E. coli: Whats next?. HOST Bill McDowell Editorial Director, Meatingplace MODERATOR Mike Fielding...

E. coli: What’s next?

HOST Bill McDowellEditorial Director,

Meatingplace

MODERATOR Mike FieldingEditor, Meatingplace

POLL QUESTIONS

Will the “Big Six” be declared to be productadulterants?• Yes• No

Should they be?• Yes• No

Non-O157 Shiga Toxin Producing E. coli: Biology, Detection and

Significance

Mohammad Koohmaraie, Ph.D.CEO – Meat DivisionIEH Laboratories & Consulting GroupLake Forest Park, WA

Presentation Outline

• Nomenclature• O157 vs. non-O157• Will Non-O157 STECs get adulterant designation? • Detection – FSIS Approach• Prevalence• Summary and conclusions

Nomenclature

E. coli O157:H7

YY

Lipopolysaccharide (LPS)

Flagella = H antigen= O antigen

H1-H56O1-O173

E. coli SerotypingO157:H7O104:H4O26:H11

Pathogenicity

• Not all E. coli strains are pathogenic.• To be pathogenic, a strain must have the

necessary properties to cause disease in human.

• These properties are called “virulence factors.”• Exactly what virulence factors are required is

unknown.

Pathogenicity• E. coli can cause human disease when they possess stx1 or

stx2.

• Individuals infected with strains producing stx2 are more likely to develop severe disease than those infected with strains carrying Shiga toxin 1.

• It is commonly thought that E. coli must contain stx1 or stx2 and eae (intimin) and its substitutes to have the highest chance of causing disease in humans – of course there are always exceptions.

Shiga

Toxin producing

Escherichia

Coli

STEC

Entro

Hemorrhagic

Escherichia

Coli

EHEC

pSTEC

Top Non-O157 Serotypes (CDC)

– O26 22% of non-O157 STEC– O111 16% of non-O157 STEC– O103 12% of non-O157 STEC– O121 9% of non-O157 STEC– O45 7% of non-O157 STEC– O145 5% of non-O157 STEC

Annual Submission of Non-O157 to PulseNet

CDC

E. coli O157 vs. Non-O157 E. coli

E. coli O157 ingested

3 - 4 days

non-bloody diarrhea, abdominal cramps

5 - 6 daysresolution

92% 8%

HUS

bloody diarrhea

1 - 2 days80%

Mead. Lancet 1998

Sequence of events in E. coli O157:H7 infection

CDC

Non-O157 STEC ingested

3 - 4 days

5 - 6 daysresolution

99%bloody diarrhea

1 - 2 days40%

non-bloody diarrhea, abdominal cramps

<1%

HUS

Sequence of events in Non-O157 infection

CDC

Food commodities implicated outbreaks of non-O157 STEC infections with known food commodity,

1990-2007

Commodity Number outbreaks Food items

Fruit-nuts 3 Juice, apple cider, berries

Dairy 2 Cheese, margarine

Leafy vegetables 1 Lettuce

No outbreaks due to beef

But it all changed in 2010 with the recall of ground beef because of human illness

associated with O26 contamination

Will Non-O157 STECs Get Adulterant Designation?

• It is a matter of when and not if any more • Less than 12 months

Detection

Testing for STEC

• E. coli O157–Unusual feature: does not ferment sorbitol

• Non-O157 STEC– Lack unusual features– A group of organisms

USDA Approach

• Meat sample• Enrich• Screen for eae + stx• If negative – STOP• If positive, PCR to determine if it is a top 6• If one of the top 6 – Sample is POSITIVE• If not a top 6 – Sample is NEGATIVE

Non-O157 Prevalence

Prevalence of Non-O157 STEC • Commercial fed cattle processing plants

• Commercial fed cattle processing plants as a function of the season of the year

• Commercial cow/bull processing plants

• Commercial lamb processing plants

• Imported raw ground beef material (trim)

• National ground beef supply

We are very appreciative of the U.S. meat industry for allowing us to use their facilities as our laboratory.

Results

Pre-evisceration (No intervention)

Final (after all interventions)

E. coli O15744.4%

(144/324 carcasses)1.8%

(6/326 carcasses)

Non-O157 STEC

54%(180/334 carcasses)

8.3%(27/326 carcasses)

