low water activity of the material, but would be eliminated during smoking. Handling material before smoking could be a source of enteric infection, whereas Salmonella spp. may contaminate and survive in olive oil used to extract cannabinoids from cannabis buds. As with Salmonella in contaminated flour and cake mixes that have caused foodborne infection when eaten raw, baking or frying of the cannabis-laced baked product will destroy this serious bacterial pathogen.
As to the control and monitoring of the fungal content of cannabis-derived products, the question is whether setting a total yeast and mold count (TYMC) limit, that is, not more than (NMT) 104 CFU/g, is sufficient to promote user safety, or if members of the genus Aspergillus should be specifically excluded because of its role in chronic pulmonary aspergillosis and the potential aflatoxin production. For example, the state of Colorado favors setting a TYMC limit, whereas the state of California favors routine screening for specific Aspergillus species (A. fumigatus, A. flavus, A. niger, and A. terreus). If it can be clearly established that Aspergillus is storage contaminant, and not an epiphytic microorganism associated with cannabis plants, then the emphasis should be placed on post-harvest drying and storage as a risk mitigation step, with water content (water activity) added to the cannabis specifications in place of screening for specific Aspergillus species. The reader is directed to a discussion of the distinction between epiphytic fungi, fungal diseases of cannabis plants, and cannabis storage fungi (24). The author recommends the microbiological standards for unprocessed cannabis as shown in Table V. (See upper right for Table V, click to enlarge.)
In contrast to buds, which are smoked, the active ingredients in the cannabis plant may be solvent extracted from the dried, powdered plant material to be smoked, vaporized, eaten, or applied topically.
The extraction method will depend largely on the cannabinoids targeted for purification and the type of concentrate wanted. The three most common extraction procedures are butane, alcohol, or supercritical carbon dioxide extractions (25).
Carbon Dioxide Extraction
These systems use carbon dioxide (CO2) pressurized to its supercritical state. This converts CO2 into a liquid that passes through a chamber containing powdered cannabis material. The filtrate can be isolated easily by reducing the pressure that evaporates the CO2, leaving a cannabis extract with no solvent.
Sophisticated extractors on the market can also incorporate fractionation to isolate individual cannabinoids, which enables process tuning to isolate desired components, and recover the CO2 for reuse.
The resulting material according to the final stages of processing may be called shatter, wax, budder, or crumble.
These systems pressurize butane, propane, or other low molecular weight hydrocarbons to a liquid state. Care must be taken as these solvents are flammable at room temperature. The liquid hydrocarbon passes through a bed of powdered cannabis material and filter, yielding a hydrocarbon extract solution of hydrocarbon and the cannabinoids. Like the CO2 method, a reduction in pressure evaporates the hydrocarbon liquid, yielding a solvent-free cannabinoid. Maintaining the pressurized hydrocarbon in the liquid state requires low temperatures. Recirculating temperature control units that can provide cooling to -60 °C (-76 °F) and below facilitates this process. Heating circulators are also incorporated to increase the liquid butane evaporation to isolate the extract and recycle the butane. The resultant concentrate is frequently described as butane hash oil.
This is the least sophisticated extract method of the three. Food-grade ethanol is used as a solvent to extract cannabis plant material. This can be used with vessels varying from reactors to barrels. A popular process has the ethanol chilled to <-20 °C (-4 °F) either in a cold room or freezer and then pumped into a container of cannabis. After a soak period, the ethanol solution is either filtered or the plant material removed in a “tea bag” fashion. The resultant ethanol mother liquor and extract is then concentrated by removing the ethanol. Typical distillation apparatus used to remove the ethanol include rotary evaporators or a vacuum distillation system. A simple form that allows the alcohol to evaporate is called quick wash isopropyl alcohol hash oil (QWISO).
Review of these three extraction and purification procedures strongly indicates that, other than bacterial spores, microorganisms will not survive contact with organic solvents, freezing temperatures without a cryo-protectant, and elevated temperatures associated with distillation. This suggests that cannabis buds and infusions, tinctures, and edibles made from oil-extracted cannabis in terms of microbiological specification should be treated differently than purified cannabinoids in terms of microbial specifications and testing because of the lower risk of microbial contamination (see Table VI). (See upper right for Table VI, click to enlarge.)
