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

Samples, culture-based assays and DNA purification

Cannabis samples were derived from seven recently-established indoor growth facilities in Massachusetts, Maine and Rhode Island. Samples were prepared and placed into culture on 3M Petrifilm^TM Rapid Yeast and Mold Count Plates (40–72 h) and Biomérieux TEMPO^® YM cards (70–76 h) at 25 ± 1.0°C, according to the manufacturer’s instructions. All samples but two were also analyzed using Biomérieux TEMPO^® AC cards to enumerate aerobic bacterial counts. For qPCR, Cannabis samples (250 ± 30 mg) were placed in Whirl-Pak^® bags and massaged in 3.55 ml Trypticase Soy Broth (TSB; American Bioanalytical) for 1 minute. DNA was then extracted using SenSATIVAx reagents (Medicinal Genomics part #420001), as described previously¹⁵ and eluted with 50 μL ddH20. DNA was similarly extracted after growth on the two culture based platforms as described above. Colonies grown on 3M plates were scraped off into 285 μL of ddH2O, and 190 μL of those samples, or samples grown in TEMPO cartridges (liquid culture), were extracted using SenSATIVAx as above. Fungal species stocks from the American Type Culture Collection (ATCC) were reconstituted and incubated at the appropriate temperature, as recommended by ATCC product documentation. Cultures of ATCC strains were then grown in 5ml TSB for 5 days at room temperature and checked visually for turbidity. Serial dilutions were plated on 3M PetrifilmTM Rapid Yeast and Mold Count Plates, incubated at room temperature, and counted after 3–5 days. Colonies were scraped off the plates and DNA was then extracted as described above.

The cannabis samples used for this study were collected within the regulatory framework for the individual State Medical Marijuana programs by ProVerde Laboratories; an accredited ISO/IEC 17025:2005 cannabis safety testing laboratory. The purified DNA, which is not a schedule I substance, was tested to verify that the hydrophilic DNA purification does not contain hydrophobic cannabinoids and is therefore in accordance with the Hemp Associates vs DEA regarding hemp fiber shipment within the United States. Since all activities that involved handling of material containing cannabinoids was within the individual state requirements, no federal (FDA or DEA) registration or permission was required.

Total yeast and mold and total aerobic bacteria qPCR assays

DNA samples extracted directly from Cannabis samples, or after growth on the two culture-based platforms, were subjected to qPCR analysis. Quantitative PCR was performed using a commercially available TYM assay (TYM-PathogINDICAtor, Medicinal Genomics, Woburn MA), or TAC assay (TAC-PathogINDICAtor, Medicinal Genomics, Woburn, MA) in a Bio-Rad CFX 96 Touch qPCR instrument, according to the manufacturer’s instructions.

Primers used for PCR and sequencing

PCR was performed using 5μL of DNA (3ng/μL) 12.5μL 2X LongAmp (NEB) with 1.25 μL of each 10 μM MGC-ITS3F and MGC-ITS3R primer or MGC-TAC_F and MGC-TAC_R primer (MGC-ITS3F: TACACGACGTTGTAAAACGACGCATCGATGAAGAACGCAGC), (MGC-ITS3R: AGGATAACAATTTCACACAGGATTTGAGCTCTTGCCGCTTCA), (MGC-TAC_F: TACACGACGTTGTAAAACGATCCTACGGGAGGCAGCAGT) and (MGC-TAC_R: AGGATAACAATTTCACACAGGGGACTACCAGGGTATCTAATCCTGTT) with 10μL ddH20 for a 25 μL total reaction. An initial 95°C 5-minute denaturation step was performed followed by 25 cycles of 95°C for 15s and 65°C for 90s. Samples were purified with 75 μL SenSATIVAx, washed twice with 100 μL 70% EtOH and bench dried for 5 minutes at room temperature. Samples were eluted in 25 μL ddH20.

Library preparation using Nextera

The 16S amplicon targeted by the MGC primers (spanning the V3 and V4 hypervariable regions) is approximately 460 bp in size, and ITS2 amplicons from different fungal species are known to vary in size from ~0.5–1 kilobases. To enable representative coverage across the entire amplicon for sequencing and analysis of each sample, we enzymatically fragmented the amplicons to ~300 bp average size. Fragmentation was accomplished and DNA libraries were constructed using the commercially available Nextera Library Prep Kit (Illumina). 6ng of purified PCR product, 5 μL of TD buffer, 0.1 μL of TD enzyme and 3.9 μL ddH20 was combined for a total of 10 μL. The reaction was incubated at 55°C for 30 minutes followed by a 10°C hold. The reaction plate was immediately removed from the thermal cycler and purified with 15 μL of Agencourt Ampure XP (Beckman Coulter), washed twice with 200 μL 70% EtOH and bench dried for 10 minutes at room temperature. Samples were eluted in 25μL 10mM Tris-HCl.

