PROGRESS IN THE CONTROL OF SHIGA TOXIN-PRODUCING - PowerPoint Presentation

PROGRESS IN THE CONTROL OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) IN BEEF THROUGH THE USDA-NIFA STEC COORDINATED AGRICULTURAL PROJECT (CAP) Rodney A. Moxley, DVM, PhDCharles Bessey Professor & STEC CAP Project DirectorSchool of Veterinary Medicine and Biomedical SciencesInstitute of Agriculture and Natural Resources University of Nebraska-LincolnLincoln, NE 68583 37th Annual Midwest Section AOACI MeetingGraduate Lincoln HotelLincoln, NebraskaJune 5, 2018

Escherichia coli Non-pathogenic strains are part of the normal intestinal flora of warm-blooded animalsPathogenic strains are NOT part of the normal floraE. coli cells are Gram-negative rodsE. coli cells are usually about 1-2 μm longE. coli colonygrowingon bloodagarE. coli cells often have surface appendages 2 mm

Infection with Shiga Toxin-Producing E. coli (STEC) Often Results in Disease Hemorrhagic colitis (bloody diarrhea)Hemolytic uremic syndrome (HUS) (anemia and kidney failure) and end-stage renal disease (ESRD)

Shiga Toxin-Producing E . coli (STEC)Refers to any E. coli strain that produces Shiga toxin (Stx).Inclusion in this category does not necessarily confirm pathogenicity in the absence of other virulence factors.Strains isolated from human patients with hemorrhagic colitis and/or HUS, and isolates positive for both stx and eae (intimin ) genes are known as enterohemorrhagic E. coli (EHEC).

E nterohemorrhagic E. coli (EHEC)Shiga toxin (Stx)– main virulence factor of EHEC– ribosome-inactivating toxin– shuts down protein synthesis– causes cell death( Croxen et al. 2013. Clin. Micro. Rev. 26:822-880):Isolates from cases of hemorrhagic colitis/HUS Isolates that contain the genes forShiga toxin (stx) virulence factors that cause A/E lesions — intimin (eae )— others hemorrhagic colitis attaching-effacing (A/E) lesions E. coli O157:H7 is the prototype of EHEC Intestine Brain hemorrhage and necrosis of endothelium and neurons E E M M N R

*Adapted from Scallan, E., et al. 2011. Foodborne illness acquired in the United States ― Major Pathogens. Emerg. Infect. Dis. 17:7-15.Estimated Annual Number of Domestically Acquired EHEC Illnesses, Hospitalizations and Deaths in the United States*EHEC O157EHECNon-O157 TotalAll illnesses92,872137,502 230,374 Foodborne illnesses 63,153 112,752 175,905 ( 76% ) Hospitalizations 2,138 271 2,409 Deaths 20 0 20 6

Annual Economic Cost of Illness Due to STEC O157 in United States* Severity CategoryEstimated National Cost (US Dollars)Estimated Cost Per Case (US Dollars)Nonfatal Cases Not hospitalized, did not see physician2,040,00035Not hospitalized, saw a physician 8,170,000 600 Hospitalized, did not have HUS 13,750,000 7,624 Hospitalized, had HUS but not end stage kidney 12,660,000 42,209 Hospitalized, had HUS and end stage kidney 70,400,000 7,044,692 Fatal Cases Did not have HUS 125,270,000 5,444,290 Had HUS 319,580,000 8,408,950 Total 551,750,000 7,510 *Adapted from Frenzen , P.D. 2007. USDA Economic Research Service, Bulletin No. 28. In today’s dollars (June 2018) adjusted for inflation since 2003.