Cannabis Oil and Waxes (Vaporized)
An electronic cigarette is a battery-operated vaporizer, which simulates the experience of smoking, without burning tobacco. Their use is commonly termed vaping. Vaping is the rapidly growing delivery method for nicotine, especially amongst younger people, and will grow amongst cannabis users. The literature reports these electronic vaping devices offer greater efficiency in nicotine delivery than smoking cigarettes, without the inherent dangers of tobacco tars, carbon monoxide, and poly aromatic hydrocarbons. The vehicle of vaping is a heat-generated aerosol of propylene glycol, glycerin, flavoring, and nicotine or cannabinoids. Whereas the combustion temperatures of smoking cannabis ranges from 600–900 °C with around 30% of the cannabinoids lost by pyrolysis, vaping heats the cannabis oils and waxes to 160–230 °C. Cannabinoids vaporize near their boiling points. The boiling points of cannabinoids occur in this range: tetrahydrocannabinol (THC) 157 °C, cannabidiol (CBD) 160–180 °C, and cannabinol (CBN) 185 °C (26). The cannabinoid extraction process, storage in a propylene glycol-glycerin mixture E-solution of low water activity, and the vaporization temperature all make it highly unlikely the heat-generated aerosol will contain viable microorganisms.
Canna-butter, Vegetable Oil, Tea, or Tincture (Edible)
Olive oil may be a common choice for cannabis extracts used for cooking. Studies of the naturally occurring microorganisms in olive oil have been limited and do not give a complete picture for olive oils produced in all of the olive-producing regions of the world (27). For example, Italian researchers (28,29) examined olive oils produced in central Italy, and conducted microbiological analysis for aerobic (standard plate count agar) and lactic acid bacteria (MRS agar), yeasts (SDA), and molds (glucose yeast extract agar with gentamicin and chlorampenicol). They reported that yeasts were consistently present both initially and during storage, molds were occasionally found, and bacteria were never found.
The molds belonged primarily to the genus Aspergillus. Using light microscopy, they found that the microorganisms and the solid particles were entrapped in micro drops of vegetation water that were suspended in the olive oil.
Teas may be made by suspending and steeping powdered cannabis in boiling water, which should eliminate vegetative microorganisms. Similarly, tincture made with greater than 50% ethanol would be free of vegetative microorganisms, so routine screening for specified microorganisms could be eliminated.
USP <1151> Pharmaceutical Dosage Forms has a three-tier classification:
For cannabis-derived products we can add a fourth tier-recreational or medicinal. For example, a paste-filled hard shell capsule containing the drug substance CBD would be classified as a pharmaceutical drug product having a gastrointestinal route of administration for medical use.
Oral Capsules and Liquids (Pharmaceutical)
Based on the recommendations in USP <1111> Microbiological Examination of Non-Sterile Products: Acceptance Criteria for Pharmaceutical Preparations and Substances for Pharmaceutical, consumers (patients) should use an appropriate microbiological quality requirement for a pharmaceutical oral dosage form containing highly purified cannabis derivatives as described in Table VII. (See upper right for Table VII, click to enlarge.)
Based on the recommendations in USP <1111>, an appropriate microbiological quality requirement for a pharmaceutical topical dosage form containing highly purified cannabis derivatives would be as described in Table VIII. (See upper right for Table VIII, click to enlarge.)
The normal microbiological profile of baked goods is mold <102 to 103 counts/g; yeast and yeast-like fungi <10 to 103 counts/g; bacteria-aerobic plate count <102 to 103 CFU/g and coliform count <10 to 102 CFU/g (30). The baking or frying process destroys most of the microorganisms in baked goods. Exceptions may be Gram-positive, spore-forming bacteria such as B. subtilis that survive the baking process and cause spoilage. Post-baking mold contamination can occur if baked goods are stored at ambient temperature for prolonged periods.
As discussed earlier, rapid drying of cannabis plant material from 60–80% to 6–12% moisture content at temperatures below 40 °C will prevent mold growth without degrading cannabinoids. The water activity will be reduced from 0.80–0.90, a range that readily supports the growth of the mold Aspergillus flavus stored at temperatures above 25 °C, to 0.40–0.50, which does not support fungal growth (31). Modeling the effect of water activity and temperature on the growth rate and aflatoxin production by A. flavus on harvested rice demonstrated an optimum growth temperature of 30 °C with growth above 0.84 with higher toxin production at higher water activities (32). Clearly water activity (content) is a critical physical attribute for the protection of powdered cannabis from storage molds and mycotoxin production. A review of the moisture sorption isotherms of spices, in absence of those for cannabis, indicates that unprotected exposure of powdered cannabis to humidity exceeding 80% will promote water adsorption and result in mold growth. A recently published article (33), reported the moist adsorption isotherms of hemp seed (Cannabis sativa L) and that drying conditions at 40 °C and 50% RH will achieve moisture content less than 6%. The resulting material will not support fungal growth. Storing powdered cannabis in paper or jute bags is a bad idea because it will not protect the product from elevated humidity.
Water activity determination has been widely applied to the food, cosmetics, and pharmaceutical industries (34,35). Standardized water activity determination methods have been published recently. These include the ASTM D8196 Standard Practice for Determination Water Activity (Aw) in Cannabis Flower and the more generic USP <922> Water Activity.