Library PCR and Illumina sequencing

17.5 μL of 2X Q5 polymerase (NEB) was added to 10μL of purified DNA with 2.5 μL of i7 Nextera index primer, 2.5 μL L of i5 Nextera index primer, 0.5 μL of ILMN1 primer (50 μM), 0.5 μL of ILMN2 primer (50 μM), 1 μL 5-methyl-dCTP (10 μM) and 0.5 μL H₂O. After an initial 72°C for 3 minutes and 98°C for 30 s, the library was amplified for 12 cycles of 98°C for 10 s, 63°C for 30 s, 72°C for 1 minute and a 10°C hold. Use of methylated nucleotides for PCR decontamination is described previously^26,27. PCR samples were purified by mixing 52.5 μL of Agencourt Ampure XP into the PCR reaction. The samples were placed on a magnet for 15 minutes until the beads cleared and the supernatant could be removed. Beads were washed twice with 200 μL of 70% EtOH. Beads were left for 10 minute to air dry and then eluted in 25 μL of 10 mM Tris-HCl. 5 μL of each PCR product was pooled and quantified with a Qubit (Thermo) for proper dilution onto MiSeq version 2 chemistry according to the manufacturers’ instructions. 2×150 bp reads were selected to obtain maximal ITS2 sequence information.

Analysis

2×150 bp reads were de-multiplexed with Illumina software bcl2fastq v2.17.1.14. Sequences were classified at the Family, Genus and Species level by discriminative k-mer analysis using CLARK-S²⁸ with the NCBI/RefSeq bacterial database and taxonomy, or UNITE²⁹ fungal database and taxonomy. Cannabis chloroplast and mitochondrial sequences were included in the bacterial and fungal databases since they amplify with the 16S rRNA primers used, and the Nextera fragmentation process used in our lib prep may incorporate high copy number sequences even without amplification. Cannabis mitochondrial sequences generally comprised a large fraction of the classified reads (up to 97%) in DNA derived from plant material. The Cannabis reads were subtracted out to enable enumeration of the bacterial species down to 1% of classified non-Cannabis reads.

Sequences were alternatively classified by BLAST analysis of operational taxonomic units (OTUs) generated by clustering at the ≥ 97% sequence similarity level using USEARCH8³⁰. Each set of paired-end reads were merged using fastq_merge pairs³¹. We used cutadapt to trim primer and adaptor regions from both ends (http://cutadapt.readthedocs.io/en/stable/guide.html). Sequences were quality trimmed to have a maximum expected number of errors per 100 bases of less than 0.1 (Q30). OTUs with membership of at least 200 sequences were included in downstream analyses, and BLAST hits with less than 97% query coverage and 97% identity were discarded. Analyses of the USEARCH OTUs were performed in R (https://www.r-project.org). Each library was normalized by the total number of OTUs found. OTUs were associated with microbes based on the name and description provided by NCBI. R² values were calculated by adjusted linear regression in R or by embedded formulas in Excel. In order to mitigate the large effect of noise in samples with low OTU counts, specificity analysis was done after pooling the un-normalized data.

Quantitative PCR and colony counts before and after culture

Summary results from the different testing platforms evaluated in this study for 15 samples with complete data are presented in Table 1. The samples were evaluated with Medicinal Genomics’ PathogINDICAtor ITS2-based TYM-qPCR and 16S-based TAC-qPCR assays directly from extracted plant material (Before), and from recovered medium after culture on the Biomérieux Tempo instrument using YM sample cards (After BMX). Samples were also evaluated directly using the Biomerieux instrument with Tempo YM and AC cards, or on 3M Rapid Total Yeast and Mold Count Plates (3M TYM). Results in bold type and shaded boxes indicate failed tests following the limits set for Massachusetts medicinal Cannabis.

Overall, the BMX TYM platform failed the highest number at 67% (10/15); the 3M TYM platform failed 60% (9/15), and the qPCR TYM failed 20% (3/15). The failure rates for the BMX AC and qPCR TAC assays were 13% (2/15) and 7% (1/15), respectively. An additional set of TYM qPCR tests were performed after growth on the BMX platform, resulting in 12/15 failures and confirming the presence of live, culturable fungi in 80% of the samples. The 3M TYM and BMX YM systems performed similarly in terms of pass/fail, with only one discrepancy, which had a value close to the failure threshold. The TYM-qPCR assay passed seven samples that failed on at least one of the two culture-based platforms. One of those (sample 4) had an elevated quantitation cycle (Cq) value approaching the failure threshold; the rest (samples 11–16) gave high Cq values, indicating very low fungal DNA levels (Table 1).

