In part I of this series, we start the discussion on the important microbiological targets for the cannabis market and understand their significance as a threat to health and safety.
Often when scientists or analysts speak of analysis and contamination in the cannabis market it is regarding chemical components such as heavy metals, pesticides, and other potentially hazardous chemical substances. In previous columns, we have taken a deep dive into the chemical aspects of cannabis analysis and now we change direction into another important study involving cannabis—microbiology. Chemical compounds are far from the only potential contamination found in cannabis products, in fact, in the world of consumer products, chemical contamination makes up for only a small amount of product recalls in comparison with microbiological contamination. In this column, we start the discussion on the important microbiological targets for the cannabis market and understand their significance as a threat to health and safety.
Biological contaminants in the form of microbes are by far one of the greatest concerns for illness. According to the World Health Organization (WHO), there are more than 300 million cases of foodborne illnesses in the world each year resulting in almost half a million deaths (1). In the US, the Centers for Disease Control and Prevention (CDC) estimates that 48 million people get sick from foodborne illnesses and up to 3000 people die from foodborne diseases (2).
As cannabis becomes legal in more states and countries, there has been a corresponding increase in the amount of testing, and the cannabis testing market to try to ensure accuracy of labelling and claims in addition to product safety from physical, chemical, and biological pathogens.
Microorganisms, or microbes, are organisms that are microscopic in size and are composed of either single cells, colonies of cells, or small multicellular structures. The ways in which human beings have classified life since the early beginnings of scientific study has changed dramatically over the centuries (and even over the last few years!). The most current consensus based on the three-domain system is that living organisms fall into three superkingdoms or domains: archaea, bacteria, and eukarya.
Archaea and bacteria are prokaryotes lacking a nucleus and membrane-bound organelles. These two domains consist of only single-celled organisms. There are few to no widespread pathogens in the Archaea domain, but they do share similarities in their genetics and metabolic pathways to some bacteria, especially E. coli. Most pathogens fall into the domains of bacteria and eukarya.
In this three-domain model, viruses are not included in one of the domains but instead are submicroscopic infectious agents that can only survive and replicate inside other cells. From the two dominant domains and viruses there are at least five types of biological agents (microbes) possible of causing disease including: bacteria, viruses, and the eukaryotes (parasites, protozoa, and fungi).
Parasites (multicellular) and protozoans (unicellular) are single celled organisms that can be free-living or parasitic living off other organisms or their detritus. There are dozens of human parasitic protozoans that cause a variety of disease from waterborne amoebas to mosquito driven Plasmodium malarias. In general, cannabis products are minimal risk targets for protozoans or human parasite contamination, instead the primary source of human pathogens are bacteria and fungi. However, protozoa, parasites and viruses can still be agricultural targets for the cannabis industry in need of analytical testing.
In a previous column (3) we looked at fungal targets and their biological by-products, mycotoxins. The mobility of fungal spores and their minute size makes fungus and molds a significant threat to agriculture and human health. Of particular concern are the toxic fungi from the phylum Ascoymcota (molds, yeasts, mildew, and so forth), which can produce mycotoxins. The same species of fungi can produce multiple mycotoxins and many mycotoxins can be produced by more than one type of fungi (see Table I).
Agricultural crops, such as botanicals and cannabis, are prone to fungal growth because of agricultural conditions and moisture content. Plant materials with more than 14% moisture can encourage mold growth. Mycotoxins, especially aflatoxins and ochratoxins, need oxygen to grow, so the reduction of the oxygen in the storage areas can retard growth.
For the cannabis industry, the focus has been placed on measuring and controlling the Aspergillus species of fungi, especially for inhalable products since these fungi and their mycotoxin product can affect the respiratory system. The fungi were named after an aspergillum (holy water sprinkler) when it was first observed under a microscope. The similarity of the shape of the fungus has also been compared to a dandelion seedhead (see Figure 1).
Aspergillus grows on rotting vegetable matter and can be easily inhaled. While there are numerous species of Aspergillus the species of most concern for the cannabis industry are Aspergillus niger, terreus, flavus, and fumigatus. In most areas where cannabis products are legal there are some requirements for testing for Aspergillus species in select products. In many cases there must be no Aspergillus species detected for a product to pass because of the highly toxic nature of the mycotoxins produced by the fungus.
