cannabis microbiome sequencing reveals mycotoxicfungi

Cannabis microbiome sequencing reveals mycotoxic fungi

Kevin McKernanCourtagen Life ScienceMedicinal Genomics

Mold and Bacterial qPCR Testing

• qPCR provides very specific microbial risks in under 24 hours

• Most Culture bases systems are failing too much material and failing to grow important risks.

• $8/test

3Confidential

Cannabis Microbiomes w/ ITS Sequencing

PathogINDICAtor

PathoSEEK Reveals Microbiomes

PathogINDICAtorqPCR

Sequencing

Microbiome IDOUT Count

R Square In PathoSEEK

Majority of microbes don’t culture

Penicillium Citrinum & Paxilli Confirmation

Confirm the Paxilline and CitrininGenes are also present

Paxilline

Citrinin

Diverse Microbiomes Discovered

DNA sequence Doesn’t = Expression

Uhlig et al.4x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

+ESI Product Ion (3.860 min) Frag=85.0V CID@20.0 (436.3 -> **) Paxilline_PIS_LongColumn-01.d

129.8

181.6

418.0

345.7 400.1

308.0 359.6 208.2 143.8 317.7 164.9 185.8 378.0 300.3 230.7 327.9 289.1 435.8 270.4

Counts vs. Mass-to-Charge (m/z)

100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450

Orser, Marquez replicate Uhlig et al at DigiPath

Design an LC-MS/MS method to detect this in a flower matrix.

Microbiological Contamination*

* SC Labs, CannMed 2016

Positive Feed Back Loop

Cyanide inhalation

High False Positive microbe tests = More pesticide use Less beneficial microbe use

http://www.medicinalgenomics.com/wp-content/uploads/2016/05/wurzer-CannMed2016.pdf

Pesticides*

* SC Labs, CannMed 2016

Experimental Design3M & Biomeriuex vs. qPCR & Sequencing

5. Sequence both 16S and 18S PCR product (before and after Tempo DNA)

Step 1. Capture DNA Before Culture

3M Film(3/5 days to Grow)

Biomerieux Cartridge(3 days)

Step 2. Culture Sample on 3M or Biomerieux Platforms

Step 3. Extract Positive Cultures in Step 2

4. Perform both 16S and 18S ITS qPCR

Purified DNA(3 Hours)

Capture DNA After Culture

Perform both 16S and 18S ITS qPCR

NextGenSequence

Questions

1. Do original microbials match post culture microbials?

2. What % of the CFU/g are actually Yeast and Mold?

1. BMX evidence for low levels of bacteria

2. BMX and 3M show high levels of TYM. qPCR demonstrates TYM after growth.

3. We have seen 3M TYM grow Staph. Will it grow Acne?

TAC Cq BMX-TAC 3M

Before 23.98 100 N/A

After 30.12

TYM Cq BMX-TYM 3M

Before N/A 37K/140K Fail

After 20.08

qPCR, BMX, 3M

Tilletiopsis_pallescens

Bacillus_coagulans

Ralstonia_syzygii_subsp._indonesiensis

bacterium_oxSCC-14

PathoSEEK TAC OUT Counts Before vs. After DNA TYM Biomerieux Culture

16S qPCR and Sequencing demonstrate bacteria grow very well in TYM cartridges

After TYM OTU Counts w/ Sequencing

Befo

re T

YM C

ultu

re O

TU C

ount

s w/

Sequ

enci

ng

PathoSEEK TAC OTU Counts Before vs. After DNA TYM Biomerieux Culture

Some Are Harmless Acne, Some Not

Kluyvera_ascorbatahttp://cid.oxfordjournals.org/content/33/7/e69.full

Raoultella_ornithinolyticahttp://jcm.asm.org/content/47/3/868.full

Klebsiella_sp.

