Overview of NARMS Program and Detecting Emerging/Novel Antimicrobial Resistance Genes Using WGS Shaohua Zhao DVM, MPVM, PhD U.S. Food and Drug Administration Center for Veterinary Medicine Office of Research Laurel, MD Disclaimer This communication is consistent with 21 CFR 10.85 (k) and constitutes an informal communication that represents my best judgment at this time but does not constitute an advisory opinion, does not necessarily represent the formal position of FDA, and does not bind or otherwise obligate or commit the agency to the 1 views expressed.
Outline Public concern of antimicrobial resistance Review US NARMS program Detecting Emerging and Novel Antimicrobial Resistance Genes in Campylobacter Using Whole Genome Sequencing WGS) Using WGS to predict resistant phenotype/application WGS in NARMS program) 2
The Public Health Action Plan Four Principal Components Surveillance Goal 1: Improve the detection, monitoring, and characterization of drug-resistant infections in humans and animals. Goal 2: Better define, characterize, and measure the impact of antimicrobial drug use in humans and animals in the United States. Prevention and Control Research Goal 1: Facilitate basic research on antimicrobial resistance. Goal 2: Facilitate the translation of basic research findings into practical applications for the prevention, diagnosis, and treatment of resistant infections. Goal 3: Facilitate clinical research to improve the treatment and prevention of antimicrobial drug resistant infections. Goal 4: Conduct and support epidemiological studies to identify key drivers of the emergence and spread of AR in various populations. Product Development
NARMS Study Population and Target Organisms Human Isolates ** Food-producing animals Retail Food Isolates 1997 2002 Campylobacter Non-typhoidal Salmonella Generic E. coli Enterococcus **also piloted MRSA, C. diff and VRE in foods Campylobacter Non-typhoidal Salmonella Generic E. coli Enterococcus 1996 Campylobacter Non-typhoidal Salmonella E. coli O157:H7 Typhoidal Salmonella Shigella Vibrio (2009) 5
Human Salmonella Surveillance Sites* 1996: 14 sites 1999: 17 sites 2002: 28 sites 2003: 53 sites *In 1996, surveillance began in 14 sites. In 2003, participation increased to nationwide: 50 state and three local health 6 departments, Los Angeles County (joined in 1996), New York City (1996), and Houston, Texas (2003).
Human Campylobacter Surveillance Sites In 1997, surveillance was initiated in five states. Additional sites joined after 1997. By 2003, participation included 10 sites: CA, CO, CT, GA, MD, MN, NM, NY, OR, and TN. 7
NARMS Retail Meat Surveillance Partnership with state FoodNet Sites CT, GA, MD, MN, TN 1/2002 CT, GA, MD, MN, TN, OR 9/2002 CT, GA, MD, MN, TN, OR NY, CA 1/2003 CT, GA, MD, MN, TN, OR NY, CA, CO, NM 1/2004 CT, GA, MD, MN, TN, OR NY, CA, CO, NM, PA 1/2008 CT, GA, MD, MN, TN, OR NY, CA, CO, NM, PA, WA, LA, MO 1/2013 Sampling scheme Each site purchases 10 packages each of chicken breasts, pork chops, ground turkey, ground beef per month All 14 sites culture for Salmonella and Campylobacter In addition, 3-4 sites (GA, OR, TN, ±MD ) culture for E. coli and Enterococcus In 2005, changed from convenience to randomized sampling Sample total = 6,720 per annum Retail Food Testing Sites
