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Official websites use. Share sensitive information only on official, secure websites. WO performed the comparative Aspergillus growth confirmation experiments. KM and DRS prepared the manuscript, and all authors were involved in revision and approval of the final manuscript. This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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. 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. 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. Plant associated microbes may present risks of infectious illness for human end consumers. However, many plant-associated microbes may provide benefits for plant cultivation in terms of growth stimulation, insect or microbial resistance, or may simply be neutral passengers 1 — 3. The microbiome of Cannabis leaves and flowers includes bacteria and fungi residing on the exterior surface of these tissues epiphytes as well as those residing within the plant tissues endophytes. While epiphytic microbes may originate from many sources like aerosols, dusts and liquids, or via human contact, endophytes typically gain entry from the rhizosphere via root junctions, and subsequent translocation through the xylem 4 , 5. Considering this and the known impact that the soil and root microbiome has on plant growth and development 6 , 7 , all sources of microbial inputs, including below ground compartments should be considered important for optimal Cannabis growth and consumer safety 8. Studies on the natural Cannabis microbiome have identified several species of culturable endophytic fungi, including Penicillium citrinum, Penicillium copticola a member of the citrinum section 9 and several Aspergillus species 10 , Similar studies looking at culturable bacterial endophytes identified nearly a dozen isolates from the Bacillus clade and two mycobacteria 1. Of those Bacillus species, B. Finally, a recent investigation of the fungal microbiome in a number of dispensary-derived Cannabis samples identified numerous species including some toxigenic Penicillia and Aspergilli While there have not been any reported cases of Cannabis -related mycotoxin poisoning resulting from Penicillium infections, there have been numerous reported cases of serious or fatal pulmonary Aspergillosis associated with marijuana smoking in immunocompromised patients 16 — A multistate outbreak of Salmonellosis has also been reported 19 , State Cannabis markets rely on a patchwork of testing regulations to protect patients and consumers. In terms of microbial testing, these vary widely from state to state. A few States require that testing laboratories follow the procedures outlined in the USP for microbiological examination of non-sterile products. Others allow testing laboratories to choose from a wide variety of technologies designed for the food testing industry. However, there is no peer-reviewed research supporting the effectiveness and validity of any of these protocols for Cannabis microbial testing. Furthermore, no studies to date have examined the impact of beneficial endophytes on the Cannabis microbiome and on microbial testing results. Sequencing and analysis of the fungal ribosomal operon internal transcribed spacer 23 , 24 ITS2 and the bacterial 16S ribosomal RNA gene V3 and V4 hypervariable regions 25 16S allowed us to identify bacterial and fungal genera and species present in each case. The results highlight some organisms of concern and demonstrate that major fungal and bacterial compositional changes occur during culture-based TYM testing. Cannabis samples were derived from seven recently-established indoor growth facilities in Massachusetts, Maine and Rhode Island. DNA was similarly extracted after growth on the two culture based platforms as described above. Colonies were scraped off the plates and DNA was then extracted as described above. 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. DNA samples extracted directly from Cannabis samples, or after growth on the two culture-based platforms, were subjected to qPCR analysis. Use of methylated nucleotides for PCR decontamination is described previously 26 , PCR samples were purified by mixing The samples were placed on a magnet for 15 minutes until the beads cleared and the supernatant could be removed. 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. Sequences were quality trimmed to have a maximum expected number of errors per bases of less than 0. Each library was normalized by the total number of OTUs found. R 2 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. Summary results from the different testing platforms evaluated in this study for 15 samples with complete data are presented in Table 1. Results in bold type and shaded boxes indicate failed tests following the limits set for Massachusetts medicinal Cannabis. Results in bold type and shaded boxes indicate failed tests. 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. While the sequencing assay provides approximate intra-sample quantitation, it does not support inter-sample quantitation 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. The qPCR Cq measured directly from extracted plant material provides the best inter-sample comparative metric. 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. 