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Shiga toxin Producing Escherichia coli-Nancy Strockbine PhD

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Eastern PA Branch-ASM, 41st Annual Symposium, November 17, 2011

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Shiga toxin Producing Escherichia coli-Nancy Strockbine PhD

  1. 1. Pathogenesis and Detection of Shiga toxin- producing Escherichia coli ─ Food Safety Issues Related to E. coli O157 and non-O157 Strains Nancy A. Strockbine, Ph.D. Chief, Escherichia and Shigella Reference Unit Enteric Diseases Laboratory Branch Presented at the Eastern Pennsylvania Branch-ASM 41st Annual Symposium“Global Movement of Infectious Pathogens and Improved Laboratory Detection Methods” Philadelphia, Pennsylvania 17 November 2011 Division of Foodborne, Waterborne and Environmental Diseases National Center for Emerging and Zoonotic Infectious Diseases
  2. 2. Outline• Pathogenesis• Epidemiology and surveillance• Detection• Food safety
  3. 3. Terminology• STEC – Escherichia coli that produce one or more Shiga toxins• EHEC– A subset of STEC that are capable of causing diarrheal disease, including bloody diarrhea and HUS
  4. 4. WHAT DO THEY CAUSE?
  5. 5. Clinical Presentation of STEC Disease in Humans • Asymptomatic infection • Nonbloody diarrhea • Bloody diarrhea/hemorrhagic colitis • Hemolytic uraemic syndrome (6-15%) – Microangiopathic hemolytic anemia – Thrombocytopenia – Acute renal failure • Chronic kidney failure in 25% of those with HUS • Neurologic symptoms seen in TTP
  6. 6. Sequence of events in STEC infection STEC O157 ingested Non-O157 STEC ingested 3 - 4 days 3 - 4 days non-bloody diarrhea, non-bloody diarrhea, abdominal cramps abdominal cramps (short lived fever) (short lived fever) 80% 1 - 2 days 45% 1 - 2 days bloody bloody diarrhea diarrhea 94% 6-15% 98% <2% 5 - 6 days 5 - 6 days (up to 2-3 (up to 2-3 weeks)resolution HUS resolution weeks) HUS
  7. 7. E. coli Pathotypes ―Flexible Genome‖• ~ 9,400 genes in pangenome UPEC/ NMEC EIEC/ Shigell EAEC a• ~ 2,200 genes in core EPEC ETEC• Drivers of genetic diversity Commensal – Phages – Plasmids 4,238 – 5,589 genes per bacterial genome – Pathogenicity Islands ~ 2,200 Rasko, DA et al. J. Bacteriol. 2008
  8. 8. How big is 1030 ?1030 phages equals mass of ~106 Blue Whales106 Blue Whales end-to-end will circle over half the Earth’s circumference
  9. 9. Shiga toxins Phage encoded toxins Act locally and systemically O’Brien AD et al. Science 226:694-696, 1984.  Receptors on intestinal epithelium and kidney endothelium  Inhibit protein synthesis  binding of toxin to vascular tissue thought to trigger coagulation cascade Two subgroups (Stx1 and Stx2)  Strains that produce Stx2 are more virulent Necessary but not sufficient to cause disease  Other virulence factors involved
  10. 10. Potential Virulence GenesGene or plasmid Predicted product or phenotypestx1 Shiga toxin 1stx2 Shiga toxin 2eae intiminEHEC-hlyA (ehxA) EHEC hemolysin (enterohemolysin)espP serine proteasekatP catalasecdt cytolethal distending toxinefa-1 EHEC factor of adherence (Efa1)saa STEC autoagglutinating adhesin (Saa)iha IrgA homologue adhesin (Iha)lfpA Major fimbrial subunit of LPF (Long polar Fimbriae)ent/espL2, nleB, nleE, nleF, genes from genomic islands OI-122 and OI-71nleH1-2, nleAirp-2 Iron-repressible protein 2fyuA Yersiniabactin receptor
