Tag Archives: Enterobacteriaceae

Beyond Compliance: Understanding and Combating Contamination

By Jill Ellsworth MS, RDN, Tess Eidem, Ph.D.
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As an emerging field in cannabis, contaminant testing remains a gray area for many businesses. The vast differences in state-by-state regulations, along with the frequent changes of previously established rules make testing a difficult, time-consuming process. But at its core, the science and reasoning behind why we test cannabis is very clear – consumer safety and quality assurance are key factors in any legal, consumer market. The implications of federal legalization make cannabis testing even more important to the future of the cannabis supply chain. Understanding the types of contaminants, their sources and how to prevent them is essential to avoiding failures, recalls and risking consumer safety.

When talking about cannabis contaminant testing there are four groups of contaminants: pesticides, heavy metals, foreign materials and microbes. The microbes found on cannabis include plant pathogens, post-harvest spoiling microbes, allergens, toxin release and human pathogens. While all of these can be lurking on the surface of cannabis, the specific types that are tested for in each state vary widely. Understanding the full scope of contaminants and looking beyond state-specific compliance requirements, cultivators will be able to prevent these detrimental risks and prepare their business for the future.

Environmental controls are essential to monitor and regulate temperature and humidity

Beyond just the health of the plant, both medical patients and adult use consumers can be adversely affected by microbial contaminants. To immunocompromised patients, Aspergillus can be life-threatening and both adult use and medical consumers are susceptible to allergic reactions to moldy flower. But Aspergillus is just one of the many contaminants that are invisible to the human eye and can live on the plant’s surface. Several states have intensive testing regulations when it comes to the full breadth of possible harmful contaminants. Nevada, for example, has strict microbial testing requirements and, in addition to Aspergillus, the state tests for Salmonella, STEC, Enterobacteriaceae, coliforms and total yeast and mold. Over 15 states test for total yeast and mold and the thresholds vary from allowing less than 100,000 colony forming units to allowing less than 1,000 colony forming units. These microbes are not uncommon appearances on cannabis – in fact, they are ever-present – so understanding them as a whole, beyond regulatory standards is a certain way to future-proof a business. With such vast differences in accepted levels of contamination per state, the best preparation for the future and regulations coming down the pipeline is understanding contamination, addressing it at its source and harvesting disease-free cannabis.

The risk of contamination is present at every stage of the cultivation process and encompasses agricultural practices, manufacturing processes and their intersection. From cultivation to manufacturing, there are factors that can introduce contamination throughout the supply chain. A quality control infrastructure should be employed in a facility and checkpoints within the process to ensure aseptic operations.

Microbial monitoring methods can include frequent/consistent testing

Cultivators should test their raw materials, including growing substrates and nutrient water to ensure it is free of microbial contamination. Air quality plays an important role in the cultivation and post-harvest processes, especially with mold contamination. Environmental controls are essential to monitor and regulate temperature and humidity and ensure unwanted microbes cannot thrive and decrease the value of the product or make it unsafe for worker handling or consumers. Developing SOPs to validate contact surfaces are clean, using proper PPE and optimizing worker flow can all help to prevent cross-contamination and are part of larger quality assurance measures to prevent microbes from spreading across cultivars and harvests.

Methods of microbial examination include air quality surveillance, ATP surface and water monitoring, raw materials testing, and species identification. Keeping control of the environment that product is coming into contact with and employing best practices throughout will minimize the amount of contamination that is present before testing. The solution to avoiding worst case scenarios following an aseptic, quality controlled process is utilizing a safe, post-harvest kill-step, much like the methods used in the food and beverage industries with the oversight of the FDA.

The goal of the grower should be to grow clean and stay clean throughout the shelf life of the product. In order to do this, it is essential to understand the critical control points within the cultivation and post-harvest processes and implement proper kill-steps. However, if a product is heavily bio-burdened, there are methods to recover contaminated product including decontamination, remediation and destroying the product. These measures come with their own strengths and weaknesses and cannot replace the quality assurance programs developed by the manufacturer.

Rapid Pathogen Detection for the 21st Century: A Look at PathogenDx

By Aaron G. Biros
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In 1887, Julius Petri invented a couple of glass dishes, designed to grow bacteria in a reproducible, consistent environment. The Petri dish, as it came to be known, birthed the scientific practice of agar cultures, allowing scientists to study bacteria and viruses. The field of microbiology was able to flourish with this handy new tool. The Petri dish, along with advancements in our understanding of microbiology, later developed into the modern field of microbial testing, allowing scientists to understand and measure microbial colonies to detect harmful pathogens in our food and water, like E. coli and Salmonella, for example.

The global food supply chain moves much faster today than it did in the late 19th century. According to Milan Patel, CEO of PathogenDx, this calls for something a little quicker. “Traditional microbial testing is tedious and lengthy,” says Patel. “We need 21st century pathogen detection solutions.”

