2017 NAMI Meat Industry Summit, San Diego, CA April 3-5, Keith E. Belk

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1 2017 NAMI Meat Industry Summit, San Diego, CA April 3-5, 2017 Keith E. Belk Professor & Monfort Chair Center for Meat Safety & Quality Department of Animal Sciences Colorado State University Fort Collins

2 Building Knowledge Concerning AMR? Fight Misinformation

3 What Antibiotic Resistance is NOT... Antibiotic Residues in your meat Images:

Antibiotic Residues U.S. National Residue Program tests for different chemical compounds & classes, including veterinary drugs, pesticides, and metals.

4 Antibiotic Residues U.S. National Residue Program tests for different chemical compounds & classes, including veterinary drugs, pesticides, and metals. In 2014, a total of 6,066 residue samples were collected & 0.16% (10) were violative (FSIS, 2015). Food Safety and Inspection Service. United States National Residue Program for Meat Poultry and Eggs FY 2014 Residue Sampling Results

Packer Math for Probability of a Mutant Superbug killing you The frequency of an AMR mutation is small 0.00000001 for a one bp change and 0.

15% of American s develop a foodborne illness each year, including viral (CDC, 2015). [P] = 0.

Number of bacteria, along with many other factors, would be considered in a formal model. Freed, Gary L.

5 Packer Math for Probability of a Mutant Superbug killing you The frequency of an AMR mutation is small for a one bp change and for two bp change (Martinez & Baquero, 2000). 10% of the U.S. is estimated to be immunocompromised (Freed, 2002). 15% of American s develop a foodborne illness each year, including viral (CDC, 2015). [P] = % chance* *This is a rough model to illustrate the low likelihood of occurrence based on only a few estimations. Number of bacteria, along with many other factors, would be considered in a formal model. Freed, Gary L. "Expected adverse events in a mass smallpox vaccination campaign." Effective Clin Pract (2002). Martinez, J. L., and F. Baquero. "Mutation frequencies and antibiotic resistance." Antimicrobial agents and chemotherapy 44.7 (2000): Center for Disease Control and Preventio

6 What is antibiotic resistance? When cattle were administered Excede, resistance did not differ between the day it was given & 26 days later. antibiotic treatment 0.01% Resistant 0.10% Resistant 0.01% Resistant

7 How is antibiotic resistance transmitted? Photo: Source: Nature Reviews Genetics, Soucy et al., 16: (2015)

Horizontal Transfer of AMR Genes Bacteriophages infect an estimated 10 23 to 10 25 bacterial

Data represent the numbers of antibiotic-resistant transformants obtained from DNA extracted

8 Horizontal Transfer of AMR Genes Bacteriophages infect an estimated to bacterial cells each second... FIG 1 Two novel lytic coliphages promote extensive plasmid DNA transformation. Data represent the numbers of antibiotic-resistant transformants obtained from DNA extracted from various phages lysates of ~108 E. coli MG1655 cells bearing ampicillin resistance plasmid Pbad24.

9 Where does it come from? VFD

10 Next Generation Sequencing Technology Human Genome Project ( ): >13 Years ~$3 Billion 3 Billion BP Our metagenomics AMR study: 5 days $900 >8 Billion BP

11 Ecological Disconnect in AMR Studies? 100s to 1000s of Cattle 1g from animals 1 to 5 Colonies 1 or 2 Species AMDs Conclusions about Risks? ~20-30 kg feces/day bacteria per gram Fecal Microbiome >1000 bacterial species Firmicutes Proteobacteria Actinobacteria Bacteroidetes E. coli What Do We Want to Represent?

12 Abundance of Resistance Genes Generally Declines During Feeding (Source: Noyes et al., 2016) More abundant at placement More abundant at shipping soil water feces spectinomycin aminocoumarin aminoglycoside MLS tetracycline phenicol β-lactam sulfonamide bacitracin fluoroquinolone permeability genes polymyxin B rifampin modulatory genes efflux pumps Class of Resistance Genes Log-Fold Change in Abundance

13 Most bacterial species are less abundant in post-fabrication samples (Source: Noyes et al., 2016) Less abundant in postfabrication samples Log-Fold Change More abundant in postfabrication samples

14 AMR genes NOT identified in postfabrication samples (Source: Noyes et al., 2016) 0.12% 0.10% feces soil water Relative Abundance 0.08% 0.06% 0.04% 0.02% 0.00% Placement Shipping Truck Holding Post-fab

15 AMR in Effluent from Hospitals vs. Farms Source: Rowe et al., 2017, J Antimicrob Chemother

16 AMR in Conventional vs Natural Products (Source: Rovira et al., 2016) 1500 Conventional 1500 CONV-D CONV-F Natural NAT-F AMRnormsparse 1000 Production Conventional Natural AMRnormsparse 1000 ORG-D Production CONV-D CONV-F NAT-F ORG-D Conventional Production Natural CONV-D CONV-F NAT-F ORG-D Production

17 Microbiome by niche (Source: Rovira et al., 2016) Feces +Bacteroidetes +Firmicutes Soil +Proteobacteria +Actinobacteria WW +Proteobacteria

18 Resistome (single facilities; n = 4) by Species

19 Bait Pull-Down w/ Enrichment: AMR Hits/1M Reads Beef Poultry Swine WWTP

Improved Detection of 48 Classes of Resistance/Rirulence lipopeptide (daptomycin, 19 hits, poultry) BUT this gene was not in bait design 13 34 1 -

20 Improved Detection of 48 Classes of Resistance/Rirulence lipopeptide (daptomycin, 19 hits, poultry) BUT this gene was not in bait design arsenic - nickel - gold - paraquat - magnesium - acid - chromium - biguanide - metronidazole - aluminum - peroxide - phenolic compound - manganese - lead

Development of Bioinformatics/Analytics: Alignment vs. Hidden Markov Model Classification Source: Lakin et al., 2017 Sequence reads classified as hits against AMR sequences using Alignment (BWA) vs.

21 Development of Bioinformatics/Analytics: Alignment vs. Hidden Markov Model Classification Source: Lakin et al., 2017 Sequence reads classified as hits against AMR sequences using Alignment (BWA) vs. HMM (Meta- Marc). Use of HMM is very important in low-abundance, but enriched, rinsate samples; it is more tolerant of variants, & so will allow us to find more genes & more with SNPs MEAT RINSATE Resistance Gene Drug Class

break Without repair Cell Death With repair Genome

22 In Bacteria: Two Plasmid CRISPR-Cas9 System Plasmid Cas9 Plasmid Cas9 Plasmid CRISPR Double-strand DNA break Without repair Cell Death With repair Genome editing or Genome engineering CRISPR-Cas System in Bacteria

23 CRISPR-Cas9 Results against E. coli O157: H7 pcrispr (Log CFU/reaction) pcrispr w/bsai (Log CFU/reaction) pcrispr w/stx (Log CFU/reaction) Repeat Repeat pcrispr (Control) pcrispr w/bsai pcrispr w/stx CRISPR-Cas System in Bacteria

24 So, what is Antibiotic Resistance? Time & DNA Transfer Microbial Ecology Group (MEG) at CSU

The Microbiome of Food Animals and the Effects of Antimicrobial Drugs

Microbial Ecology Group The Microbiome of Food Animals and the Effects of Antimicrobial Drugs Paul S. Morley DVM, PhD, DACVIM Professor of Epidemiology and Infection Control / Colorado State University