  11. 11. Virulence profile and clinical manifestation in559 Danish STEC patients 1994-2005 Other 100% D 80% PD 60% BD 40% PBD 20% HUS 0% stx2 + stx1 + stx1 + stx2 stx1 + stx1 eae stx2 + eae stx2 eae Courtesy Flemming Scheutz, WHO Collaborating Center for Escherichia and Klebsiella, SSI
  12. 12. Stx1 : 4 subtypes a - d 7-8 variantsPairwise (OG:100%,UG:0%) (FAST:2,10) Gapcost:0%VT1 translated sequences 100 96 97 98 99 Stx1a-S._dysenteriae-3818T a Stx1a-S._sonnei-CB7888 Stx1b-O111-CB168 Stx1b-O157-EDL933 b Stx1b-O48-94C Stx1b-O111-PH Stx1c-O174-DG131-3 c Stx1d-ONT-MHI813 d Courtesy Flemming Scheutz, WHO Collaborating Center for Escherichia and Klebsiella, SSI
  13. 13. Pairwise (OG:100%,UG:0%) (FAST:2,10) Gapcost:0% Disc. unk.vtx_TRANSL 10082 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 vtx2d-O157-7279 d vtx2d-O174-EC1720a vtx2d-O91-a-B2F1 vtx2d-O91-b-B2F1 vtx2d-O8-C466-01B Stx2 : vtx2d-C_freundii-LM76.. vtx2d-O6-NV206 vtx2d-O22-KY-O19 vtx2d-O73-C165-02 7 subtypes vtx2a-O157-EDL933 vtx2a-O26-FD930 vtx2a-O157-SF a a-g vtx2a-O48-94C vtx2a-O26-126814 vtx2a-E_cloacae-95MV2 vtx2c-O157-E32511 vtx2c-O157-FLY16 c vtx2c-O157-C394-03 vtx2c-O157-469 vtx2c-O174-b-031 35 variants vtx2g-O2-7v vtx2g-O2-S86 vtx2g-Out-S-8 g vtx2b-O111-S-3 vtx2b-O96-S-6 vtx2b-O22-3143-97 vtx2b-ONT-5293-98 vtx2b-O118-EH250 vtx2b-O16-6451-98 b vtx2b-O174-a-031 vtx2b-O111-PH vtx2e-O139-412 vtx2e-O22-3615-99 e f vtx2e-O101-E-D43 vtx2f-O128-T4-97 Courtesy Flemming Scheutz, WHO Collaborating Center for Escherichia and Klebsiella, SSI
  14. 14. Shiga toxin 2 (stx2) subtype and clinical presentation Subtype Non-HUS * HUS* stx2a 60 11 stx2c 49 1 stx2d-activatable 4 stx2d 39 stx2e 2 stx2-variant 3 stx2 + stx2c 23 7 stx2 + stx2d 1 2x stx2-activatable 4 stx2c + stx2-activatable 1 Total 186 19stx2 OR* 32.5 > stx2c OR* 4.7 for HUS*) OR: odds ratio; multivariant analysis adjusted for age Ethelberg et al. 2004 EID: vol 10 Courtesy Flemming Scheutz, WHO Collaborating Center for Escherichia and Klebsiella, SSI
  15. 15. Lifestyle options of Shiga toxin-converting bacteriophages Lysogenic cycle Phage DNA integrates into host chromosome Lytic cycle Bacteriophages replicate, toxin production is amplified, cells lyse and release Shiga toxin and phage progenyElectron micrographs by R. Hendrix Slide courtesy Louise Teel, USUHS
  16. 16. Induction of expression of the late gene cluster of lambdoid phagesBasic lambda genome structure… …toxin genes here X att int xis cIII N cI cro cII O P Q stxA/B S R Rz head genes tail genes C1 repressor RecA • Damage to the host cell DNA triggers the SOS response • Expression of the bacterial RecA protein is up-regulated • RecA cleaves the phage repressor of the lytic cycle • Downstream genes, including the toxin genes, get transcribed Slide courtesy Louise Teel, USUHS
  17. 17. Norfloxacin-induced Stx phage being released from a bacterium Allison, HE Future Microbiol. 2:165-174, 2007
  18. 18. Escherichia coli O104:H4 Outbreak in Germany, May 2011Proposed scheme for the origin of a new E. coli pathotype--Enteroaggregative hemorrhagic Escherichia coli Brzuszkiewicz E. et al. Arch Microbiol. 2011
  19. 19. HOW COMMON ARE NON-O157 STEC?