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. These data imply highly reproducible genomic surveys of the amplified DNA present. 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. These samples represent different strains from the same grow and likely share similar soil environments. 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. 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. Representative results from two of those samples, numbers 2 and 14, are shown in Figure 1. 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. They were not detected at significant levels after growth on the 3M platform. Some of these species, and others, were observed to grow differentially on the BMX and 3M platforms. Factors that may contribute to this are the presence of chloramphenicol Cm and possible low oxygen levels in the BMX platform. 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. The remaining three samples did not include any fungi that could be classified at the species level. 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. Instead, substantial growth of Trichoderma species, primarily T. 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. 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. 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. The one other outlier in these data is Candida glabralta. The correlation between CFU per gram of plant material and Cq is 0. Aspergillus demonstrates log scales lower growth at RT than most other yeast. Data associated with the article are available under the terms of the Creative Commons Zero 'No rights reserved' data waiver CC0 1. The samples selected for this study were derived from seven newly established indoor Cannabis growth facilities located in a humid coastal environment Eastern Massachusetts, Maine and Rhode Island. They were enriched for samples that failed on either or both the 3M and BMX platforms, which are commonly used to test for bacteria, yeast and mold in the industry. Quantitative PCR was evaluated as a third approach to hopefully resolve discrepancies. The high failure rate observed in this study should not be taken as representative of industry-wide averages, which have been reported elsewhere 15 , The sample set provided an opportunity to investigate the diversity of species that grow in different culture-based platforms as well as to characterize the microorganisms that were responsible for the sample failures. Metagenomic sequencing data were collected on 15 samples, directly from plant material and after culture on both the 3M and BMX platforms. The sequencing results demonstrate substantial shifts in presence and abundance of bacterial and fungal species after growth on the two platforms. Thus both of the culture-based platforms are detecting and enumerating only a subset of the species present, and the final composition of microbes after growth is markedly different from the starting sample. Most concerning is the frequent identification of bacterial species in systems designed for the exclusive quantification of yeast and mold, as quantified by elevated TAC Cq values after culture in the BMX TYM medium. These observations call into question the specificity claims of these culture-based testing platforms. The presence of bacterial colonies on TYM growth plates or cards may falsely increase the rejection rate of Cannabis samples for fungal contamination, and induce growers to increase the use of fungicides unnecessarily. The CLARK-S classification software has been reported to have very high sensitivity and precision for sequence assignments 28 , 36 , Nevertheless, further work is required to confirm these species assignments and to check for the presence of toxins that may be produced by these microbes. The observations certainly call into question the wisdom of species-agnostic microbial quantitation for a product like medicinal Cannabis, which is used by many seriously ill or immunocompromised patients. Cross-platform comparisons demonstrate that certain bacteria and fungi grow well on 3M plates, but not on BMX, or vice versa. There are certainly differences in terms of the media. For example, BMX medium includes chloramphenicol to suppress bacterial growth, and uses sealed growth chambers that may limit oxygen availability. The observation of anaerobic Clostridium species such as C. Clostridium botulinum was only detected at very low levels before growth on BMX medium, and was not detected on 3M plates. Previous white papers have suggested C. However, C. Additionally, proximity between cultivation and processing may lead to contamination of finished products such as emulsified oils or concentrated extracts containing water. Media such as these provide anaerobic conditions and nutrients sufficient for C. This is most threatening to indoor cultivation facilities which also process, store, and package finished products on site, often in sub-optimal storage conditions. The fact that the organism was observed to proliferate in the BMX system suggests that its presence, even at low levels, could be a potential concern in emulsified Cannabis oil formulations or edible products that are stored in closed containers. Initially, it was suspected that the significant TYM qPCR and read counts might derive from dead cells, perhaps as a result of growers attempting to