Standard-setting organizations including the Association of Official Analytical Chemists (AOAC), USP, American Botanical Product Association (ABPA), Association of Public Health Laboratories (APHL), International Organization for Standardization (ISO), U.S. Department of Agriculture (USDA), and the FDA recommend microbiological screening methods that can be used for cannabis-derived products.
The most critical test methods would be the total yeast and mold count and tests for the absence of Aspergillus. Most selective fungal isolation media suppress the overgrowth with bacteria by high carbohydrate concentration, low pH, and the inclusion of antibiotics in the media. SDA, the original formulation or the Emmons modification, without any antibiotic, has historically been used as the standard medium for the primary isolation of fungus and is still widely used (36). SDA is cited in USP <61>, <2021>, and the FDA Bacteriological Analysis Manual. An experienced mycologist can readily identify A. fumigatus by its color and colony morphology and mode of sporulation when grown on SDA, and even differentiate it from A. niger, A. flavus, and A. terreus, but the average microbiologist would not.
Perhaps a simpler approach is the use of Aspergillus flavus and parasiticus Agar (AFPA) that was developed by Australian mycologists (37,38) as described by Beuchat in 1984. The formulation of this medium (33) was 20 g of yeast extract, 10 g of peptone, 0.5 g of ferric ammonium citrate, 0.1 g of chloramphenicol, 0.002 g of dichloran (2,6-dichloro-4-nitroaniline; 1.0 mL of a 2% ethanolic solution), and 15 g of agar per liter of distilled water. The pH of the medium was adjusted to 4.5 before sterilizing at 121 °C for 15 min. Sterilized media were cooled to 45–55 °C and poured to a depth of 5–7 mm into petri plates. The plates were allowed to remain at ambient temperature for 18–26 h before serial dilutions (0.1 mL) of test samples were spread over the surface with sterile glass rods. The total number of colonies of yeasts and molds as well as the number of colonies showing orange-yellow reverse coloration indicative of potentially aflatoxigenic aspergilli were recorded after 42–44 h of incubation.
The use of next generation sequencing (NGS) was used to survey of the fungal communities found in dispensary-based cannabis flowers by internal transcribed spacer (ITS2) sequencing, and demonstrate the sensitive detection of several toxigenic penicillium and Aspergillus species (39). The suitability of NGS in routine microbiological testing laboratories may be practical in the near future as the price drops and the technology is simplified.
All cannabis testing laboratories should be certified as to complying with an appropriate standard such as the ISO 17025:2017 General Requirements for the Competence of Testing and Calibration Laboratories.
The critical processing steps are growing, harvesting, milling, and drying cannabis buds. These steps share many of the risk factors associated with leafy vegetables.
The microbial risk associated with the extraction and purification steps are not clearly defined. The expectation is that the formulation and manufacturing processes for pharmaceutical-grade cannabis-derived products will be regulated by the FDA and will comply with current good manufacturing practices.
Aflatoxins are secondary metabolites produced primarily by the fungi Aspergillus flavus and Aspergillus parasiticus in agricultural foodstuff such as peanuts, maize grains, cereals, and animal feeds. The Food and Agricultural Organization (FAO) estimated that as much as 25% of the world’s agricultural commodities are contaminated with mycotoxins, leading to significant economic losses. Moreover, aflatoxins are highly toxic, mutagenic, teratogenic, and carcinogenic. Assay methods can be found in USP Chapter <561> Articles of Botanical Origin. The prevalence of mycotoxins in cannabis buds is largely unknown (40,41), but will be related to the post-harvest moisture content (water activity) of stored material. An earlier publication (42), reported the A. flavus and A. parasiticus produced aflatoxin when inoculated onto cannabis plants.
Another related issue is whether aflatoxins and other mycotoxins are extracted from the plant material and copurified with the cannabinoids. Mycotoxins will probably not be extracted from dry plant material using nonpolar solvents like butane, and may not survive a distillation procedure. However, definitive information appears to be lacking. Table IX summarizes key chemical and physical characteristics of common mycotoxins. (See upper right for Table IX, click to enlarge.)
The cannabis industry is a rapidly emerging enterprise governed by a patchwork of state regulations and little or no federal involvement. Microbial contamination risks associated with the wide range of cannabis-derived products are not fully defined. More comprehensive risk assessments, product testing histories, epidemiological investigations of reported human infection, and improved regulatory science will all be needed to develop appropriate in-process controls and finished product testing programs.
Tony Cundell is a New York-based consulting microbiologist serving the pharmaceutical industry. Direct correspondence to: email@example.com
T. Cundell, Cannabis Science and Technology 2(4), 36–49 (2019).