  20. 20. Surveillance systems National surveillance: passive  National Notifiable Disease Surveillance System  Public Health Laboratory Information System  CDC National E. coli Reference Laboratory  PulseNet Sentinel surveillance: active  Foodborne Disease Active Surveillance Network (FoodNet)
  21. 21. FoodNet10 sites , 46 million persons (15% of US population)
  22. 22. Incidence of reported STEC O157 and non-O157 STEC infections, by year, FoodNet, 1996-2009 2.5 STEC O157 Non-O157 STECCases per 100,000 population 2 1.5 1 0.5 0 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 Year Healthy People 2010 objective is 1 case/100,000 persons
  23. 23. Incidence of O157 STEC and non-O157 STEC Cases at FoodNet Sites, 2009 3 2.5Number of cases/100,000 population 2 STEC** 1.5 O157 STEC non- 1 O157 0.5 0 CA CO CT GA MD MN NM NY OR TN Overall 2009
  24. 24. Burden of Illness Surveillance detects the tip of the iceberg Detecting an illness depends on probability of…  ill person seeking medical care  stool sample requested  stool sample received  necessary tests performed  test result positive  infection reported
  25. 25. Proportion of Annual Foodborne Illness in the United States by Pathogen Salmonella spp., nontyphoidal Clostridium perfringens, foodborne Campylobacter spp. Staphylococcus aureus, foodborne Shigella spp. STEC non-O157 Yersinia enterocolitica Bacillus cereus, foodborne STEC O157 V. parahaemolyticus ETEC, foodborne Vibrio spp., other Other diarrheagenic E. coliStreptococcus spp. group A, foodborne S. enterica serotype Typhi Listeria monocytogenes Brucella spp. V. vulnificus Vibrio cholerae, toxigenic Mycobacterium bovis Clostridium botulinum, foodborne 0 200,000 400,000 600,000 800,000 1,000,000 1,200,000 Number of illnesses Scallan et al. 2011 EID 17(1)7-15
  26. 26. Hospitalization rate, % Listeria monocytogenes V. vulnificus Clostridium botulinum, foodborne S. enterica serotype Typhi Mycobacterium bovis Brucella spp. STEC O157 Vibrio cholerae, toxigenic Vibrio spp., other Yersinia enterocolitica Salmonella spp., nontyphoidal V. parahaemolyticus Shigella spp. Campylobacter spp. STEC non-O157 Staphylococcus aureus, foodborne Other diarrheagenic E. coli ETEC, foodborne Clostridium perfringens, foodborne Bacillus cereus, foodborneStreptococcus spp. group A, foodborne 0 10 20 30 40 50 60 70 80 90 100 Scallan et al. 2011 EID 17(1)7-15
  27. 27. Death Rate, % V. vulnificus Clostridium botulinum, foodborne Listeria monocytogenes Mycobacterium bovis Vibrio spp., other Yersinia enterocolitica V. parahaemolyticus Brucella spp. Salmonella spp., nontyphoidal STEC O157 STEC non-O157 Staphylococcus aureus, foodborne Shigella spp. Clostridium perfringens, foodborne Campylobacter spp. Vibrio cholerae, toxigenicStreptococcus spp. group A, foodborne S. enterica serotype Typhi Other diarrheagenic E. coli ETEC, foodborne Bacillus cereus, foodborne 0 5 10 15 20 25 30 35 40 Scallan et al. 2011 EID 17(1)7-15
  28. 28. E. coli O157:H7 Gel
  29. 29. BioNumerics Server Client BNServerwith database Client • Upload & download of information • Internet based