Sampling at Slaughter HACCP* (1997-Current) sources: carcass swabs, rinses, ground product New in-plant cecal sampling (2013-Current) Swine Cattle Chicken Turkeys Campylobacter x Salmonella x x x x E. coli x Enterococcus x Swine (Hogs, Sows) Cattle (Beef, Dairy) Young Chicken Campylobacter x x x x Salmonella x x x x E. coli x x x x Enterococcus x x x x Young Turkeys *HACCP: Hazard Analysis Critical Control Point- samples collected to assess Salmonella (and now Campylobacter) contamination and i.e. interventions where appropriate. Sampling became risk based in 2006. 9
Interpretive Criteria Used for Antimicrobial Susceptibility Testing of Salmonella and E. coli Breakpoints (µg/ml) Antimicrobial Class Antimicrobial Agent Susceptible Intermediate Resistant Aminoglycosides Gentamicin 4 8 16 b-lactam/b-lactamase Inhibitor Combinations Kanamycin 16 32 64 Streptomycin 32 N/A 64 Amoxicillin Clavulanic Acid 8 / 4 16 / 8 32 / 16 Cephems Cefoxitin 8 16 32 Ceftiofur 2 4 8 Ceftriaxone 1 2 4 Folate Pathway Inhibitors Sulfisoxazole 256 N/A 512 Trimethoprim Sulfamethoxazole 2 / 38 N/A 4 / 76 Macrolides Azithromycin 16 N/A 32 Penicillins Ampicillin 8 16 32 Phenicols Chloramphenicol 8 16 32 Quinolones Ciprofloxacin Salmonella 0.06 0.12-0.5 1 -Breakpoints adopted from CLSI, except for azithromycin and streptomycin, which have no CLSI breakpoints. The breakpoints for azithromycin and streptomycin are NARMSestablished breakpoints developed for resistance monitoring. They should not be used to predict clinical efficacy. -Sulfamethoxazole was tested from 1996 through 2003 and was replaced by sulfisoxazole in 2004 - The revised ciprofloxacin breakpoint for invasive Salmonella from the CLSI M100- S22 document, published in January 2012, is used. The revised breakpoints were applied to all non-typhoidal Salmonella. In previous NARMS reports, breakpoints from the CLSI M100-S21 were used. E. coli 1 2 4 Nalidixic acid 16 N/A 32 Tetracyclines Tetracycline 4 8 16 10
EUCAST Interpretive Criteria Used for Antimicrobial Susceptibility Testing of Campylobacter Breakpoints (µg/ml) jejuni coli Antimicrobial Class Antimicrobial Agent Concentration Range (µg/ml) Susceptible Resistant Susceptible Resistant Aminoglycosides Gentamicin 0.12-32 2 4 2 4 Ketolides Telithromycin 0.015-8 4 8 4 8 Lincosamides Clindamycin 0.03-16 0.5 1 1 2 Macrolides Azithromycin 0.015-64 0.25 0.5 0.5 1 Erythromycin 0.03-64 4 8 8 16 Phenicols Florfenicol 0.03-64 4 8 4 8 Quinolones Ciprofloxacin 0.015-64 0.5 1 0.5 1 Nalidixic acid 4-64 16 32 16 32 Tetracyclines Tetracycline 0.06-64 1 2 2 4 11
Resistance among Salmonella Isolated from Humans Resistance to 3 agents increased from <2% in the 1940s to 28% in the 2000s among this collection 12
Percent Resistance MDR among Salmonella from Humans, Poultry and Meats 60% 50% (R >3 classes ) 40% 30% 20% 10% 0% 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 Year Humans Retail Chicken Ground Turkey Chickens Turkeys 13
MDR among Salmonella Heidelberg 14
Antibiotic Resistant Gene Database Antimicrobial Class Number of Genes Aminoglycoside Resistance 282 Bacitracin Resistance 307 Beta-lactam Resistance 1460 Bicyclomycin Resistance 58 Chloramphenicol Resistance 154 Fosfomycin Resistance 62 Fusaric_acid Resistance 4 Glycopeptide Resistance 241 Lincosamide Resistance 21 Macrolide & MLS Resistance 207 MAR and Efflux Genes 1372 Nucleoside Resistance 11 Polymyxin Resistance 29 QAC Resistance 45 Quinolone Resistance 96 Rifamyxin Resistance 31 Streptogramin Resistance 47 Streptothricin Resistance 7 Sulfonamide Resistance 34 Tetracenomycin C Resistance 10 Tetracycline Resistance 313 Thiostrepton Resistance 2 Trimethoprim Resistance 150 Viomycin Resistance 1 Grand total 4944
Strategies to detect genes to the level of: Family Phylogroup Specific variants