8Most Outbreaks of EHEC O157 Infection are Foodborne pp. 1293-1301

Adapted from Scallan, et al. 2011. Emerg. Infect. Dis. 17:7-15.Data collected from 2000-2008Adapted from Painter, et al. 2013. Emerg. Infect. Dis. 19:407-415.Data collected from 1998-2008Attribution of Food Sourcesto STEC Infections Courtesy of Dr. Philip Bronstein, USDA-FSIS9

Expanded List:64 FR 2,803; Jan. 19, 1999 FSIS deemed adulterated E. coli O157:H7-contaminated raw non-intact beef products and intact cuts that are to be further processed into non-intact products before being distributed for consumption.EHEC O157:H7 is an Adulterant in Raw Non-Intact Beef Under the Federal Meat Inspection Act10Raw Ground Beef:September 29, 1994Michael R. TaylorAdministrator, USDA-FSIS “Change and Opportunity: Harnessing Innovation to Improve the Safety of the Food Supply” –1994 American Meat Institute Annual Convention—Texas Food Industry Ass’n, et al. v. Espy, Civil No.94 CA748IN, Declaration of Michael R. Taylor, Nov. 15, 1994 —Texas Food Industry et al. v. Mike Espy et al., 870 F. Supp. 143; Dec. 13, 1994An adulterant , according to FMIA, renders product injurious to health (21 U.S.C. §601(m)(1))

$BillionDemand for beef ………….1.58 Boneless beef prices……..0.17Packer expenditures……..0.85 (Capital, operating costs, recalls)Research………………………..0.07Total……………………….2.67Ten-Year (1993-2003) Cost of E. coli O157:H7 to the Beef IndustrySteve Kay, Meat and Poultry magazine $2.67 billion in 2003 is equivalent to $3.64 billion in today’s dollars.

On September 20, 2011, USDA declared these 6 serogroups adulterants in non-intact, raw beef (Federal Register, Vol. 76, No. 182, 58157-58165). EHEC O26, O45, O103, O111, O121, O145Are Adulterants* in Non-Intact Raw Beef“Top 6” or “Big 6”Six O groups comprising 6 serogroups and 13 serotypes are responsible for ~71% of the cases of non-O157 STEC infection in the US.O26:NMO26:H11 O111:NMO111:H8O103:NMO103:H2O103:H11O103:H25O145:NMO121:H7O121:H19 O45:NMO45:H2 Brooks et al . 2005. J. Infect. Dis. 192:1422-9. *Isolates must be positive for both Stx and intimin to be confirmed as adulterants.

Shiga-toxigenic Escherichia coli (STEC) in the Beef Chain: Assessing and Mitigating the Risk by Translational Science, Education and Outreach Long Term Goal:To reduce the occurrence and public health risks from STEC O26, O111, O103, O121, O45, O145, O157:H7, and O104:H4 in beef using a quantitative microbial risk assessment platform.USDA National Institute of Food and Agriculture Coordinated Agricultural Project (CAP) Grant Jan. 1,2012 – Dec. 31,2018 (will request extension to 12/31/2019)

Pillar 1 - Live cattle & beef producersPillar 2 - Slaughter, fabrication, meat processing & processorsPillar 3 - Retail products, food service & consumersObj 1 - STEC detection: reagents, sampling, assays, technology, partnershipsObj 2 - STEC biology: microbiology, ecology, epidemiology, modifiable risk, best targetsObj 3 - STEC risk reduction interventions: value, feasibility, cost-benefit, impacts Obj 4 - STEC risk analysis : risk assessment (QMRA) Obj 5 - STEC education, outreach, and evaluation : beef chain STEC CAP Objectives

Quantitative Microbial Risk Assessment (QMRA) Conceptual Model Overview

18 institutions52 collaborators STEC CAP Participating Institutions

Stakeholder Advisory Board Partners American Beef Processors of Oregon, LLCMIDWEST VETERINARY SERVICES INC

STEC CAP Executive Management Team and Project Manager Executive Management Team:Rodney A. Moxley (PD) rmoxley1@unl.eduHarshavardhan Thippareddi harsha.thippareddi@uga.eduDaniel L. Gallagher dang@vt.eduJohn B. Luchansky John.Luchansky@ars.usda.gov Randall K. Phebus phebus@ksu.eduUNL Project Manager : Jill Hochstein jhochstein@unl.edu Rod Moxley Harsha Thippareddi Dan Gallagher John Luchansky Randy Phebus Jill Hochstein

Refereed Journal PublicationsGrant Year CalendarYearPublished12012 322013 14 3 2014 15 4 2015 21 5 2016 21 6 2017 19 7 2018 14 † Total 107 † Includes those in press .