  30. 30. Shiga toxin gene distribution among 19,402 STEC from the US, 2006-2010 by SerogroupNo. isolates from PulseNet and CDC Ref Lab Serogroup * Includes 120 O groups
  31. 31. Prevalence of STEC Serogroups in the US from 2006-2010 n = 19,402 4500No. isolates from PulseNet and CDC Ref Lab 4000 O157 3500 O26 3000 O103 2500 O111 2000 1500 O45 1000 O121 500 O145 0 Other 2006 2007 2008 2009 2010 * year Total * Includes 120 O groups
  32. 32. Geographic Distribution of 1342 STEC O157 isolates from 2006-2010 = 1-250 isolates = 251-500 isolates = 501-750 isolates = 751-1000 isolates = > 1001 isolates
  33. 33. Geographic Distribution of 1342 STEC O26 isolates from 2006-2010 = none reported = 1-30 isolates = 31-60 isolates = 61-90 isolates = 91-120 isolates = > 121 isolates
  34. 34. Geographic Distribution of 1116 STEC O103 isolates from 2006-2010 = none reported = 1-25 isolates = 26--50 isolates = 50--75 isolates = 75-100 isolates = > 101 isolates
  35. 35. Geographic Distribution of 985 STEC O111 isolates from 2006-2010 = none reported = 1-25 isolates = 26--50 isolates = 50--75 isolates = 75-100 isolates = > 101 isolates
  36. 36. Geographic Distribution of 348 STEC O45 isolates from 2006-2010 = none reported = 1-10 isolates = 11-20 isolates = 21-30 isolates = 31-40 isolates = 41-50 isolates
  37. 37. Geographic Distribution of 277 STEC O121 isolates from 2006-2010 = none reported = 1-10 isolates = 11-20 isolates = 21-30 isolates = 31-40 isolates = 41-50 isolates
  38. 38. Geographic Distribution of 253 STEC O145 isolates from 2006-2010 = none reported = 1-10 isolates = 11-20 isolates = 21-30 isolates = 31-40 isolates = 41-50 isolates
  39. 39. HOW ARE STEC TRANSMITTED?
  40. 40. Key factors in STEC transmission Reservoir is the intestinal tract of animals  Especially cattle Very low infectious dose  <100 organisms Multiple modes of transmission  Foodborne  Animal contact  Waterborne  Person-to-person contact Most infections are not outbreak-related  ~19% of E. coli O157 infections, ~9% of non-O157 STEC infections
  41. 41. Proportion of illnesses by mode of transmission in 344 STEC O157 outbreaks, 1998-2007 Illnesses in outbreaks Mode of transmission (n=7,864 illnesses) % Foodborne 69 Waterborne 18 Animals or their environment 8 Person-to-person 6
  42. 42. Non-O157 STEC outbreaks: modes ofTransmission—United States, 1990-2008 Non- Non- Mode of transmission O157 O157 No. %Foodborne 9 33Person-to-person 7 26Water 4 15Animal contact 4 15Mixed modes 1 4Unknown 2 7Total 27 100
  43. 43. Outbreak of STEC O145 Infections May 2010 33 cases in 5 states  Michigan, New York, Ohio, Pennsylvania, and Tennessee  First recognized multistate outbreak of non-O157 STEC 40% hospitalized, 10% developed HUS  As severe as illness caused by E. coli O157:H7 Caused by contaminated Romaine lettuce
  44. 44. Exposures associated with sporadic non-O157 STEC infections Australia  Corned beef, camping, occupational contact with animals Germany  Children: touching a ruminant, playing in a sandbox  Adults: eating lamb and spreadable sausage United States  Minnesota: recent international travel?  FoodNet: study under development
  45. 45. HOW SHOULD LABS DETECT STEC?