Detection of AR genes PCR/Sequence Microarray Whole Genome Sequencing (WGS) 18
Accelerating Technology & Plummeting Cost Next Generation Sequencing $795 in 1977 (=$2,800 in current $) Lower cost = more innovative and more samples
$3,500 Cost per bacterial genome $3,000 454 $2,500 $2,000 $1,500 Illumina Miseq $1,000 $500 $0 2007 2008 2009 2010 2011 2012 2013 $70/genome in 2014 $40/genome in 2015 w/ Illumina NextSeq Technology Lower cost = more innovative and more samples
From WGS to Antibiotic Resistance Genotype BLAST aac(3)-iia, aada1, aph(3')-ia cata1, teto Local Blast AR Gene Database Acquired AR genes DNA from Single colony Sequencing With Illumina Miseq Assembly CLC Genomics Workbench Sequences alignment gyra gene 23S rrna gene AR Genotype Point mutations related to AR 21
Detecting Novel Gentamycin Resistance Genes in Campylobacter Isolated from Human, Retail Chicken and Food Animals in NARMS Program 22
Aminoglycosides They are highly potent, broad-spectrum bactericidal antibiotics, commonly used in the treatment of infections caused by aerobic G- bacteria as well as some selected G+ bacteria Common aminoglycoside antibiotics Gentamicin Tobramycin Amikacin Kanamycin Neomycin Streptomycin Inhibit protein synthesis 23
Aminoglycosides Resistance The primary mechanism of resistance to aminoglycoside antibiotics is enzymatic inactivation by three major aminoglycoside-modifying enzymes: aminoglycoside acetytransferases (AACs) aminoglycoside nucleotidyltransferases (ANTs) aminoglycoside phosphotransferases (APHs) More than 300 aminoglycoside resistance genes have been identified 24
Prevalence of GEN R Campylobacter coli from Different Sources 25
PFGE and AST Profiles of Gen R Campylobacter Isolates from Humans and Retail Chicken Gen R and Tet R C.coli from retail chicken and humans aph(2 )-Ig 26
Novel Aminoglycosides Resistance Genes Seven mono-functional aminoglycoside 2 - phosphotransferase genes: aph(2 )-Ib aph(2 )-Ic aph(2 )-Ig aph(2 )-If aph(2 )-If1 aph(2 )-If3 aph(2 )-Ih Two bi-functional aminoglycoside2 - phosphotransferase genes: aac(6 )-Ie/aph(2 )-Ia aac(6 )-Ie/aph(2 )-If2 27
Percentage of Amino Acid Identity in the APH(2 ) Family *Bifunctional AAC/APH, only the APH part of the enzyme is used to construct the phylogenetic tree. 28
Timeline of Gen R Campylobacter from Humans (2000-2011) and Retail Chicken (2007-2013) Humans Gen R C.coli Gen R C.jejuni 2007 2012 Retail Chicken 2000 2004 Gen R C.coli Gen R C.jejuni Two Gen R genes (aph-if and aph-ig) were shared between human and RT Chicken isolates aph-ig Humans 2008 2009 2012 Gen R C.jejuni Humans aph-if 2003 2013 2004 29
PFGE and AST Profiles of Gen R C. coli aph-if3 Cluster C: aph-if by PCR and WGS Cluster D: aac(6 )-Ie/aph(2 )-Ia Cluster E: aph-ig aph-ic
PFGE and AST Profiles of GEN R C.jejuni Cluster A: aph-if by PCR aph-ih by WGS (n=7) A aph-if aph-ib aph-if B aph-ig Cluster B: aph-if by PCR aph-ih by WGS (n=2) 31
Comparison of AR Gene Clusters in MDR Plasmid pn29710-1 with pcg8245 and SX81 Track 1: AR island in SX81 Track 2: AR gene cluster in pcg8245 Track 3: AR gene cluster in pn28710 Track 4: ptet Track 5: GC content of pn29710-1 32
Summary Gen R has increased rapidly in Campylobacter in the U.S. 9 variants of Gen R genes were identified 7 were identified for the first time in Campylobacter 5 were novel aminoglycoside resistance genes Human isolates contained more diverse Gen R genes than retail chicken isolates PFGE and Gen R genotypes indicated that contaminated retail chicken could serve as a source of Gen R C. coli infections in humans. WGS is a powerful tool to detect resistance genotypes. 34
