Developments in Microbial Identification in Cannabis: Dawn of a Molecular Era
The conidiophore of the fungal organism Aspergillus fumigatus. A. fumigatus is one of the microbiological contaminants some cannabis regulators require labs to test for. Obtained from the CDC Public Health Image Library via Wikimedia Commons. Author: CDC/Dr. Libero Ajello (PHIL #4297),
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The recent growth in the cannabis industry, both for medical and recreational use, has brought to light the importance of microbial testing for cannabis. This includes the presence of microbes that were present in the plant’s environment, and microbes introduced by the handling of cannabis during processing. Ensuring the quality of cannabis is of utmost importance: as medical cannabis, it will be used by patients whose immunity may be compromised (such as cancer patients) this making them particularly susceptible to microbial infection. Even for recreational use, some contaminating microbes may be human pathogens, which could harm healthy individuals. In addition, the native microbiome affects plant fitness, and cannabis is no exception. As cannabis becomes more widely accepted, improved methods of cannabis agriculture will be needed to meet the increasing demand.
Conventional microbiological methods: Lessons learned from the food industry
A number of techniques for estimating microbial burden have long been established and widely applied, such as those used for quality control purposes within the food industry. Guidelines for these methods of “microbial enumeration”, i.e., determination of the number of viable microorganisms in a sample, have been laid out by the United States Pharmacopeia (USP), in chapter <61>, “Microbiological examination of nonsterile products: microbial enumeration tests.” Identification of microorganisms can be achieved via tests outlined in chapter <62>, “Microbiological examination of nonsterile products: tests for specified microorganisms,” while techniques for determination of mycotoxins are outlined in chapter <561>, “Articles of botanical origin.” Taking a page out of the food industry’s book, the states that have legalized cannabis have similarly made a battery of microbial tests mandatory. Although the specifics vary, they include tests for microbial enumeration, such as total aerobic microbial count (TAMC) and total yeast/mold count (TYMC), as well as specific detection of Shiga toxin-producing E. coli, Salmonella spp, certain aspergilli, coliform counts, and mycotoxins.
TAMC is a well-established microbiology test that estimates the number of viable aerobic bacteria in a sample. The sample is serially diluted and applied to solid growth media, and the number of colonies that have grown are counted following incubation. TYMC is a similar test, except this counts the number of viable yeasts or mold spores within a sample. Both TAMC and TYMC are measured by colony-forming units (CFU) - the number of colonies that have formed, generally from a certain mass or volume of sample. Bacterial identification is performed by culturing on a variety of selective growth media, formulated to sustain the growth of only certain microbes. A number of commercially available kits and automated counting systems have become available to facilitate the acquisition of TAMC, TYMC, and bacterial identification. Finally, mycotoxins are secondary fungal metabolites that can elicit illness or even death in humans. Mycotoxin quantification can be achieved using analytical tools, such as with chromatographic-mass spectrometric (GC-MS) methods or immunological methods.
Harnessing molecular biology: Microbial testing in cannabis
Although relatively straightforward and inexpensive, TAMC, TYMC, selective growth media, and mycotoxin quantification are not very sensitive (i.e., they do not identify all contaminated cannabis) nor are they very specific (i.e., they do not always correctly identify the contaminating microorganism). “Some total count tests are good preliminary measures of quality control but they are not good determinants of whether a product is safe,” explained Kyle Boyar, who works as a Field Applications Scientist at Medicinal Genomics, a biotechnology company in Woburn, Massachusetts. “However, more states are requiring identification of specific pathogens, and this is the direction cannabis testing is heading in.”
Medicinal Genomics develops molecular-based detection methods, which should be more sensitive and specific than conventional methods. “We have created a quantitative real-time PCR (qPCR) protocol to identify and quantify microbes in cannabis. The method directly tests cannabis samples, so it does not require culturing, which saves time. This also eliminates bias from the culturing step, which could favor the growth of certain microbes compared to others, leading to a skewed analysis of the microbes present in a sample” Boyar elaborated. “qPCR is also a targeted and highly specific molecular method. On the other hand, selective growth media, despite their name, are simply not that selective. We’ve seen microorganisms such a Pseudomonas grow and trigger failures on yeast/mold media, although it isn’t a yeast or mold. Conversely, it won’t grow on bacterial media, despite being a bacterium. So, we’re finding that the selective growth media platforms don’t pick up all the threats that are there.” The advent of molecular methods, as in medicine, is revolutionizing microbial testing, bringing faster and more accurate assessment of cannabis quality.
Harnessing molecular biology: Cannabis microbiome and plant fitness
It has been known for nearly a hundred years that the native microbiome affects plant fitness. “It is well-established that microbes colonize plant roots, and can have tremendous effects on plant physiology, such as increased growth, resistance to pathogens, or metabolic activity,” discussed Max Winston, Ph.D., the lead author on a recent study of the cannabis microbiome. However, this has been investigated much less for cannabis, although it stands to be of tremendous commercial value. Balanced microbiomes and improved plant fitness translate to greater crop yields, and as the demand for cannabis increases, so too must agricultural production.
“One of the difficult parts of translating this relevant and important fact to improve agricultural practices at scale is the vast diversity of microbes that live in the soil under different environmental conditions,” Dr Winston explained. “Understanding who the players are in these different conditions and how they colonize the plant was the focus of this paper, and really the first step towards identifying robust microbe-plant interactions that could have a real, positive impact on cannabis agriculture.” Researchers are investigating how to leverage the plant microbiome to boost plant productivity, function as endogenous fertilizers, and as biocontrol agents, a greater understanding of which will lead to better crop management practices. “We’re still in the nascent stages,” concluded Dr Winston. “However, we’ve taken the initial steps forward.”
The microbiologist’s arsenal of tools contains more methods that may eventually prove useful. For example, matrix-assisted laser desorption ionization time of flight (MALDI-TOF) has revolutionized clinical microbial testing in patient biosamples. Although not yet applied for testing cannabis, this untargeted method could potentially detect unsuspected microbes in samples, but unfortunately like conventional methods, requires a culturing phase. Smaller, portable DNA sequencers have dramatically lowered sequencing costs and may also make microbial identification more accessible. Although commercial microbial cannabis is increasingly available, there are very few peer-reviewed studies. As improved methods of microbial detection develop, or as a deeper understanding of the microbiome on plant fitness progresses, controlled studies will be needed to assess novel technologies.