Metagenomic sequencing and analysis results

The sequencing data generated for this project are available at the NCBI short read archive; see Dataset 1 (Table I) for accession numbers and URLs. A summary of the CLARK-S classification results for each of the 15 samples, directly from plant material (before), or after culture on the 3M or BMX platforms, is provided in Dataset 1 (Table II: CLARK-S output for bacterial species analysis with read counts, Table III: matrix file with % classified reads at the species level for all TAC samples, Table IV: matrix file with % classified reads down to 1% at the species level from selected TAC samples used to generate charts, Table V: CLARK-S output for TYM analysis with read counts, Table VI: matrix file with % classified reads for all TYM samples, Table VII: matrix file with % classified reads down to 1% from selected TYM samples used to generate charts, Table VIII: matrix file with % classified reads down to 1% at the genus level from the same selected TAC samples as in Table IV).

While the sequencing assay provides approximate intra-sample quantitation, it does not support inter-sample quantitation³². The sequencing procedure utilizes two PCR steps instead of the single PCR step used in qPCR (and does not utilize an internal probe for signal generation). Sample quantities are normalized prior to the Nextera reaction to ensure consistent shearing. These procedures are optimized to yield 1 million reads or more per sample for high sensitivity, but the read numbers are not proportional to microbial counts in the starting samples. Instead, the classified read counts and percentages simply indicate the genera or species present at detectable levels and their approximate proportions (with the caveat that the target amplicons from some species may amplify with lower efficiency owing to primer mismatches or extremes of G+C content). The qPCR Cq measured directly from extracted plant material provides the best inter-sample comparative metric. BLAST results from clustered OTUs were used to confirm prevalent species assignments on a case-by-case basis, but the results are not presented here owing to the very large number of OTUs generated by the USEARCH software (>12,000 across the full sample set).

Sequencing reproducibility: 14 frozen samples were amplified with ITS2 primers and sequenced 30–60 days apart; 13 of the comparative R square values for classified fungal species were greater than 0.999 and the remaining one was 0.966. Similarly, 20 frozen samples were amplified with 16S primers and sequenced 30–60 days apart; 18 of the comparative R square values for classified bacterial species were greater than 0.999 and the remaining two averaged was 0.998. These data imply highly reproducible genomic surveys of the amplified DNA present. No Template Controls (NTC) were also tested, producing very high Cq readings (>35) and very few classified reads (251 with TAC primers and 61 with TYM primers) controlling for the possibility of labware contamination contributing to the observed signals.

Specificity: To verify the specificity of the analysis for accurate discrimination between bacterial and fungal genera, we ran CLARK-S against the bacterial and fungal databases separately at the genus level using either 16S or ITS2 reads. There were 13,913,520 16S reads classified as bacterial, 2,293 16S reads classified as fungal, 6,220,745 ITS2 reads classified as fungal, and 241,351 ITS2 reads classified as bacterial (Dataset 1, Tables V and IX–XI; Table IX: genus level CLARK-S read counts for 16S reads against the fungal database, Table X: genus level CLARK-S read counts for ITS2 reads against the bacterial database, Table XI: genus level CLARK-S read counts for 16S reads against the bacterial database). From this we calculate the specificity (true neg/(false pos + true neg) of 16S analysis as 0.963 [=ITS2 reads classified as fungal/(ITS2 reads classified as fungal+ITS2 reads classified as bacterial)] and that of the ITS2 analysis as 0.9997 [=16S reads classified as bacterial/(16S reads classified as bacterial+16S reads classified as fungal)].

Pairs of samples from three of the seven growers were highly similar in their combined bacterial and fungal species prevalence as indicated by high correlation coefficients (CC): CC=0.92 for samples 1 and 2, CC=0.94 for samples 11 and 12, and CC=0.97 for samples 6 and 14. There was also moderate correlation between samples 6, 14 and 9, a third sample from the same grower: CC=0.66 for samples 6 and 9, CC=0.64 for samples 9 and 14. These samples represent different strains from the same grow and likely share similar soil environments.

Bacterial growth on culture-based TYM platforms

Six samples (numbers 11–16) failed in the BMX TYM test, but passed the MGC qPCR TYM test with low signals (Cq >40). Five of those (numbers 11, 12, 14–16) had elevated qPCR TAC signals, suggesting that the growth of bacteria could be contributing to colony counts and failures in the culture-based TYM tests. Sequencing results for each of those samples, before and after culture in BMX medium, confirm the presence of actively growing bacteria, and reveals the bacterial genera that are primarily responsible for the TAC-qPCR signals: Bacillus and Clostridium in sample 11 (~73% of classified reads, collectively), and Bacillus, Clostridium and Ralstonia in samples 14–16 (78–83% of classified reads, collectively in the each of the three samples). A different set of genera were observed after culture on 3M media: Ralstonia and Leifsonia in sample 11 (86% of classified reads, collectively), and Xanthomonas, Ralstonia and Streptococcus in samples 14–16 (61–75%, collectively in each sample).