  46. 46. Clinical laboratory recommendations, 2009 Simultaneously culture all stools submitted from patients with acute community-acquired diarrhea or suspected HUS for O157 and assay for non-O157 STEC with a test that detects Shiga toxin Report and send E. coli O157 isolates and Stx+ broths to a public health laboratory as soon as possible
  47. 47. Why test all stool samples for STEC? Selective testing practices miss many STEC infections  Children • Over half of infections occur in older adolescents and adults • Highest mortality rate in persons ≥60 years old  Summer months • ~50% of infections occur in non-summer months • Outbreaks can occur year round  Bloody diarrhea • Some patients do not have bloody diarrhea STEC might be detected as often as other bacterial enteric pathogens
  48. 48. Why simultaneously culture for E. coli O157 and assay for Shiga toxin? Most sensitive approach to detect all STEC infections Rapidly distinguishes O157 from non-O157 STEC infections Isolates are obtained in a timely manner
  49. 49. Proposed best practice benefits patient care and public health Patient care  Facilitates early clinical management decisions to reduce risk of HUS • Avoidance of antibiotics and anti-diarrheals  Early identification of E. coli O157 can further influence management decisions  Avoidance of unnecessary procedures Public health  Allows for prompt confirmation and subtyping by public health labs to detect and control of outbreaks  Allows for monitoring of epidemiological trends
  50. 50. Clinical Diagnosis of STEC infection StoolSpecimen Test Culture • Shiga toxin for O157 or H7 STEC • ID as E. coli Streak to Test in Culture for Selective/differential O157 latex Send STEC O157 and non-O157 agar reagent positive broths to public STEC health lab 16-24 hours Shiga toxin or stx geneEnrichment broth detection Stx/stx+ broth
  51. 51. Isolation of STEC from Stx-positive broths by PHLs Shiga toxin-positive broth Selective plate: CT-SMAC or CHROM O157 Nonselective plate: SMAC or WSBM Screen suspect colonies in O157 latex reagent IF NEGATIVE SMAC or WSBM Sweep of Growth or Isolated colonies (or pool 5 colonies) Shiga toxin assay or PCR for stx1, stx2 Serogrouping and PFGE
  52. 52. •Detects E. coli O104• Available in Europe; not yet in the US•Tests for 15 bacteria,viruses and parasites inunder 5 hours
  53. 53. Seeplex® System Seeplex® System Seeplex® is a breakthrough multiplexing PCR technology that enables a new standard in simultaneous multi- Seeplex® is a breakthrough multiplexing PCR technology that enables a new standard in simultaneous multi- pathogen detection. Seegene applies its novel and proprietary Seeplex® system utilizing its DPO™ (Dual Priming pathogen detection. Seegene applies its novel and proprietary Seeplex® system utilizing its DPO™ (Dual Priming Oligonucleotide) technology to create multi-pathogen tests delivering maximum specificity, reproducibility and Oligonucleotide) technology to create multi-pathogen tests delivering maximum specificity, reproducibility and sensitivity. sensitivity. DPO™ Technology DPO™ Technology DPO™ technology is a fundamental tool for blocking extension of non-specifically primed templates generating DPO™ technology is a fundamental tool for blocking extension of non-specifically primed templates generatingconsistently high specificity. The strength and utility of this DPO™ technology can be be successfully incorporated consistently high specificity. The strength and utility of this DPO™ technology can successfully incorporated into into molecular diagnostics systems such multiplex diagnostics and SNP genotyping systems. molecular diagnostics systems such as as multiplex diagnostics and SNP genotyping systems.
