Bridging Science and Industry:Metagenomics

Joe HeinzelmannDirector of Business Development, Food safety GenomicsNeogen

Three types of DNA Testing

100-150 BP targets: is this gene sequence present

PCR

Targeted probes against

single alleles and SNP

Array Based Genotyping

Determining the specific

sequences of genomes or gene targets

NGS

Three Genomic Testing Categories

Pathogen Outbreak and Food Safety

Investigations

Pathogen Outbreak

Spoilage Determination

and Root Cause

Analysis

Microbiome

Identity Testing and Food Fraud Prevention

Food Fraud

WGS is one application of Sequencing

WGSID and

relatedness

Next Generation Sequencing• 16S• TAS• GMO Testing• AMR• Microbiome• Shotgun metagenomics

The Listeria Initiative

Targeted Amplicon Sequencing vs WGS• Generate data specific to the application• Limit liability and unknowns by targeted data

generation• Lowers the cost and turn around time

TAS WGS

Targeted Amplicon Sequencing vs WGSNeoSeek Salmonella Serotyping• 12,000 base pairs• No additional,

information

Whole Genome Sequencing• ~5,000,000 base pairs• Contains additional

information

Targeted Sequencing Whole Genome

NeoSeek Salmonella Serotyping

Targeted Amplicon Sequencing• Serotype from enrichment• Sequence type• Multiple serotypes from one

broth

In summary, NeoSeek will:• 12,000 base pair analysis• Serotype up to 3 from one broth• Sequence type for additional

comparison

WGS vs 16s Metagenomics• WGS • 16S Metagenomics

WGS

Traditional (PFGE) Traditional

16s metagenomics

What is 16S?• 16S is a component of a

ribosomal unit. It is specific for prokaryotes.

• The ribosome is in every living cell, and translates DNA into amino acids.

The 16S rRNA gene: the most widely used molecular clock for bacteria ~54 recognized Phyla

16S

Traditional Taxonomic Classification

WGS vs 16S meta vs Shotgun metaWGS

16S Metagenomics

Shotgun Metagenomics

The Great Plate Count Anomaly

Traditional plate count methods see a fraction of the total microbial picture

Examples of Hard to ID Organisms• Photobacterium• Clostridium• Erwinia• Leuconostoc• Lactobacillus• Paenibacillus• Rhizobium

Anaerobic

VBNCID’ingcolonies on TSA

16S profile

Dilution and isolation• Misses VBNC• Introduces Biases

Slants and initial ID• Prone to human

interpretation• Errors and trained

microbiologist

Biochemical ID• Inaccuracies,

limited scope • Time consuming

and expensive

Human Health• Human Microbiome Project

• Animal Health

• Soil Microbiome

• Big Ideas:• Microbial Community Structure has an incredibly

important impacts

Soil microbiome and Environmental• Managing the elements

of the microbiome can lead to production and sustainability gains

Animal Health and Production• Body weight and growth depend on a functional

and efficient GI. How is that defined?

Antimicrobial Resistance• Results of this study suggest that

parenteral metaphylactic treatment of cattle with tulathromycin had minimal, if any, detectable short-term impact on the fecal resistome and microbiome of commercially raised feedlot cattle when evaluated using shotgun metagenomic sequencing. This is important because of critical concerns about public health in relation to AMD use in food-producing animals and also because this is an important drug for treatment and control of life-threatening respiratory disease in feedlot cattle. This study was conducted in a commercial feedlot operation to improve the practical relevance of our findings, but this also introduces important limitations. USDA data suggests that over 70% of feedlot cattle in the U.S. receive low doses of tylosin, a macrolide drug, infeed for prevention of liver abscesses (USDA, 2013).

Investigating Effects of Tulathromycin Metaphylaxis on the Fecal Resistome and Microbiome of Commercial Feedlot Cattle Early in the Feeding Period, Front. Microbiol., 30 July 2018, Belk, Morely, et al.

Key Applications for 16S • Spoilage Troubleshooting

• Plant Mapping

• Time dependent Microbiomes

Spoilage Investigations with 16S

Overview• Sample multiple sites• Sample multiple raw materials• Compare spoiled vs. normal• Understand facility microbiota• Root cause analysis

Data set # 1

Vials

Step 1: contamination investigation

Key Bacteria:StaphPseudomonasMeiothermusBrevibacterium

Step 2: Potential source of contamination

Organism Potential SourcesStaph Humans, raw materialsPseudomonas Ubiquitous, water sourcesMeiothermus ????Brevibacterium Actinomycetales, soil

bacterium, likely from air particles, human skin

Step 3: Correlation and dataset review

Sanitation Effectiveness

Pre-cleaning comprised of staph, brevibacterium, and bacillus

Post cleaning Staphylococcus removed. Samples have higher relative abundance of brevibacterium

Pet Food Production• Mapping microbial changes in the process• Key changes based on extrusion and

temperature changes• Comparing Day 1 to Day 4

Monitoring microbial changes in production

Day 4: Left to right represents raw

material through finished product

From Left to right: Raw material

through finished products

Beginning Process comparison

End of process comparison

Phase 2 Objectives• Objective 1:

• To observe the effect of time/seasonality on the microbiome observed in Phase 1.

• Objective 2:• To compare microbiome of 2 production facilities

(similar in design and equipment) with different geographies (east coast vs west coast).

Phase 2 Background• Background:

• Samples:• Collected at 2 production locations (Gaston and Lathrop)• Collected on 3 sample days (day 1, 4 and 6)• Five total samples per collection day:

• 2 equipment contact surface swabs• 3 product samples (work in progress, fines and finished

product)

Gaston: Days 1-3

Lathrop: Days 1, 3 and 6

Day 1 Day 3 Day 6

Biomapping the Supply Chain

Advanced Analytics

Milk Microbiome and facility biomapping

Advanced microbial mapping: Dairy production

Looking for spoilage organisms, sources, and potential root causes

Microbial diversity by process

Time Dependent Microbiomes• Communities of bacteria to understand:

• Digestion and managing effluent • Odor control of ponds

• Microbial fermentations / digestions• Optimization • Contamination control• Input management

Microbial enrichment dynamics• Determining best use of traditional microbial

enrichments• FDA analysis of enrichments with various media

on ice cream

Ottesen et al. BMC Microbiology (2016) 16:275

Beef Shelf Life and Storage Conditions

• Metagenomic approach• Received 95% vacuum package ground beef bricks• Stored at 1C, 5C, 7C and 10C• Purified bacterial DNA over time• 16S sequenced

1C5C

7C10C

Boselivac, NGS Applications beyond WGS, IAFP Symposium 2018

Beef Shelf Life and Storage Conditions• Metagenomic approach

Lactococcus

EnterococcusLactobacillus

Serratia

Leuconostoc

1C 5C 7C 10C

Day: 0 3 9 17 23 3 9 17 3 9 12 3 6 9

Boselivac, NGS Applications beyond WGS, IAFP Symposium 2018

Beef Shelf Life and Storage Conditions• 16S Metaganomics• 85% lean ground beef chubs, storage conditions

and time

0 5 9

5C

4 6 9

10C

0

2C

4 8 12

5C

4 8 12

7C

3 4 6

10C

3 4

Pseudomonas

Lactococcus

Photobacterium

LactobacillusSerratia

Day:

Boselivac, NGS Applications beyond WGS, IAFP Symposium 2018

Thank you!

jheinzelmann@neogen.com517-372-9200