Toxic and lethal dosages for mycotoxins can be quite small for acute poisonings. Ochratoxin A has a tolerable daily intake designated by the WHO of 5 ng/kg of body weight per day. Ochratoxin A is very toxic with an LD50 of 20-25 mg/kg of body weight. WHO recognizes products containing more than 1 mg/kg of aflatoxins as potentially dangerous or life-threatening. The Food and Drug Administration (FDA) has limits for mycotoxins in human and animal feed up to 20 µg/kg for direct human exposure.
While fungi and mycotoxins are of concern in products that can be inhaled, the larger danger to human health from contamination of cannabis products come from bacteria. On the US CDC list of the top 30 human food and waterborne pathogens, 22 pathogens were bacteria, and five each were viruses and parasites. The majority of bacteria targets of concern for the cannabis industry are from three families of bacteria:Enterobacteriaceae, Listeriaceae, and Staphylococcaeae (see Table II).
These bacteria of concern can be classified in multiple ways depending on their family, morphology, status as an anaerobic or aerobic organism, and ability to stain under different reagents—just to name a few categories. Morphology is one of the primary methods of differentiating families of bacteria. There are five basic morphologies of bacteria: spherical (cocci), rod (bacilli), comma or bent rod (vibrios), spiral (spirilla), or corkscrew (spirochaetes) (see Figure 2).
The bacteria that are important targets for cannabis are almost all bacilli or rod-shaped bacteria, which can be detected by negative gram stain. Gram staining is a method of labelling bacteria into gram-negative and gram-positive groups by taking advantage of the chemical and physical properties of the bacterial cell walls. Positive cells have a thicker cell wall that retains the crystal violet stain while negative cells can wash off the stain. Different counterstains and additives can strengthen or bind the stain to the cell membranes allowing for preliminary identification of certain bacterial groups.
In a variety of regulations, the target is called bile-tolerant gram-negative bacteria (BTGN) which includes gram negative bacteria of the Enterobacteriaceae family including E. coli and Salmonella. Another type of test measure that can be required is total coliform, which is a different measurement of Enterobacteriaceae species such as E. coli, Enterobacter, and Klebsiella species (see Table II).
All these bacteria potentially have cause for concern in cannabis products, however, the most regulations are created around the quantitation and detection of Salmonella and E. coli. Salmonella causes almost 80 million illnesses worldwide with hundreds of deaths each year. Salmonella are rod-shaped, gram-negative bacteria from the Enterobacteriaceae family. There are two species of Salmonella (S. enterica and S. bongori) with six subspecies and more than 2600 serotypes or strains; some (mostly typhoidal strains) can cause severe illness or death (see Figure 3).
Salmonella infections are caused by the ingestion of food that has been contaminated by feces (animal or human) either in the agricultural or handling phase of preparation. The serotypes can be divided into two main groupings: typhoidal or nontyphoidal. Typhoidal serotypes are transferred from human to human and invade the bloodstream. Nontyphoidal serotypes are zoonotic (transfer animal to human) and invade the gastrointestinal tract.
Escherichia coli is a second important target for microbiological analysis in cannabis.This bacterium is a gram-negative, rod-shaped bacteria found commonly in the intestine of warm-blooded organisms (see Figure 4). Most strains are either harmless or can be beneficial to the hosts while other strains are pathogenic and cause food poisoning.
Worldwide there are more than 300 million infections and 200,000 deaths due to some forms of E. coli that produce diarrhea and gastrointestinal illness. There are six pathogen types of the bacteria associated with diarrhea:enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), diffusely adherent E. coli (DAEC), and Shiga toxin-producing E. coli (STEC). There are further serotypes of some of these pathogen types determined by the presence of O and H surface antigens. There are about 200 known serotypes. E. coli is one of the most diverse groups of bacteria sharing only about 20% of its genetic code amongst all the strains.
One of the most important targets because of its toxicity is the Shiga toxin E. coli strain (STEC), especially the serotype O157:H7. The Shiga toxin produced by this strain of the bacteria causes severe abdominal pain, diarrhea, and life-threatening conditions such as dysentery, hemolytic uremia, hemorrhagic colitis, kidney failure, and death.
Due to the severe nature of the consequences of these microbiological infections most regulatory bodies for cannabis have proposed testing for microbiological contamination in cannabis products.