  54. 54. Seegene Diarrhea ACE Detection for Stool1 Viral and 2 Bacterial Panels (14 Agents in 6 hr) C ||Diarrhea-B2 ACE Detection C Diarrhea-B2 ACE Detection 11 Aeromonas spp. Aeromonas spp. 22 C. perfringens C. perfringens 33 Aeromonas spp. Aeromonas spp. 44 E.coli : H7 E.coli : H7 E.coli : O157 E.coli : O157 55 E.coli : H7 E.coli : H7 E.coli : O157 E.coli : O157 E.coli : H7 E.coli : H7 66 VTEC VTEC Y.enterocolitica Y.enterocolitica 1~7: Clinical 1~7: Clinical 7 7 samples samples
  55. 55. Detection of STEC in Foods http://www.fsis.usda.gov/PDF/MLG_5B_00.pdf Sample enrichment Genomic DNA extractionTaqMan-based multiplex real-time PCR assay: stx1, stx1, eae (intimin) and 16S rRNA If positive O-antigen identification (real-time PCR) Immunomagnetic separation Selective plating confirmation
  56. 56. Food Safety• September 20, 2011 FSIS announced six STEC serogroups (O26, O45, O103, O111, O121 and O145) will be adulterants on raw, non-intact beef products in the same manner as E. coli O157:H7• FSIS will apply its adulteration decision when testing is initiated March 5, 2012
  57. 57. Summary STEC can cause non-bloody or bloody diarrhea and HUS Horizontal gene transfer is common -- phage play an important role Prevalence varies geographically Primary reservoir ruminants, especially cattle Simultaneous culture for E. coli O157:H7 and an assay that detects Stx or stx genes is the most sensitive approach for all STEC STEC O26, O45, O103, O111, O121 and O145 FSIS will be regulated by FSIS like E. coli O157:H7 starting March 2012
  58. 58. Pathogenesis and Detection of Shiga toxin-producing Escherichia coli ─ Food Safety Issues Related to E. coli O157 and non-O157 Strains Nancy A. Strockbine, Ph.D. Chief, Escherichia and Shigella Reference Unit Enteric Diseases Laboratory Branch Division of Foodborne, Waterborne and Environmental Diseases National Center for Emerging and Zoonotic Infectious Diseases Centers for Disease Control and Prevention Phone: (404) 639-4186 FAX: (404) 639-3333 E-mail: Nancy.Strockbine@cdc.hhs.gov The findings and conclusions in this report are those of the author and do not necessarily represent the official position of the Centers for Disease Control and Prevention. Enteric Diseases Laboratory Branch
  59. 59. Enteric Diseases Laboratory Branch Peter Gerner-Smidt, M.D., D.M.S., Branch Chief John Besser, Ph.D., Deputy Branch Chief Sherricka Simington, Branch manager Nicole Rankine, QMS manager 4 FTE, 2 non-FTE National Enteric National Antimicrobial National Botulism PulseNet USA Team National EntericReference Laboratory Resistance Surveillance Laboratory Laboratory Diagnostics Team Team Preparedness Team and Outbreak Team Patricia Fields, Ph.D. Jean Whichard, D.V.M., Ph.D. Susan Maslanka, Ph.D. Efrain Ribot, Ph.D. Deborah Talkington, Ph.D. 12 FTE, 8 non-FTE 4 FTE, 5 non-FTE 5 FTE, 3 non-FTE 13 FTE, 7 non-FTE 7 FTE, 1 non-FTE Campylobacter and NARMS Surveillance Botulism Public Health PulseNet Database Unit Epidemic Helicobacter Unit Unit Research Unit Kelley Hise, M.P.H. InvestigationsCollette Fitzgerald, Ph.D. Kevin Joyce Brian Raphael, Ph.D. Laboratory Unit Cheryl Bopp, M.S.Escherichia and Shigella, Unit NARMS Applied Botulism Outbreak PulseNet Methods Immunodiagnostics Nancy Strockbine, Ph.D. Research Unit Investigation Unit Development and Unit Jean Whichard, D.V.M., Carolina Luquéz, Ph.D. Reference Unit Deborah Talkington, Ph.D. Ph.D. Efrain Ribot, Ph.D. Salmonella Unit Patricia Fields, Ph.D.Listeria ,Yersinia , Vibrio and otherEnterobacteriaceae Unit 2-1-2010 Cheryl Tarr, Ph.D.

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