Cannabis Culture: an Overview of Microbe Testing in the Cannabis Industry

Although cannabis is becoming increasingly legal at the state level in the United States, it remains illegal at the federal level. This means that there are no universal federal regulations and that each state has its own individual testing standards, usually carried out by independent labs.

The microbiology department is an important part of any testing lab. Here, a range of potentially dangerous microbes are tested for, including bacteria such as Escherichia coli, and fungi such as Aspergillus. There are currently no standardized methods for microbiology testing in the US, and products have been recalled in multiple states including Oregon and Arizona due to fungal and bacterial contamination.

Lack of standardized microbiology testing can have dire consequences, as patients who seek relief using medical cannabis are often immunocompromised, putting them at much greater risk of serious illness or even death from contaminated cannabis products. Nationwide testing standardization would allow collaborations between industry experts across state lines, reduce competition between testing labs, and increase product quality and trust in the industry throughout the US.

Types of microbial contaminants

Cannabis plants and products can play host to a wide variety of bacterial and fungal microbes that are harmful to humans. There are two main ways in which microorganisms can cause harm to humans. The first is through active infection, where rapid replication of the microbe results in damage to cells, toxicity, and dangerous over-activation of the immune system. The second is due to toxins produced by some species of microorganisms. These toxins can cause illness even if the bacterial or fungal cell that produced the toxin is no longer viable.

Molds and fungal contamination pose the greatest risk to human health in cannabis product. Two of the most common fungal species found in cannabis – Penicillium and Aspergillus – are known to produce aflatoxins, while another common fungal species – Fusarium – produces mycotoxins known as fumonisins. These toxins are considered carcinogens, and can have been linked to toxicity and cell death in the liver, kidneys, and neurons. The effects of these toxins are affected by various factors, such as the dose and the method of exposure (ingestion or inhalation), as well as the health of the infected person. Most mycotoxins are chemically stable and can survive processing for products such as edibles or concentrates. There are four species of Aspergillus known to cause illness in humans: A. fumigatus, A. flavus, A. terreus and A. niger. Although most testing regulations require labs to test for these four species, several states have chosen not to mandate testing for Aspergillus specifically, testing instead for general indicators of microbial growth. This is concerning, as Aspergillus contaminated cannabis can cause allergic reactions in healthy people, and has been directly linked to pulmonary aspergillosis, a severe illness in immunocompromised patients.

Most of the molds and fungi found in cannabis are naturally occurring. However, a Fusarium species, F. oxysporum was intentionally spread across the US in the 1970s and 1980s as a biological control to limit illegal cannabis farms by causing cannabis wilt. Unfortunately, this fungi is now a major pest in legal cannabis farming operations, as well as potentially causing negative health effects in humans.

Bacterial contamination of cannabis products is less common than fungal, but several studies have found pathogenic species of bacteria lurking among more benign species in cannabis cultivars. Escherichia coli, Listeria monocytogenes, Salmonella, and Clostridium are potential human pathogens that are commonly associated with cannabis. E. coli¸ particularly the Shiga toxin-producing strains, can cause a variety of illnesses, including gastrointestinal symptoms like vomiting and diarrhea, and meningitis. L. monocytogenes and Salmonella species both cause foodborne illnesses with gastrointestinal symptoms, that can be particularly dangerous in people with weakened immune systems, while Clostridium botulinum produces the potentially fatal botulinum toxin.

As yet, there is little evidence to suggest that bacterial contamination of cannabis has directly led to infections, even in immunocompromised people. However, the presence of these bacteria demonstrates the potential for cannabis-acquired infections, particularly for bacteria like L. monocytogenes that can survive processing and freezing and still be viable in edible and concentrated products.

The challenges of current testing methods

When it is mandated, microbial testing of cannabis and cannabis products can be time-consuming and require trained analysts. Traditionally, total organism counts like total yeast and mold counts are performed using culture-based techniques. These tests involve taking samples of the cultivar, spreading them onto culture plates, then counting the total number of a type of organism. Reading plates requires highly skilled analysts, who can encounter issues with plant material becoming attached to a culture plate, causing a color change, and affecting the readout. In general, the cut-off for failure of this test is 10,000 colony-forming units (CFU) per gram or greater. However, as this kind of test doesn’t differentiate between harmless and pathogenic organisms, passing the test doesn’t necessarily mean that a cannabis sample is free from harmful microbes.

Additionally, Aspergillus, the most harmful potential contaminant, has been shown to grow poorly in culture-based methods, meaning that this pathogen is consistently under-reported in microbial testing. Water activity (the amount of water present in a sample) is also measured in several states as a proxy for microbial contamination, as if cannabis buds are not dried to water activity levels below 0.70 Aw, they can support microbial growth.

Time is also a factor in microbial testing. Traditional culture plating methods can take up to seven days to produce a result. Consequently, more and more labs are moving towards molecular methods such as quantitative polymerase chain reaction (qPCR) methods, which are not only much faster but can also specifically detect harmful microbes at very low levels. However, this method has come under fire due to concerns over the potential accuracy of qPCR for the quantification of yeasts and molds. Plating methods can be used as confirmation for microbes of concern like Aspergillus, but as discussed, these come with their own issues.

Could the future of microbial testing for cannabis lie in mass spectrometry (MS)? In the clinical world, MS is regularly used to assess microbial presence in human samples, by looking for protein fragmentation patterns that are characteristic of particular organisms. While MS is highly accurate and produces results rapidly, the instruments tend to be extremely expensive and require highly specialized training to use. At present then, MS may be out of reach of most testing laboratories.

The future of microbiology testing

As the legal market for cannabis spreads, microbial testing is going to play an increasingly important part in maintaining a safe, trustworthy market. Many states now require labs to be ISO 17025 certified, demonstrating that they can operate competently and generate valid results. Further regulations already present in drug and food industries such as good manufacturing practices (GMP) (which comprise robust operating procedures, laboratory standards, and quality management systems), may be increasingly relevant to the cannabis industry as federal legalization looms. Ultimately, poorly managed microbial testing protocols that result in false negatives can be just as dangerous as no testing at all, stressing the case for nationwide testing standardization to improve quality, collaboration, and trust in the industry.