Whole-Genome Sequencing for Surveillance of AMR in ... Whole-Genome Sequencing for Surveillance of AMR

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  • Whole-Genome Sequencing for Surveillance of AMR in

    Foodborne Bacterial Pathogens Errol Strain

    Senior Advisor for Science Informatics National Antimicrobial Resistance Monitoring System (NARMS)

    FDA Center for Veterinary Medicine

    CASSS Meeting 2/20/2022

  • 2

    Disclaimer The views expressed in this presentation are the thoughts and opinions of the author and do not represent the views or policies of the FDA. Center for Veterinary Medicine

    • Office of Research, Laurel MD • Changes in the antimicrobial

    susceptibility of enteric (intestinal) bacteria – food, animals, humans

    • Track & limit spread of resistance

  • 3

    Overview 1. What is NARMS 2. Whole-Genome Sequencing (WGS) and

    Food Safety 3. Genotype = Phenotype 4. NCBI Pathogen Detection

  • 1. What is NARMS?

  • 5

    The Review on Antimicrobial Resistance Chaired by Jim

    O’Neill December 2014

    Global Burden of rise in

    Antimicrobial Resistance

    (AMR) – 2.5-3% reduction in

    GDP by 2050

    FDA\NARMS – Monitor burden of

    resistance from food animals

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    • NARMS is a collaborative program of state and local public health departments and universities, the FDA, the Centers for Disease Control and Prevention (CDC), and the U.S. Department of Agriculture (USDA).

    • This national public health surveillance system tracks changes in the antimicrobial susceptibility of enteric (intestinal) bacteria found in ill people (CDC), retail meats (FDA), and food animals (USDA) in the United States.

    • The NARMS program helps promote and protect public health by providing information about emerging bacterial resistance, how resistant infections differ from susceptible infections, and the impact of interventions designed to limit the spread of resistance.

    • NARMS data are used by FDA to make regulatory decisions designed to preserve the effectiveness of antibiotics for humans and animals.

  • Random stratified sampling in 21 States

    ORA Imported Foods

    Random cecal sampling of national production

    at slaughter

    Eastern FSIS Laboratory

    Animal PopulationRetail Meats

    21 States Labs

    Human Population

    Physician Visit

    Local Lab

    State Lab

    Campylobacter

    Salmonella

    Enterococcus

    E. coli

    HACCP isolates

    FSIS

    Data Integration

    Integrated Report

    (E. coli O157:H7, S. Typhi, Shigella, Vibrio)

    Integrated Surveillance of Antimicrobial Resistance in Foodborne Bacteria:1996

    Chicken Turkey Pork Beef

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    • Cover all 4 U.S census regions: W,

    MW, NE and S; • Collect samples in

    area that represent 22.67% of the US

    population; • Test samples from

    states that encompass 58.44%

    of the U.S population;

    State Public Health Departments and Universities 2002 CT, GA, MD, MN, TN, OR 2003 CT, GA, MD, MN, TN, OR, NY, CA 2004 CT, GA, MD, MN, TN, OR, NY, CA, CO, NM 2008 CT, GA, MD, MN, TN, OR, NY, CA, CO, NM, PA 2012 CT, GA, MD, MN, TN, OR, NY, CA, CO, NM, PA, WA, LA, MO, 2017 CT, GA, MD, MN, TN, OR, NY, CA, CO, NM, PA, WA, LA, MO, IA, SC, KS, ND/SD, TX/OK, NC 2020 CT, GA, MD, MN, TN, OR, NY, CA, CO, NM, PA, WA, LA, MO, IA, SC, KS, ND/SD, TX/OK, OH, HI

    Retail Meat Sampling

  • 9

    NARMS Panels (2018)

    Salmonella Campylobacter E. coli Enterococcus Amoxicillin-Clavulanic Acid X X Ampicillin X X Azithromycin X X X Cefoxitin X X Ceftiofur X X Ceftriaxone X X Chloramphenicol X X X X Ciprofloxacin X X X X Clindamycin X Daptomycin X Erythomycin X X Flavovmycin X Florenicol X Gentamicin X X X X Kanamycin X Lincomycin X Linezolid X Meropenem X X X Nalidixic Acid X X X Nitrofurantoin X Penicillin X Streptomycin X X X Sulfamethoxazole-Sufisoxazole X X Telithromycin X Tetratracycline X X X X Tigecycline X Trimethoprim-Sulfamethoxazole X X Tylosin X Vancomycin X

  • 10

    Veterinary Laboratory Investigation and

    Response Network (Vet-LIRN)

    • 2017-2018 pilot to evaluate the feasibility of using Vet-LIRN veterinary diagnostic laboratories to monitor the

    antimicrobial susceptibility of three veterinary pathogens:

    Escherichia coli and Staphylococcus pseudintermedius in dogs

    and Salmonella enterica in any host. Approximately 5,000

    isolates from clinically sick animals were collected and tested

    • Twenty Vet-LIRN Source diagnostic laboratories collected isolates and tested the susceptibility using Clinical and

    Laboratory Standards Institute (CLSI) methods. Additional

    information about the pathogen (the organ it came from, the

    animal species, which part of the country) was reported.

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    Pettengill JB, et al. Distribution of antimicrobial resistance genes across Salmonella enterica isolates from animal and non-animal foods. Submitted.

  • 2. WGS and Food Safety

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    Why Develop a WGS based Network?

