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

Abstract

Background: The presence of bacteria and fungi in medicinal or recreational Cannabis poses a potential threat to consumers if those microbes include pathogenic or toxigenic species. This study evaluated two widely used culture-based platforms for total yeast and mold (TYM) testing marketed by 3M Corporation and Biomérieux, in comparison with a quantitative PCR (qPCR) approach marketed by Medicinal Genomics Corporation. Methods: A set of 15 medicinal Cannabis samples were analyzed using 3M and Biomérieux culture-based platforms and by qPCR to quantify microbial DNA. All samples were then subjected to next-generation sequencing and metagenomics analysis to enumerate the bacteria and fungi present before and after growth on culture-based media. Results: Several pathogenic or toxigenic bacterial and fungal species were identified in proportions of >5% of classified reads on the samples, including Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Ralstonia pickettii, Salmonella enterica, Stenotrophomonas maltophilia, Aspergillus ostianus, Aspergillus sydowii, Penicillium citrinum and Penicillium steckii. Samples subjected to culture showed substantial shifts in the number and diversity of species present, including the failure of Aspergillus species to grow well on either platform. Substantial growth of Clostridium botulinum and other bacteria were frequently observed on one or both of the culture-based TYM platforms. The presence of plant growth promoting (beneficial) fungal species further influenced the differential growth of species in the microbiome of each sample. Conclusions: These findings have important implications for the Cannabis and food safety testing industries.

Keywords: 3M-Petrifilm; Biomérieux-TEMPO; Cannabis; Illumina; PathogINDICAtor-qPCR; metagenomics; microbiome; safety.

Figure 1.. Genomic profiles of before and after culturing.

Comparison of classified read percentages for bacterial 16S DNA on samples 2 and 14, before and after culturing on 3M and BMX media. The results represent all species observed down to 1% of classified reads. Large shifts in species prevalence are seen after growth on the two culture-based platforms.

Figure 2.

A) TYM platform discordance before and after growth. Results from sample 4 showing the percentage of reads classified into fungal genera based on sequencing of TYM ITS2 amplicons directly from the plant (Before), or after growth on the 3M or BMX platforms. The lower part of the figure shows the colonies observed on 3M media (left) and appearance of the BMX YM card (right) after growth. B) Poor growth of Aspergillus species. In 12/15 cases where Aspergillus species are detected by ITS2 sequencing, they do not grow on 3M or BMX media (results from sample 6). The lower part of the figure shows the colonies observed on 3M media (left) and appearance of the BMX YM card (right) after growth. C) Trichoderma antagonism. Penicillium species are present in material extracted directly from the plant in sample 16, but are displaced by Trichoderma after growth on 3M or BMX media.

Figure 3.. The following 11 species were grown at RT.

Candida catenulata: ATCC 10565, Candida sphaerica: ATCC 8565, Candida krusei: ATCC 28870, Candida albicans: ATCC 10231, Candida glabralta: ATCC 15545, Yarrowia lipolytica: ATCC 18944, Rhodotorula mucilaginosa: ATCC 4557, Debaryomyces hanseii: ATCC 10623, Trichothecium Roseum: ATCC 90473, Aspergillus japonicus: ATCC 16873, Aspergillus flavus: ATCC 16870. Aspergillus demonstrates log scales lower growth at RT than most other yeast. “Expected” is the inferred CFU count from the Cq measurement using the formula CFU/g = 10 ^{[(42.185 – Cq Value)/3.6916]}.