Why is Chloramphenicol problematic for microbial testing on cannabis and hemp?

Chloramphenicol is an antibiotic used in DRBC plates to prevent bacterial growth. Unfortunately, chloramphenicol also inhibits the growth of dangerous fungi (Aspergillus, Penicillium, and Fusarium)

Chloramphenicol is an antibiotic. This can be helpful to eliminate the bacteria which can form obscuring colonies in 24 hours while yeast and molds can take 5-10 days to grow.

Unfortunately, chloramphenicol also inhibits the growth of certain fungi, particularly the most dangerous fungi on cannabis: Aspergillus, Penicillium, and Fusarium. Below are peer-reviewed studies that demonstrate this effect:

• Activity of antibiotics against Fusarium and Aspergillus

• Evaluation of Amphotericin B and Chloramphenicol as Alternative Drugs for Treatment of Chytridiomycosis and Their Impacts on Innate Skin Defenses

• In vitro anti-yeast activity of chloramphenicol: A preliminary report

• Chloramphenicol inhibition of Pythium ultimum and Rhodotorula glutinis

Comparing DRBC to PDA and qPCR

To confirm the peer-reviewed literature, we acquired organisms from ATCC and plated them on
Potato Dextrose Agar (PDA) and DRBC.

ATCC glycerol stocks performed better than Microbiologic pellets.
Microbiologic desiccated pellets required more time to grow on DRBC (12 days)

As expected the Aspergillus species either did not grow or took 12 days to grow on DRBC. PDA was able to detect all the Aspergillus species.

Aspergillus and Penicillium are some of the most commonly found pathogens on cannabis, making the results of these experiments relevant and concerning for the industry. Below are peer-reviewed studies that confirm the presence of these species on cannabis. 

• Pathogens and Molds Affecting Production and Quality of Cannabis sativa L.

• Flower and foliage-infecting pathogens of marijuana (Cannabis sativa L.) plants

• Cannabis microbiome sequencing reveals several mycotoxic fungi native to dispensary grade Cannabis flowers

• Metagenomic analysis of medicinal Cannabis samples; pathogenic bacteria, toxigenic fungi, and beneficial microbes grow in culture-based yeast and mold tests

• A microbiome assessment of medical marijuana

AOAC Sponsored Independent Study

We shared this data with the AOAC and they commissioned an independent review. This was performed by a 3rd party cannabis testing laboratory under AOAC's guidance. The study looked at three separate plating mediums.

In the results from this study below, you can see a LOG scale more colonies on PDA with Chloramphenicol (CAMP) than with DRBC, and a LOG scale more organisms on PDA without any selection. The difference in CFU counts may be due to some bacterial growth, but some is also attributable to yeasts and molds that are sensitive to Chloramphenicol.

The fact that the choice of plating method will produce such wildly different results is problematic for plating methods being the "gold standard". It is also difficult for another method, such a qPCR, to show equivalence.  Please see the following articles for more about this debate:

• After a Century as the "Gold Standard", It's Time for Plating to be Retired
• Benchmarking qPCR to Culture: PDA vs. DRBC
• 5 Reasons Why Culture Plating Should NOT Be Used for Microbial Safety Testing on Cannabis

Conclusions

These data demonstrate that using DRBC as a gold standard for benchmarking other TYM detection technologies is flawed. The failure of DRBC to detect the most dangerous human pathogenic organisms found on cannabis was noted with organisms ordered directly from ATCC. This problem is further exacerbated in real-world cannabis samples as these organisms are usually endophytes which live inside the plant. It is difficult to extract Aspergillus from within the cannabis plant and still maintain its viability. However, in this case, even if the Aspergillus were viable, it wouldn’t grow on DRBC.

The use of chloramphenicol selection in cannabis microbial testing presents a clinical risk to cannabis patients and patient safety.