Correlation between Antimicrobial Resistance Phenotype and Genotype in Campylobacter and Salmonella Campylobacter spp: Sample size: 104 isolates Source: Retail meat (n=74), Humans (n=40) Representative MDR patterns of human isolates recovered from 2000-2011 and retail meat isolates from 2004-2013 Salmonella Sample size: 285 isolates Source: Retail meat (n=181) and Humans (n=104) Representative unique combinations of resistance pattern, source and serotype from 2011 to 2012 35
Resistance genes database at FDA/CVM Drug class Gene count Cluster count Aminoglycoside 297 120 Beta-lactam 1253 178 Fosfomycin 14 10 Fusaric acid 4 2 Glycopeptide 234 88 Lincosamide 19 8 Macrolide & MLK 174 61 Metronidazole 13 8 Olaquindox and phenicol 2 2 Phenicol 147 40 QAC 12 8 Quinolone 80 13 Rifamyxin 29 7 Streptogramin 26 16 Streptothricin 7 3 Sulfonamide 34 5 Tetracenomycin C 4 4 Tetracycline 271 55 Thiostrepton 1 1 Trimethoprim 116 39 Viomycin 1 1 Total 2738 669 36
Campylobacter Resistance Phenotypes and Genotypes strain AST Gene GyrA 86 23S 2074 23S 2075 N13165 CIP, GEN, NAL, TET, ant(6), aph(3')-iiia, blaoxa-61, sat4, teto, I A A N14784 AZI CIP CLI ERY NAL TEL TET aph(3')-iiia blaoxa-61 teto I A G N14840 AZI CLI ERY TEL TET aph(3')-iiia blaoxa-61 teto T A G N15262 AZI CLI ERY TEL T T A N15870 AZI CIP CLI ERY NAL TEL TET aph(3')-iiia teto I A G N1630 CIP NAL TET blaoxa-61 teto I A A N1636 AZI CIP CLI ERY NAL TEL TET aph(3')-ic teto I A G N18323 AZI CLI ERY TEL TET aph(3')-ic teto T A G N18725 AZI CLI ERY TEL TET aph(3')-iiia blaoxa-61 teto T A G N20320 AZI CIP CLI ERY NAL TEL TET blaoxa-61 teto I A G N20344 GEN, TET, aph(2'')-ic, aph(3')-iiia, blaoxa-61, teto, T A A N20402 GEN, TET, aph(2'')-ic, aph(3')-iiia, blaoxa-61, teto, T A A N23169 AZI CLI CIP ERY NAL TEL TET aph(3')-iiia blaoxa-61 teto I A G N23392 AZI CLI ERY TEL TET aph(3')-iiia teto T A G N26070 AZI CIP ERY NAL TEL TET aph(3')-iiia blaoxa-61 teto I A G N26697 AZI CLI CIP ERY NAL TEL TET blaoxa-61 teto I A G N26699 AZI CLI ERY TEL TET teto T A G N279 AZI CLI ERY TEL TET teto T A G N287 AZI CLI ERY TEL TET aph(3')-iiia blaoxa-61 teto T A G N3506 AZI CLI ERY TEL TET aph(3')-ic teto T A G N3508 AZI CLI ERY TEL TET aph(3')-ic teto T A G N39665 GEN TET aade ant(3'') aph(2'')-ig aph(3')-iiia sat4 teto T A A N39671 GEN TET aade ant(3'') aph(2'')-ig aph(3')-iiia sat4 teto T A A N39677 GEN TET aade ant(3'') aph(2'')-ig aph(3')-iiia sat4 teto T A A N40944 GEN TET aade ant(3'') aph(2'')-ig aph(3')-iiia blaoxa-61 sat4 teto T A A 37
Drug class Corrections of Resistance Phenotypes and Genotypes in Campylobacter Number of resistance and susceptible isolates Correlation (%) Gentamicin 40 R /34 S 98.6 Tetracycline 68 R /6 S 100 Quinolone 24 R /50 S 100 Macrolide 35 R /39 S 100 Lincosamides 35 R /39 S 97.3 Keolides 34 R /40 S 95.9 Total 74 98.6 Correlation: The tested phenotype and the genotype matched bidirectional 38