All of the samples underwent a change in species composition after growth on the BMX or 3M yeast and mold platforms. Three of the 15 samples (numbers 5, 15 and 16) produced a similar distribution of species on the BMX and 3M platforms, with correlation coefficients (CC) of 0.41–0.82. The results from the remaining 80% of the samples, however, were strikingly different on the two platforms (CC: -0.03-0.21). Representative results from two of those samples, numbers 2 and 14, are shown in Figure 1.

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.

Significant levels of Bacillus coagulans and Clostridium botulinum (a toxigenic pathogen) were observed together in two thirds of the samples (numbers 6–9 and 11–16) after incubation in the hermetically sealed cards of the BMX TYM platform. These organisms were detected before growth at very low levels (0.5% or less), indicating the presence of viable cells or spores in the samples. They were not detected at significant levels after growth on the 3M platform.

Other potentially pathogenic bacterial species that were detected at proportions of >1% of classified bacterial reads on plant material before growth include: Acinetobacter baumannii, Acinetobacter pitti, Corynebacterium diphtheriae, Coxiella burnetii, Escherichia coli, Propionibacterium acnes, Pseudomonas aeruginosa, Ralstonia pickettii, Salmonella enterica, Staphylococcus aureus, Stenotrophomonas maltophilia, and Streptococcus pneumoniae. Some of these species, and others, were observed to grow differentially on the BMX and 3M platforms. Species that grew well on 3M but not BMX included S. maltophilia and Leifsonia xyli; those that grew well on BMX but not 3M included C. botulinum, B. coagulans, Pseudomonas fluorescens and C. tetani. Factors that may contribute to this are the presence of chloramphenicol (Cm) and possible low oxygen levels in the BMX platform. S. maltophilia is Cm sensitive and P. fluorescens is Cm resistant. C. botulinum and C. tetani are obligate anaerobes and B. coagulans is a facultative anaerobe.

Differential growth of toxigenic and beneficial fungi

The concordance between the two culture based platforms was much higher overall for fungi than for bacteria. The distribution of fungal species observed after growth on the BMX and 3M platforms was highly similar for nine of the 15 samples (cc 0.98-1.0), and low to moderate for another three samples (cc: -0.02-0.49). The remaining three samples did not include any fungi that could be classified at the species level. The following toxigenic fungi were detected levels at >1% of classified reads in at least one sample: Aspergillus fumigatus, Aspergillus ostianus, Aspergillus sydowii, Penicillium citrinum, Penicillium commune, and Penicillium steckii.

We expected that all fungal species would grow effectively on the 3M and BMX TYM platforms, but there were some notable exceptions. First, we observed that although Aspergillus species were present in 15 plant samples (average proportion: 25% of classified reads), they were only detected at low levels in three samples after culturing on either 3M or BMX media (average proportion: 1.1% or 0.4% of classified reads, respectively). Representative results from two such samples are shown in Figure 2. Second, Penicillium was the most prevalent genus observed before and after growth on both platforms, with the most prevalent species classifications being P. citrinum and P. olsonii. However, although Penicillium species were present at significant levels in sample 16 (76% of classified reads; Figure 2C), they did not grow well on either platform in this sample (2.7–5.6% of classified reads). Instead, substantial growth of Trichoderma species, primarily T. hamatum, was observed (80–90% of classified reads). T. hamatum is one of several Trichoderma species that have been shown to inhibit the growth of Penicillium and other toxigenic fungi^33,34. Apparent competitive growth inhibition of Penicillium species was also observed in sample 4 where there was substantial growth of Fusarium species (23–72% of classified reads; Figure 2A), and in samples 1, 2 and 7 where there was substantial growth of Saccharomyces species (57–82% of classified reads).

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.

While the qPCR and sequencing assays are capable of detecting free DNA, all of the samples tested in this study appear to contain live spores or microbes. Even in the one sample (number 6) where the TYM-qPCR Cq did not decrease after growth in BMX media, the proportions of fungal species changed and TAC qPCR demonstrated growing bacteria with a 10 Cq decrease (from over 40 to 30.4) after culture.

Comparative growth of Aspergillus species and other fungi on 3M media

To further evaluate the ability of Aspergillus species to grow on 3M Rapid TYM Petri-Films, we plated 10 fungal monocultures from ATCC stocks and measured the concordance between qPCR Cq and 3M CFU (Figure 3). The Aspergillus species CFU counts are approximately three orders of magnitude lower than expected based on Cq estimates that were developed and optimized by plating cultured cells of other species. Excluding the two Aspergillus species, the correlation between CFU/g and Cq is 0.71. The one other outlier in these data is Candida glabralta. The correlation between CFU per gram of plant material and Cq is 0.99 across the remaining eight different fungal species.

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]}.