Bile-tolerant gram-negative bacteria can survive in the human stomach (bile-tolerant) and they have a protective cell wall that causes them to turn red when subjected to the gram staining process (gram-negative). This combination of characteristics makes BTGN bacteria potentially harmful to humans.
Gram-negative bacteria thrive in virtually all environments that support life. They are an important medical challenge because the outer membrane that protects them from gram staining also protects them from many antibiotics, detergents, and anti-microbial enzymes produced by the immune system. In fact, when immune cells lyse gram-negative bacteria cells it can cause a toxic reaction that can include an increased respiratory rate, and low blood pressure — a life-threatening condition known as septic shock.
According to the United States Pharmacopeia’s website,
“There is no strict definition of this group of micro-organisms. They are defined operationally as those micro-organisms that show growth in the stated conditions on Violet Red Bile Glucose Agar medium. They include, Gram negative bacteria that grow in the presence of bile salts, non-lactose fermenting but able to utilize glucose, e.g., some Bile Tolerant Gram Negative Bacteria includes members of the family Enterobacteriaceae, Pseudomonads and Aeromonas.”
Proficiency testing suppliers don’t offer much guidance as to defining which organisms fit the BTGN description. For example, NSI Lab Solutions uses the same two organisms (E. coli and Klebsiella pneumoniae) for its proficiency testing samples for aerobic bacteria, coliform, Enterobacteriaceae, and BTGN.
It is also hard to find culture-based methods that are specifically designed for BTGN. Most manufacturers recommend labs use their Enterobacteriaceae media.
Because there is no strict definition for BTGN, and the operational definition is based on growth in a specific medium, developing a DNA-based test is difficult.
Our research led us to design our PathoSEEK Bile-Tolerant Gram-Negative assay to include enterobacteriaceae species as well as Pseudomonas aeruginosa.
The American Herbal Pharmacopeia and the United States Pharmacopeia both list BTGN in their monographs. There are several regulatory agencies that use those documents to set their testing requirements, including:
Peer-reviewed studies have shown that qPCR testing methods, such as the PathoSEEK Microbial Safety Testing Platform, are able to more accurately detect and quantify bacteria species present on a cannabis sample than culture-based methods. The PathoSEEK Bile-Tolerant Gram-Negative test looks for a specific DNA sequence that exists in all BTGN bacteria. Because of this, the PathoSEEK Bile-Tolerant Gram-Negative test can detect all BTGN species, regardless of whether they grow in culture. Culture-based tests can only detect the species that grow in a given medium and timeframe. Conversely, qPCR can more effectively exclude off-target microbial species, such as yeasts and molds, which have been shown to grow in bacteria culture medium.
Labs can use our PathoSEEK Microbial Safety Testing Platform to test for Bile-Tolerant Gram-Negative bacteria species in a variety of cannabis matrices. In fact, the PathoSEEK® Platform is the only method designed for and validated on cannabis flower, extracts, and a variety of infused products.
Important note: The P. aeruginosa assay has been shown (via Sanger sequencing of the amplicons) to amplify Pseudomonas rhizosphaerae, an off-target species. P. rhizosphaerae has been found on some hemp plants in Canada. We have designed our Pseudomonas assay to target closely-related P. aeruginosa strains that are pathogenic to humans and listed in the Cystic Fibrosis literature. The exclusion criteria for these primers did not include microbes that are not available in tissue banks, such as P. rhizosphaerae. The health risks of beneficial microbes still need to be scrutinized for agricultural products that are inhaled. To date, we are not aware of any clinical reports of harm from P. rhizosphaerae.
P. rhizosphaerae is not commercially available from ATCC (American Type Culture Collection) for purchase and identification, thus it is difficult to ascertain if it forms colonies and to what extent this species is inflating the CFU/g estimates from the P. aeruginosa assay. We are actively investigating primers that exclude this beneficial microbe and are open to collaboration with laboratories that have specific experience culturing and quantifying this microbe.
|PathoSEEK® Bile-Tolerant Gram-Negative Detection Assay v2
|PathoSEEK® BTGN / Coliform / Entero Control