sterilize the plant material. Elevated Cq values due to ribosomal DNA copy number amplification does not seem a likely explanation because the estimated copy numbers of several Aspergillus species are similar to those of other fungi 40 , While the presence of spores with a slow germination rate 42 could explain the results on plant material, it does not explain the qPCR result using active cultures. Another factor could be the obligate hyphal growth nature of Aspergillus species 43 , wherein each colony forming unit may contain hundreds of interconnected hyphal cells. These findings are surprising, and therefore a third culture-based system, manufactured by Biolumix, was tested for its ability to detect A. The result was negative. The failure of three different culture-based platforms to detect Aspergillus species suggests the need for caution in the use of such platforms. McClenny 45 recommends longer times and higher temperatures to accurately detect Aspergilli with culture based methods. Aspergillus is arguably the most significant fungal threat in Cannabis cultivation. Aspergillosis has been reported in numerous immunocompromised patients and, to date accounts for the only clinical reports of fatalities associated with an infectious organism linked to Cannabis consumption 16 — 18 , 46 — Vonberg et al. Growers may pasteurize Cannabis samples to avoid failing culture-based microbial testing, but Aspergillus spores are pasteurization resistant 50 , as are the toxins they produce 51 , so pasteurization does not eliminate the potential risk from these organisms. Another interesting observation is the apparent growth inhibition of Penicillium species P. Other classified species that failed to grow in some of those samples include Furcaspora eucalypti and Tilletiopsis pallescens. Organic growth practices often utilize beneficial bacterial or fungal endophytes 52 to promote crop growth and to enable lower chemical fungicide use. The State of Nevada has issued guidelines for allowable pesticides for use in Cannabis cultivation that include various Trichoderma and Bacillus species However, in most states, the use of such beneficial microbes may be precluded by the requirement for stringent yeast and mold testing that does not discriminate between beneficial and harmful microorganisms. More specific nucleic acid based testing techniques can resolve this. Finally, as observed in a previous study on the Cannabis fungal microbiome in a different sample set 15 , P. This species has been isolated as a growth promoting endophyte in Cannabis and several other plant species 10 , 11 , 57 — However, the high prevalence of P. These data have several limitations. Quantitative inter-sample comparisons cannot be performed with the sequencing data at present due to the lack of internal controls to help calibrate any pooling or sampling issues throughout the workflow. The qPCR data can be used to estimate inter-sample bacterial or fungal burden but these data do not always resolve to the genus or species level. Intra-sample comparisons can nonetheless provide information on the relative proportions of bacterial or fungal species. Sampling from BMX cards was straightforward, since it uses a liquid culture medium, but 3M sampling was subject to bias in scraping off colonies from culture plates. Additionally, the use of Nextera shearing and primer amplification may introduce some biases due to transposon integration preferences. Culture based techniques used to measure the microbial burden and establish safety of Cannabis have several shortcomings. States adopt and implement regulations at different tolerance thresholds for bacteria and fungi without specifically detailing standardized methods or coordinating inter-laboratory ring testing. Yeast and mold counts from the culture-based platforms tested here are confounded by the growth of bacteria - even when antibiotics like chloramphenicol are included. The microbiome in the plant material tested changes radically after culturing, such that the microbes and counts that are finally observed bear little or no resemblance to those of the starting sample. This is a serious issue, which clearly has implications beyond Cannabis safety testing. The 3M and BMX platforms tested here are also used widely in the food testing industry. Perhaps the most concerning observation is that one of the most regulated of fungal pathogens, Aspergillus - the only microbe to ever be associated with clinical harm concerning cannabis - grows poorly, and is therefore severely under-reported by current culture-based platforms. The differential growth of other toxigenic fungi, depending on the companion species present, further influences the results. Bacterial pathogens are not uncommon, and beneficial bacteria are also capable of influencing the growth or inhibition of other flora. We have demonstrated that molecular testing is capable of accurately quantifying and identifying a wide spectrum of microorganisms present on Cannabis samples, while avoiding false positives due to the presence of bacteria for fungal testing. Molecular testing is rapid and is capable of distinguishing between harmful and beneficial microbes — permitting the use of the latter in organic cultivation practices to eliminate the need for reliance on chemical fungicides. FResearch: Dataset 