Stationary for 18 h at 42°C 55 of 55 strains tested had reduced growth in TSB-NVRBT, compared to TSB 2 strains (*) had poor growth @42°C Mean CFU/ml Log10 reduction = 1.18 (P < 0.0001, paired t-test) **Strain #4 = O26 (MT#10).*Strain #31 = O157:H7(S2006 #4).Growth of STEC-7 Strains in TSB-NVRBT vs TSB*Possé et al. J. Appl. Microbiol. 2008. 105:227–35. Gentry L. Lewis

Rodney MoxleyZachary Stromberg Gentry LewisDavid MarxNovobiocin (5 mg/L) K tellurite (0.5 mg/L)EC Broth – contains lactose and bile salts

Possé et al. developed a differential agar to detect EHEC O26, O103, O111, and O145 in three colony colors [red-purple (O26), blue-purple (O103, O111), and green (O145)] ( FEMS Microbiol Lett. 2008; 282:124-131).We determined that the remaining three EHEC-7 serogroups also yield these three colors on Possé differential medium (Lewis et al., CRWAD, 2011): EHEC O45 (blue-purple), O121 (red-purple, blue-purple, or green) , and O157 (red-purple).We modified this medium to contain reduced novobiocin and potassium tellurite to enhance the detection of EHEC-6 (Foodborne Pathog. Dis. 2015;12:631-638). Difco MacConkey Agar Base (40 g/L) (per liter: Peptone 17 g, Proteose Peptone 3g, Bile Salts No.3 1.5 g, Sodium Chloride 5g, Agar 13.5 g, Neutral Red 0.03 g, Crystal Violet 0.001 g)Sucrose (6 g/L)Sorbose (6 g/L)Bile Salts No.3 (3.5 g/L)X-gal (0.05 g/L)IPTG (0.05 g/L)Novobiocin (8.0 mg/L)Potassium Tellurite (2.5 mg/L) Possé Differential Agar (Possé) Modified Possé Agar (mPossé) Difco MacConkey Agar Base (40 g/L) (per liter: Peptone 17 g, Proteose Peptone 3g, Bile Salts No.3 1.5 g, Sodium Chloride 5g, Agar 13.5 g, Neutral Red 0.03 g, Crystal Violet 0.001 g) Sucrose (6 g/L) Sorbose (6 g/L) Bile Salts No.3 (3.5 g/L) X-gal (0.05 g/L) IPTG (0.05 g/L) Novobiocin ( 5.0 mg/L) Potassium Tellurite ( 0.5 mg/L) Possé Differential Agar

Enterohemolytic colonies were screened for EHEC.STEC Heart Infusion Washed Sheep Blood Agar with Mitomycin C (SHIBAM)(FDA BAM Medium M195) Lin et al. J. Food Prot. 2012;75:2028-2030. Washed Blood Agar with Mitomycin C (WBAM)Sugiyama et al. 2001. Lett. Appl. Microbiol. 33:193-195.CHROMagar™ O157 with novobiocin and K+ tellurite, or cefsulodin, cefixime and K+ telluriteMoxley et al. 2009. Foodborne Pathog. Dis. 6:879-884.CHROMagar™ STEC Gouali et al. 2013. J. Clin. Microbiol. 51:894-900.Mauve colonies were screened for EHEC. Green to blue colonies were screened for EHEC. USMARC STEC differentiation agar (SDA) Kalchayanand et al. 2013. J. Food Prot. 76:192-199. Mauve colonies were screened for EHEC. Other Agar Media for Culture-Based Detection