Transcript ICRS-2016 - Medicinal Genomics

Cannabis Microbiome Sequencing Reveals Penicillium Paxilli and the
Potential for Paxilline Drug Interactions with Cannabidiol
Kevin McKernan1, Jessica Spangler1, Yvonne Helbert1, Ryan Lynch1, Adrian Devitt-Lee1, Lei Zhang1, Wendell Orphe1, Ted Foss1, Chris Hudalla2, Matthew Silva2, Cindy Orser3
,Douglas Smith1
Affiliations: 1Medicinal Genomics Corporation, Woburn, MA; 2ProVerde Laboratories, Milford, MA, 3DigiPath Labs
Introduction
Background MGC Microbial Assays
MGC’s current PathogINDICAtor® assays for use with SenSATIVAx® DNA Extraction are listed below:
Presence/Absence Tests
• E.coli
• E.coli STEC only
• Salmonella
• Aspergillus (A.niger, A.fumigatus, & A.flavus species)
Examples of PathogINDICAtor® Assay Data
Internal
Control
An ideal microbial detection system would discern
pathogenic from beneficial microbes. This can be
challenging to do with selective medias and culturing
conditions but can easily be accomplished with DNA
based methods. We propose a 2 step method that first
depletes or captures beneficial microbial DNA
(Bacillus, Trichoderma etc.) onto a magnetic particle.
The supernatant of this particle thus contains the nonbeneficial microbes and can be tested by qPCR using
ITS primers, as described in McKernan et al.
Microbial
DNA
Figure 4: Sequencing of 16S amplicons
(bacteria) from 3M Yeast and Mold (TYM)
plates demonstrates media selective growth in
ROI1 and ROI3. ROI1 demonstrate Bacteria
that selectively grow on 3M TYM petri films.
R0I3 demonstrate bacteria that grow BMX TYM
Tempo cartridges but do not grow on 3M TYM
petri films. Of note, BMX Tempo cartridges are
known to contain Chloramphenicol. ROI2
demonstrates bacteria that grow in both
platforms. Note the scales on the charts are
dynamic.
Internal
Control
Figure 2: Sample with no microbe
DNA present. (Plant DNA = Green,
Microbial DNA detection = Blue)
Figure 1: Sample containing plant
and microbe DNA. (Plant DNA =
Green, Microbial DNA = blue). X-axis
is cycles (every 1.5 minutes). Y-axis
is relative fluorescence units in a log
10 scale.
Sample 1: Comparison of fungal growth on BMX and 3M using TYM primers
Sample 2: Comparison of fungal growth on BMX and 3M using TYM primers
0.06
0.06
Trichoderma
Aspergillus
Penicillium
Unspecified
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Experimental Design
0.04
0.03
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Trichoderma
Aspergillus
Penicillium
Unspecified
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Normalized 18S OTU counts on 3M using TYM
This qPCR system can be coupled with a next
generation sequencing assay known as the
PathoSEEK™ microbial identification test for
Cannabis and Hemp that sequences the ITS PCR
products to generate Operational Taxonomic Unit
(OTUs) estimates for each species. We utilize these
methods to quantitate the microbial diversity present
on Cannabis samples both before and after culturing
of microbes using both 3M and Biomerieux products.
This data reveal substantial bacterial growth in
commercial Yeast and Mold detection platforms and
itemization of beneficial and harmful microbes in
current testing regimes.
Threshold Tests
• Total Yeast & Mold (18S)
• Total Aerobic Bacteria (16S)
• Total Enterobacteriaceae (16S)
• Total Coliform
Normalized 18S OTU counts on 3M using TYM
Traditional Cannabis microbial safety testing relies on
counting colony forming units (CFU/g) that grow on a
petri dish, 3M film or in a culture based system
(Marcu, 2013). These systems take 3-5 days to
culture fungi or microbes to detectable limits and
cannot discern harmful microbes from beneficial
microbes and often fail to detect fungi that synthesize
compounds contra-indicated in the medicinal use of
cannabinoids (McKernan et al.). As a result of this lack
of specificity, overuse of fungicides is common in
Colorado and California in 2016 (Wurzer, 2016).
0.04
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0
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Normalized 18S OTU counts on BMX using TYM
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0
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Normalized 18S OTU counts on BMX using TYM
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Sample 3: Comparison of fungal growth on BMX and 3M using TYM primers
We explored ITS PCR both before and after culturing on 3M Total
Yeast and Mold (3M-TYM) and Biomerieux Tempo cartridges (BMXTYM) to monitor the microbial diversity before and after culturing.
To our surprise many bacterial species are identified growing on the
Yeast and Mold cultures generating False Positive Yeast and Mold
results. Many species grow specifically on one culture platform or the
other.
Comparison of bacteria present on Sample 1 before and after culturing
Conclusions
Accurate microbial testing should be capable of
speciating risks and discounting beneficial
microbes. Failure to do so encourages
fungicidal use that is likely more dangerous
than the microbial risks.
Trichoderma
Aspergillus
Penicillium
Unspecified
0.03
Normalized 18S OTU counts on 3M using TYM
Figure 5: Sequencing of 18S amplicons (fungi)
from 3M and BMX TYM plate indicates some
consistency between TYM tests for growing
yeast and molds, but highlights the importance
of filtering out beneficial microbes like
Trichoderma.
0.035
0.025
0.02
0.015
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0.005
0
0
0.005
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0.015
Normalized 18S OTU counts on BMX using TYM
0.02
0.025
Future Directions - Beneficial Microbial DNA Depletion
0.01
Bacillus
Ralstonia
Enterobacter
Unspecified
0.009
Normalized OTU counts after culturing
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0
0
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Normalized OTU counts before culturing
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0.009
0.01
Figure 3: Sequencing Before and After culturing reveals differential
growth of bacteria on Total Yeast and Mold BMX assays. Of note is
the rapid growth of Bacillus often used as beneficial microbes. The
rapid change in 16S contributions during culturing implies a
“Heisenberg uncertainty” in microbial testing platforms where the
act of measuring the risks, alters the accurate quantitation of risks.
Research and Development Contact
Sales Contact
References
Jessica Spangler
Medicinal Genomics Corporation
Email: [email protected]
John Geanacopoulos
Medicinal Genomics Corporation
Email: [email protected]
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