Salmonella Resistance Phenotypes and Genotypes (Retail Meat) CVM_NUMB ER AST GENE N29307 Pan susceptible N29309 Pan susceptible N29310 AMP blatem-1 N29313 AMC AMP AXO FIS TET TIO blacmy-2 sul2 teta N29315 AMC AMP AXO FOX FIS STR TET TIO aph(3'')-ib aph(6)-id blacmy-2 blatem-1 sul2 teta N29317 AMP GEN STR TET aac(3)-iia aada aph(3'')-ib aph(6)-id blatem-1 teta N29321 AMP GEN KAN FIS STR aada2 aadb aph(3')-ia blatem-1 sul1 N29323 AMC AMP AXO KAN FIS TET TIO aph(3')-ia blacmy-2 sul2 teta N29338 AMP GEN STR TET aac(3)-iia aada blatem-1 teta N29339 AMP KAN STR FIS TET aph(3')-ia aph(3'')-ib aph(6)-id blatem-1 sul2 tetb tetc tetd N29343 KAN FIS TET aph(3')-ia sul2 teta N29350 AMP CHL FIS STR TET aada2 blacarb-2 flor sul1 tetg N29351 AUG AMP FOX TIO AXO GEN blacmy-61 N29355 Pan susceptible N29357 GEN FIS STR aac(3)-vi aada1 sul1 N29360 TET tetb tetc tetd N29362 AMP GEN TET aac(3)-iia aada blatem-1 teta N29363 STR TET aph(3'')-ib aph(6)-id tetb tetc tetd N29367 Pan susceptible N29369 AMP TET blatem-1 tetb tetc tetd N29377 AMP GEN STR TET aac(3)-iia aada blatem-1 teta tetb tetc tetd N29378 FIS STR TET aph(3'')-ib aph(6)-id sul2 teta N29379 AMC AMP AXO CHL FOX GEN FIS STR TET TIO aac(3)-vi aada1 aph(3'')-ib aph(6)-id blacmy-2 flor sul1 sul2 teta 39 N32052 AMP GEN TET aac(3)-iia aada blatem-1 teta
Salmonella Resistance Phenotypes and Genotypes (Clinical Isolates) CVM AST Patterns gene GyrA 83 GyrA 87 43743 ASSu aada12 blatem-1 sul1 S D S 43744 ASSuT aph(3'')-ib aph(6)-id blatem-1 sul2 teta S D S 43745 ACSSuTGen aac(3)-vi aada1 aada2 blacarb-2 flor sul1 tetg S D S 43746 ASSuTKan aph(3')-ia aph(3'')-ib aph(6)-id blatem-1 sul2 tetb tetc tetd S D S 43747 CT cata1 teta S D S 43748 Pan susceptible S D S 43749 ASSuTNal aph(3'')-ib aph(6)-id blatem-1 sul2 teta S Y S 43750 SuGen aac(3)-vi aada1 sul1 S D S 43751 Pan susceptible S D S 43752 STKan aph(3'')-ib aph(3')-ii aph(6)-ic aph(6)-id ble tetb tetc tetd S D S 43753 Pan susceptible S D S 43754 ASSuSxt aph(3'')-ib aph(6)-id blatem-1 sul2 dfra8 S D S 43755 ASSuNal aph(3'')-ib aph(6)-id blatem-1 sul2 S Y S 43756 ACipNal blatem-1 F Y I 43757 Sxt aac(3)-vi aada1 dfra1 S D S 43758 ASuGen aac(3)-vi aada1 blahera-3 sul1 S D S 43759 ACSSuT aada2 blacarb-2 flor sul1 tetg S D S 43760 Pan susceptible S D S 43761 ASSuTNalSxt aph(3'')-ib aph(6)-id blatem-1 sul2 teta dfr5 S Y S 43762 CSSuT aph(3'')-ib aph(6)-id flor sul2 teta S D S 43763 SuTSxt qnrs sul1 teta dfra1 S D S 43764 AAuCxCfFox blacmy-2 S D S 43765 CSuTKanNalSxt aac(3)-iv aada1 aph(3')-ia aph(4)-ia flor sul1 teta dfra14 S Y S 43766 ACSSuTAuCf aph(3'')-ib aph(6)-id blacmy-2 flor sul2 teta S D S 43837 SuSxt aada1 sul1 dfra1 S D S 43838 ACSSuTAuCxCfFoxCipNalSxt aada2 aph(3'')-ib aph(6)-id blacmy-2 bleo oqxb oqxa flor qnrb19 sul2 teta dfra12 S D S 40 ParC 80
Summary Based on current knowledge and technology, WGS predicts resistance very well 98-100% correlation for the drug classes beta-lactam, tetracycline, chloramphenicol, sulfonamide, trimethoprim/sulfamethoxazole, macrolides and quinolone 92-97% correlation for aminoglycoside, lincosamides and keolides A comprehensive and accurate database of ARG is critical Reasons for disconnect AST interpretation standard experimental and analytical error variable gene expression level unknown mechanisms 41
Benefits of a WGS Strategy in NARMS WGS has potential to serve as a single assay of NARMS surveillance and supplant multiple methods 1. Classical serotyping 2. PFGE and other molecular typing methods 3. In vitro antimicrobial susceptibility testing 4. Multiple PCR assays to detect resistance genes and plasmid typing And to provide: 1. Genome surveillance 2. Virulence profiles 3. Markers for source attribution 4. Better understanding of emerging resistance trends, origin, dissemination and selection pressure 5. Cost saving 42
Acknowledgement FDA: USDA: CDC: NARMS retail meat arm working group NARMS animal arm working group (ARS and FSIS) NARMS human arm working group CDC PulseNet FoodNet/State Public Health Laboratories 43