    Tracking and Tracing of food pathogens • Insufficient resolution of current tools

    -matching clinical to environmental -improve the environmental database

    • Faster identification of the food involved in the outbreak

    • Limited number of investigators vs. facilities and import lines

    • Global travel • Global food supply

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    Farm/Supplier Processing Facility/ Distributor

    Food/Feed Patient

    Laboratory

    WGS and Food Safety

  • Microbiology Laboratory Workflow

    GenomeTrakr Labs & Collaborators

    Salmonella

    Listeria

    E.coli and Shigella, Campylobacter jejuni, Klebsiella pneumoniae, Mycobacterium tuberculosis, Acinetobacter baumannii, Neisseria, Pseudomonas aeruginosa, Enterobacter, Clostridioides difficile, Vibrio parahaemolyticus, Vibrio cholerae, Legionella pneumophila, Cronobacter, Serratia marcescens, Staphylococcus pseudintermedius, Klebsiella oxytoca, Citrobacter freundii, Vibrio vulnificus, Clostridium botulinum, Clostridium perfringens, Providencia alcalifaciens, Elizabethkingia anopheles, Morganella morganii, Photobacterium damselae, Corynebacterium striatum, Kluyvera intermedia

  • 16

  • 17

    > 300K Foodborne Pathogens

  • 3. Genotype = Phenotype

  • 19

    Genotype-Phenotype Correlation

    Isolate N33849PS

    Phenotypic susceptibility

    NAL FIS CHL TET STR

    Sequencing/ genotyping GyrA (S83L) sul2 floR tetA strA/strB

  • 20

    Predicting Resistance from the Genome

    Bacterium Gen/Phe correlation Reference

    99.70% Zankari et al., 2013, J Antimicrob Chemother

    99.00% McDermott et al., 2016, Antimicrob Agents Chemother

    97.10% Stoesser et al., 2013, J Antimicrob Chemother

    98.50% Tyson et al 2015., J Antimicrob Chemother

    Campylobacter spp. 99.20% Zhao et al 2015., J Antimicrob Chemother

    Staphylococcus aureus 98.80% Gordon et al 2014., J Antimicrob Chemother

    Pneumococcus 98.00% Metcalf et al 2016, Clin Microbiol Infect

    Enterobacteriaceae (B-lacs) 100.00% Shelburne et al, 2017 Clin Infect Dis

    95.30% Phelan et al 2016. Genome Med

    92.30% Walker et al 2015. Lancet Infect Dis

    Salmonella enterica

    Escherichia coli

    Mycobacterium

  • 21

    Resistance Genes

    21

    Resistance Gene Antimicrobial Class Resistance Phenotype aac(3)-Id, aac(3)-IId, aac(3)-IV, aac(3)-VI Aminoglycosides Gentamicin aadB Aminoglycosides Gentamicin, Kanamycin aph(3')-la, aph(3')-II Aminoglycosides Kanamycin aph(4)-la Aminoglycosides Hygromycin B aadA, 1, 2, 5, 7, 12, 13, 24, aac(6')-lb, aph(6)-lc, aph(3')-

    lb (strA), aph(6)-Id (strB)

    Aminoglycosides Streptomycin

    blaCMY-2, blaCTX-M-1, blaCTX-M-14b, blaSHV-2a, blaFOX-6 β-lactams Amoxicillin-clavulanic acid, Ampicillin, Ceftriaxone, Cefoxitin,Ceftiofur

    blaCARB-2, blaHERA-3, blaLAP-1, blaOXA2, blaTEM-1 β-lactams Ampicillin

    ble, blmS Bleomycin sul1, 2, 3 Folate Pathway Inhibitors Sulfisoxazole dfr5, 7, dfrA1,8, 12, 14, 15, 17. Folate Pathway Inhibitors Trimethoprim-Sulfamethoxazole mphA Macrolides Azithromycin mel, mphB, mphE Macrolides Erythromycin catA1, cmlA, floR Phenicols Chloramphenicol

    oqxA, oqxB

    Phenicols,Olaquindox, Quinolones Olaquindox

    qacH QAC Disinfectants, ethidium bromide qnrA, qnrB19, qnrS Quinolones Reduced susceptibility to NAL, CIP tetA, B, C, D, G, M, O, X Tetracyclines Tetracycline linB Lincosamides Lincomycin

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    Correlation between Antimicrobial Resistance Phenotype and Genotype in Salmonella (n=640)

    ANTIBIOTIC Phenotype: Resistant Phenotype: Susceptible

    Genotype: resistant

    Genotype: susceptible

    Genotype: resistant

    Genotype: susceptible

    Sensitivity Specificity

    GENTAMICIN 99 6 5 530 94.3% 99.1% STREPTOMYCIN 257 3 35 345 98.8% 90.8% AMOXY/CLAV 114 2 0 524 98.3% 100.0% CEFOXITIN 93 2 21 524 97.9% 96.1% CEFTIOFUR 113 0 4 523 100.0% 99.2% CEFTRAXONE 116 0 1 523 100.0% 99.8% AMPICILLIN 241 1 1 397 99.6% 99.7% SULFA 244 1 0 395 99.6% 100.0% TRM/SULFA 19 3 0 618 86.4% 100.0% AZTREONAM 1 0 0 639 100.0% 100.0% CHLORAMPHENICOL 44 0 1 595 100.0% 99.8% CIPROFLOXACIN 4 0 0 636 100.0% 100.0% NALIDIXIC ACID 13 2 0 625 86.7% 100.0% TETRACYCLINE 349 0 0 291 10