1. Raw data of metagenomic analysis of medicinal Cannabis samples, The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This article represents an area of research that needs more attention. My only concerns are minor, and are regarding the figures in the article. It would be great if there were more reproducibility indicated within the figures, as this article will be highly read and potentially utilized in a growing industry. I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We would like to extend our sincere gratitude for the opportunity to provide an open peer review for the work of McKernan and colleagues on the Cannabis microbiome and uses of metagenomics to shed light on the microbial complement of the Cannabis phyllosphere. As strong proponents of open science, we engage to provide an objective assessment of the work presented here and to make suggestions aimed at improving the clarity and readability of the present work. The microbiome the collection of microbial genomes present on an organism or in an environment has emerged as an additional dimension in addition to genomic, epigenomic, metabolomic and phenotypic data… from which one can harness cryptic information that may contribute to a particular biological phenomenon. In their paper, McKernan et al. As such, much of our comments relate to improving the transparency of their results. Below, please find minor comments, which we would like to authors to consider:. We are in agreement with review 1 Ethan Russo that the abstract could be improved if word limit permits. We particularly think that the concluding statement could incorporate a stronger statement about the application of their approach in the Cannabis industry. A short statement on why the comparison between culture-based platforms and DNA-based detection is relevant e. Perhaps the considerations about Trichoderma could be saved for the discussion. Some brief background on the two culture platforms 3M, BMX would help frame the need for other novel technologies in microbial detection. As a general comment to all sections from hereon, it would be helpful to have the same sub-headings as much as possible logically flow from methods to results and into the discussion points:. If there is sufficient evidence that the novel approach outperforms the old is another question that seems rather elusive in the current paper. It would be valuable to share some information about their standard curve and how they derived their Cq values of 21 and 26 cycles for TYM and TAC assays respectively. The authors should consider perhaps using other multivariate statistics than bivariate correlation coefficients. While sample size is likely limiting, are there other similarities between samples for common origins? Or grown in similar conditions? Table 1 could be presented in a clearer way: Table description and content the column headers are redundant; too much text in the table description; instead of sample nr. The presentation of the results using excel bar plots , while understandable, is not that efficient at presenting the data at hand. Without overstepping, we suggest looking at multivariate plots that would be more suited to drive their points home. It seems counterintuitive that qPCR, being more sensitive than plating approach, would fail the lowest number of samples out of all approaches: Is the BMX positive bias toward C. In that vein, it would be helpful to describe your strategy to assess false positives, i. Any negative control with botulism? Failure thresholds are subjective in nature, please expand on how the Cq threshold is superior, what microbial load e. We found that while a large and varied bacterial assemblage was identified here, it would be important to note that modern Cannabis such as the 15 samples presented here have likely gone through several genetic and microbiotic bottlenecks. While the Cannabis domestication process is convoluted and masked by prohibition, it is likely that the same pattern is observed in Cannabis. Characterizing the genetic profiles of Cannabis, along with the microbiome of wild Cannabis accessions will likely yield enhanced inference in terms of the underlying mechanisms related to plant growth and disease tolerance. A larger part of the discussion should be dedicated to the community composition shift before and after culturing. Especially some considerations about the biological relevance of this shift: i. It would be good to more explicitly separate what the authors think are artifacts caused by different methodologies community shifts with biologically relevant phenomena. If the authors found polymorphisms in OTUs, they may want to suggest the application of the Cannabis microbiome to provide higher resolution to clustering exercises in highly related or poly-hybridized Cannabis accessions. This may also be used to trace the origin of particular dispensary samples to a cultivator or methodology of plant growth as using hydroponics, soil, aquaponics, etc. While the authors discuss the presence of C. In Canada until recently, only Cannabis flowers were prescribed as medicinal Cannabis. This study highlights some drawbacks of using this type of Cannabis for medical purposes, particularly when used in immune-compromised individuals, and indirectly supports the use of Cannabis extracts that can be dosed