Agricultural products (including cannabis) around the world are routinely evaluated for contaminants and impurities such heavy metal ions, pesticides, mycotoxins, and residual solvents. This is true for all agricultural products that are consumed by human beings. Heavy metals are tested by inductively coupled plasma-mass spectrometry (ICP-MS) or inductively coupled plasma-optical emission spectroscopy (ICP-OES). Residual solvents, which are small, volatile impurities, are tested by gas chromatography (GC). Pesticides and mycotoxins, which are products of pathogens like Aspergillus, are routinely tested by liquid chromatography (LC). Many countries have specific requirements for biological contamination such as microorganisms. The presence of live microorganisms is assessed in several ways. The best quantitative results are obtained by quantitative polymerase chain reaction (qPCR), but other methods exist, such as cell culture techniques, and enzyme-linked immunosorbent assays (ELISA).
There are numerous ways to detect microorganisms in agriculture products, but the most common methods include cell culture, chromogenic media, ELISA assays, and polymerase chain reaction (PCR). Let’s discuss each of these in more detail.
The methodology for the detection and quantitation of microorganisms has traditionally been performed by utilizing various forms of cell culture. Many different culturing media have been used over the years that are enriched with specific concentrations of nutrient broths mixed with agar for support and grown in individual dishes or plates. The microorganisms are allowed to grow for specific durations of time (hours to days) and the direct number of colonies are counted per milliliter of fluid (CPM) or through viable cell counting to determine cell proliferation using colony forming units/mL (CFU).
In more recent years, much attention has been given to the use of chromogenic media for use in plating microorganisms. This application combines the use of synthetic substrates for the detection of enzymatic microbial activities with colorimetric reactions, allowing for more specific detection of species or sub-species, often with only visual analysis. Cell culturing requires a good working knowledge of scientific technique and requires designated laboratory space for laminar flow hoods, orbital shakers, incubators, and other necessary equipment. Moreover, microbiologists that have become experts in these techniques are reticent to accept some of the newer methodologies, so some debate exists in cannabis and throughout pathogen detection as to whether cell culture is indeed the “gold standard” of microbiology. Moreover, since culturing is a manual and time-consuming process, the need for more rapid results has pushed the development of other assays, namely ELISA and qPCR.
ELISA kits are enzyme-linked immunosorbent assays designed for both the rapid detection and quantitation of target proteins, antibodies, and other soluble targets of interest including cytokines, growth factors, and signaling molecules, as well as transcription factors and post-translational gene regulators (RNAi). ELISAs are highly sensitive, with a low limit of detection typically in the range of 0.1-0.01 ng. Their use for detecting microorganisms involves using a heat-inactivated bacterial strain or a target protein at known concentration for positive control and allows users to automate their sampling and produce far more replicates for statistical derivation in a typically 96-well format using a multiplate reader and minimal additional equipment needs. Additionally, the reactions do not require a high degree of technical expertise, making them more suitable for use in the cannabis space.
PCR, and now the use of qPCR, has allowed for the detection and quantitation of genes found with less than 10 copies in each sample. The combined use of fluorophores also allows a researcher to quantify five genes simultaneously, with one as a reference or “housekeeping” gene. This ensures that the deoxyribonucleic acid (DNA) amplified occurs in known relative concentrations to a DNA baseline, thereby minimizing human error. qPCR is slowly becoming the “go to” technology for operators in the cannabis space for microorganism detection and quantitation by virtue of such rapid generation of results and diversity of application. Despite having a reputation of being error prone, numerous studies have shown that qPCR-based microorganism detection protocols are faster and more sensitive than classical culturing or plating techniques for many organisms and the process can be automated and routinely performed with a modest amount of equipment and technical expertise.
Cannabis, like every other agricultural product, is tasked with providing for the safety of the consumer by limiting or eliminating pathogens. The agricultural practices and handling processes in the cannabis industry can lead to increased exposure to microbes. There are significant health concerns regarding microbiological contamination of products made into edible foods, inhalation products, and medicines for critically ill patients with potentially compromised immune systems. It is critical to understand the type, nature, and detection of the microbiological species to separate the potentially harmless serotypes and species from the toxic variants. In the next column, we will continue into a deeper discussion of the methods for detection and quantitation of the diverse types of microbiological targets along with their strengths and weaknesses as analytical techniques.
Patricia Atkins is a Senior Applications Scientist with Spex, an Antylia Scientific company and has been a member of many cannabis advisory committees and working groups for cannabis including NACRW, AOAC and ASTM.
Atkins, P., MeasuringMicrobiology, Part I: A Look at the Microbiological Contaminants in Cannabis, Cannabis Science and Technology, 2023, 6(5), 16-22.