Uncultured bacterium

Propionibacterium (Acne)

Enterobacter_sp._10-17

Plant Background DNA

Kluyvera_sp

Befo

re T

YM C

ultu

re O

TU C

ount

s w/

Sequ

enci

ng

After TYM Culture OTU Counts w/ Sequencing

18S Shows P.citrinum GrowthBe

fore

TYM

Cul

ture

OTU

Cou

nts w

/ 18

S Se

quen

cing

After TYM Culture OTU Counts w/ 18S Sequencing

PathoSEEK TAC OTU Counts Before vs. After DNA TYM Biomerieux Culture

fungal_endophyte

Delftia_sp._JC257

Penicillium_citrinum

Penicillium_sp._2433_FbLBPenicillium_citrinum

fungal_endophyte

fungal_endophyte

Penicillium_sp._568_FbG

Penicillium_citrinumPenicillium_sp._568_FbG

18S and Aspergillus Failure to Grow

PathoSEEK TYM OTU Counts

funga_endophyte

Aspergillus_niger

Aspergillus_nigerAspergillus_niger

Fusarium

Befo

re T

YM C

ultu

re O

TU C

ount

s w/

18S

Sequ

enci

ng

After TYM Culture OTU Counts w/ 18S Sequencing

16S primers on 3M petri film vs Biomerieux

uncultured_bacterium

Aureimonas_sp._PP-WC-4G-234

uncultured_bacterium

uncultured_bacterium

bacterium_CICC_23843

Bacillus_coagulans

uncultured_bacteriumRalstonia_pickettii

Discordant Before vs After data with culture

Raises a Hiesenberg Uncertainty problem for regulators

-The act of measuring microbial load, changes the microbial load.

But do the differing culture systems agree with one another?

18S qPCR, 3M petri film vs Biomerieux

Penicillium_citrinum

fungal_endophyte

fungal_endophyte

Penicillium_citrinum

Penicillium_brevicompactum

Correl = .992

Correl =0.196

Correlations with Bacteria are weak

18S qPCR specificity

• When 18S qPCR & Sequencing is performed how many times do we see 16S sequence hits?

Future Directions

• If we want to reduce Pesticide usage, we must have specific microbial testing

• Beneficial microbes are known to reduce the need for pesticides.

• Beneficial microbes should not be included in a CFU/g count.

Nevada On The Shoulder of Giants

Current Microbial Tests Lack Specificity

• Culture based CFU/g has no bearing on safety…

• Bacteria Grow on/in Yeast and Mold culture (FP)– Staphyloccus (Human introduced)– Pseudomonas (ubiquitous, harmless except Aeruginosa)– Propionibacteria (Human introduced- Acne)– Bacillus – Likely beneficial

• Many forms of visible mold don’t grow at all (FN)

Culture systems failed to detect some Penicillium species (FN) and some aspergillus species

Lets get cannabis on the same footing!

FDA is Moving to Sequence Based Safety Testing

DNA Sequence Verification of Cannabis Strains

• Genetic Fingerprints of Cannabis Products in the market place.

• Kannapedia.net

• Links to METRC and Blockchain based proof of existence databases.

Blockchains Enable Encryption Proof Time Stamps

21e20b9db09c2af093ef926a3ec77efaec1fc2df

Create Encryption Key of SNP file with SHA-256

Spend BTC with Key.Key is placed into a block and the time stamp is distributed to millions of computers and can never be reversed.

Customer can demonstrate in Court that a Key in a time-stamped Block unlocks their genetic file and therefore the file had to exist at the time of the block

Acknowledgements

Medicinal Genomics• Yvonne Helbert• Jessica Spangler• Ryan Lynch• Wendell Orphe• Lei Zhang• Jeremy Rualo• Adrian Devitt-Lee• Christine Sutera• Lei Zhang• Ted Foss• Doug Smith

DigiPathCindy OrserGlen Marquez

ProverdeChris HudallaMatthew Silva

SCLabsJosh WurzerAlec Dixon