effectively with minimal risks of exposure to toxicogenic microbes. We sincerely hope that the authors will find our review useful and we remain available for further discussion through the F research platform. We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The purpose of this study was to investigate the composition of microorganisms found on cannabis samples and compare the ability of different culture based testing platforms with a qPCR method. Although the study provides some valuable data into some of the short comings of culture based methods it has some experimental design weaknesses that make it difficult to draw strong conclusions from this data set. Since the purpose of the study is to discuss the difference between a qPCR based microbial testing platform with culture based methods the introduction should focus more on discussing this in other industries. For example it's becoming well known that only a small percentage of organisms that exist in nature are easily cultured on the most common forms of media used. Rapid advances in sequencing are allowing metagenomic analysis of soil and plant microbiomes which also demonstrates the limitations of culturing methods. Issues like specificity between qPCR and culture based methods should be highlighted. Plant material - Nowhere in the methods section is any information provided about the cannabis plant material. Was it cannabis flowers? Were they dried? Was the sample homogenized in anyway? Information about all the samples used in this study should be summarized in a table or in a section within the methods part of the manuscript. Table 1- This table highlights one of the main criticisms I have with this study. There are no replicates. These results seem to be based off a single analysis of each sample. Therefore we can't conclude anything about the reproducibility of the qPCR platform compared to the other platforms. It is also interesting to note that most of the culture based methods detected levels of fungi that would be considered failures while most of the qPCR samples detected only low levels of fungal DNA. It is difficult to follow from reading through the text of the manuscript which samples were analyzed by metagenomic sequencing. Every sample analyzed by metagenomic sequencing and a summary of their results, in terms of what species were detected and their approximate amounts, should be summarized somewhere in the manuscript for ease of reference and completeness of data presentation. Why those samples were chosen should be discussed. Throughout the results section numbers of samples are discussed but we don't know if those are the same samples or which samples shown in Table 1. Which 15 plant samples? This kind of vague reference to samples needs to be corrected and be made more clear. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. This is a very interesting, well written and designed account comparing the accuracy and utility of genetic microbial testing as compared to standard microbiological culture techniques. All aspects of study design, methods and conclusions are well explained and defended, and should easily allow replication if comparable techniques are applied. I would suggest expansion of the study's implications in the abstract if the word count will permit this. In , Vancouver Coastal Health in British Columbia reported transmission of meningococcal cases by sharing of joints, and perhaps this pathogen deserves scrutiny given its ubiquity in young adults very likely to be engaging in social cannabis usage. The legal analysis permitting cross-border transmission of DNA from cannabis material has important implications for greater adoption of similar analytical techniques, which certainly seems warranted given the advantages in accuracy in distinguishing beneficial, commensal and symbiotic microbiota from pathogens, and the speed of this approach to the issue. This section collects any data citations, data availability statements, or supplementary materials included in this article. As a library, NLM provides access to scientific literature. Find articles by Kevin McKernan. Find articles by Jessica Spangler. Find articles by Yvonne Helbert. Find articles by Ryan C Lynch. Find articles by Adrian Devitt-Lee. Find articles by Lei Zhang. Find articles by Wendell Orphe. Find articles by Jason Warner. Find articles by Theodore Foss. Find articles by Christopher J Hudalla. Find articles by Matthew Silva. Find articles by Douglas R Smith. Smith courtagen. Accepted Oct 3; Collection date Open in a new tab. Raw data of metagenomic analysis of medicinal Cannabis samples All data files supporting this work are provided. Click here for additional data file. Find articles by Jahan Marcu. Competing interests: No competing interests were disclosed. Jahan Marcu : Referee. PMC Copyright notice. Find articles by Philippe Henry. Find articles by Lukas Wille. Philippe Henry : Referee. Lukas Wille : Co-referee. Find articles by Justin Fischedick. Justin Fischedick : Referee. Find articles by Ethan Russo. Ethan Russo : Referee. Associated Data. Similar articles. Add to Collections. Create a new collection. Add to an existing collection. Choose a collection Unable to load your collection due to an error Please try again. Add Cancel.

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