Milan Patel first joined the parent company of PathogenDx back in 2012, when they were more focused on clinical diagnostics. “The company was predominantly built on grant funding [a $12 million grant from the National Institute of Health] and focused on a niche market that was very specialized and small in terms of market size and opportunity,” says Patel. “I realized that the technology had a much greater opportunity in a larger market.”

Milan Patel, CEO of PathogenDx
Photo: Michael Chansley

He thought that other markets could benefit from that technology greatly, so the parent company licensed the technology and that is how PathogenDx was formed. Him and his team wanted to bring the product to market without having to obtain FDA regulatory approval, so they looked to the cannabis market. “What we realized was we were solving a ‘massive’ bottleneck issue where the microbial test was the ‘longest test’ out of all the tests required in that industry, taking 3-6 days,” says Patel. “We ultimately realized that this challenge was endemic in every market – food, agriculture, water, etc. – and that the world was using a 140-year-old solution in the form of petri dish testing for microbial organisms to address challenges of industries and markets demanding faster turnaround of results, better accuracy, and lower cost- and that is the technology PathogenDx has invented and developed.”

While originally a spinoff technology designed for clinical diagnostics, they deployed the technology in cannabis testing labs early on. The purpose was to simplify the process of testing in an easy approach, with an ultra-low cost and higher throughput. Their technology delivers microbial results in less than 6 hours compared to 24-36 hours for next best option.

The PathogenDx Microarray

Out of all the tests performed in a licensed cannabis testing laboratory, microbial tests are the longest, sometimes taking up to a few days. “Other tests in the laboratory can usually be done in 2-4 hours, so growers would never get their microbial testing results on time,” says Patel. “We developed this technology that gets results in 6 hours. The FDA has never seen something like this. It is a very disruptive technology.”

When it comes to microbial contamination, timing is everything. “By the time Petri dish results are in, the supply chain is already in motion and products are moving downstream to distributors and retailers,” Patel says. “With a 6-hour turnaround time, we can identify where exactly in the supply chain contaminant is occurring and spreading.”

The technology is easy to use for a lab technician, which allows for a standard process on one platform that is accurate, consistent and reproduceable. The technology can deliver results with essentially just 12 steps:

  1. Take 1 gram of cannabis flower or non-flower sample. Or take environmental swab
  2. Drop sample in solution. Swab should already be in solution
  3. Vortex
  4. Transfer 1ml of solution into 1.5ml tube

    A look at how the sample is added to the microarray
  5. Conduct two 3-minute centrifugation steps to separate leaf material, free-floating DNA and create a small pellet with live cells
  6. Conduct cell lysis by adding digestion buffer to sample on heat blocks for 1 hour
  7. Conduct Loci enhancement PCR of sample for 1 hour
  8. Conduct Labelling PCR which essentially attaches a fluorescent tag on the analyte DNA for 1 hour
  9. Pipette into the Multiplex microarray well where hybridization of sample to probes for 30 minutes
  10. Conduct wash cycle for 15 minutes
  11. Dry and image the slide in imager
  12. The imager will create a TIFF file where software will analyze and deliver results and a report

Their DetectX product can test for a number of pathogens in parallel in the same sample at the same time down to 1 colony forming unit (CFU) per gram. For bacteria, the bacterial kit can detect E. coli, E. coli/Shigella spp., Salmonella enterica, Listeria and Staph aureus, Stec 1 and Stec 2 E.coli. For yeast and mold, the fungal kit can test for Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger and Aspergillus terreus.

Their QuantX is the world’s first and only multiplex quantification microarray product that can quantify the microbial contamination load for key organisms such as total aerobic bacteria, total yeast & mold, bile tolerant gram negative, total coliform and total Enterobacteriaceae over a dynamic range from 100 CFU/mL up to 1,000,000 CFU/mL.

Not all of the PathogenDx technology is designed for just microbial testing of cannabis or food products. Their EnviroX technology is designed to help growers, processors or producers across any industry identify areas of microbial contamination, being used as a tool for quality assurance and hazard analysis. They conducted industry-wide surveys of the pathogens that are creating problems for cultivators and came up with a list of more than 50 bacterial and fungal pathogens that the EnviroX assay can test for to help growers identify contamination hotspots in their facilities.

Using the EnviroX assay, growers can swab surfaces like vents, fans, racks, workbenches and other potential areas of contamination where plants come in contact. This helps growers identify potential areas of contamination and remediate those locations. Patel says the tool could help growers employ more efficient standard operating procedures with sanitation and sterilization, reducing the facility’s incidence of pathogens winding up on crops, as well as reduction in use of pesticides and fungicides on the product.

Deploying this technology in the cannabis industry allowed Milan Patel and the PathogenDx team to bring something new to the world of microbial testing. Their products are now in more than 90 laboratories throughout the country. The success of this technology provides another shining example of how the cannabis market produces innovative and disruptive ideas that have a major impact on the world, far beyond cannabis itself.