STEC Prevalence inImported and Domestic

Boneless Beef TrimUsed for Ground Beef

STEC

AUS NZ URU DOM

n 220 223 256 487

Isolate positive samples 9 4 40 28

% 4.1 0.9 15.6 5.7

# of HUS-Related Serotypes

0 2 6 5

Frequency of STEC isolationin boneless beef trim by country of origin

AUS NZ URU DOM O33:H11 O26:H8 O2:H25 O116:H36 O5:H36 O117:H+

O73:H35 O26:H11 O6:H30 O130:H11 O8:H19x3 O132:H+

O113:H36x3 O64:H9 O6:H34 O163:H19x3 O20:H19 O132:H38

O113:H51 O163:H19 O8:H3 O163:H26 O55/83:H15 O142:H34

O147:H7 O8:H19x2 O168:H+ O73:H+ O150:H2/35

O171:H+ O15:H27x3 O174:H11 O73:H18 O165:H-

ONT:H+ O20:H19x4 O174:H28x2 O79:H7 O171:H2

ONT:H2 O39:H14 O174:H36x2 O83:H+ O172:H10

O55/83:H15 ONT:H+x2 O83:H38 O174:H36

O74:H28x2 ONT:H11x2 O83/132:H2 OX25:H11

O82:H8 ONT:H18 O88:H38 ONT:H2

O82:H15 ONT:H19x2 O113:H4 ONT:H7

O83:H8x2 ONT:H32 O113:H51 ONT:H32

O83:H11 ONT:H34 O116:H21 ONT:H51

O88:H38x2 ONT:H46x4 OR:H-

O113:H21 ONT:H51

O113:H36 ONT:H52

Serotypes of STEC isolated by country

Underlined serotypes have been associated with human illness.Bolded serotypes have been associated with Hemolytic Uremic Syndrome (HUS).

A National Survey of the Prevalence of non-O157 Shiga toxin-producing E. coli in Ground Beef

BIFSCo Database Microbiological Regions

1

2

3

4 5

6

7

8

Ground Beef Non-O157 STEC

• 4133 samples• Supplies by ground beef manufacturers • over a 24 months period• 24.3% (1006/4133) of the samples were

positive for Shiga toxin (stx1 or stx2).• In all, 0.2% of the samples (10/4133) could be

considered a food safety threat.

Control

Hide is the principal source of mostpathogens. An effective control in order ofsignificance is:

1st - Dressing practices2nd - Effective interventions• Effective – scientific support• Works in your hands – validation • Must make contact the potential pathogens

3rd - Effective test and hold

Summary and Conclusions

• STEC are a natural part of the animal microflora.

• Some Non-O157 STEC can cause severe disease in humans.

• Non-O157 STEC is found at high frequency in pre-harvest samples (feces and hides).

• The prevalence on carcass depends on the adequacy of dressing practices.

Summary and Conclusions

• A thoughtful and comprehensive approach should be used to develop a national policy with respect to the control of non-O157 STEC.

• This approach should be such that remove the most virulent E. coli from the food supply not just some and not just any.

• In this way we are truly reducing the burden to public health of pathogenic E. coli without undue economic hardship.

Mike MullenCorporate Account ManagerFor more information contact:[email protected] www.ecolab.com or 800-392-3392

The Sinful Six Interventions to Minimize the Risk for Beef Processors: A Research and Application Update

Harshavardhan Thippareddi, Ph.D.Associate Professor andExtension Food Safety MicrobiologistDept. of Food Science and TechnologyUniversity of Nebraska, Lincoln, NE 68583

Outline

• Introduction• Prevalence of STECs• Antimicrobial Interventions for STECs• Non-O157 STEC Challenges for the processor• Non-intact Beef – STECs• Summary

Innovative Interventions Developed and Applied by the Beef Industry

• 1993 E. coli O157:H7 Pacific Northwest– Knife trimming and water washing

• 2009 – Multiple meat processing interventions– Sequential– Hurdle Technologies– Primary and Secondary– At all phases of meat processing

Outline


• Cattle STEC prevalence data varies:– 0-19%, dairy cattle1,2,3,4

– 19.4 – 56.3% 5, beef cattle feces/hides• Food prevalence data very limited:– Pre-evisceration beef carcasses >50%5,6

– Retail ground beef, 2.3%7

1Wachsmuth et al., 1991 2Wells et al., 1991 3Cray et al., 1996 4Thran et al., 20015Barkocy-Gallagher, et al., 2003 6Arthur et al., 2002 7Samadpour et al., 2006 Dr. Hagen, 2007

Non-O157 Shiga toxin producing E. coli – From FSIS Public Meeting, 2007

Outline


Antimicrobial Interventions for Slaughter, Fabrication and Grinding

Antimicrobial Agents: Classification

Direct Food Additives►Sod. or Pot. Lactates, Buffered sodium citrate, sod.

Diacetate and Lactoferrin, Irradiation►Considered ingredients, need to be labeled as

such Secondary Direct Food Additives

►Peroxy acids, ASC, Ozone► No labeling requirement

Slaughter:– Chemical dehairing– Hot water rinses– Steam pasteurization– Steam vacuum– Chemical rinses– Lactoferrin

Antimicrobial Interventions

Fabrication:– Organic acid rinses– Sanova– Ozone– Per-acetic acid– Lauric Arginate– Lactoferrin


Trim for Grinding:– Organic acid rinses– Ozone– Per-acetic acid– ASC– Multiple hurdle technology– High Pressure Processing

Ground Beef:– Irradiation


Chlorine, Organic acids most commonly used– Organic acids – lactic, acetic, citric

Improves microbiological quality of carcasses Other chemicals include Per-oxy acetic acid,

Acidified sodium chlorite, CPC

Chemical Rinses

Antimicrobials - VarietyHigh Inoculum Low Inoculum

Survival Reduction Survival Reduction

Non-Trt 5.8 - 4.1 -

W@25°C 4.6 1.2 3.5 0.6

AC, 0.001% 5.4 0.4 3.4 0.8

AA, 2% 4.2 1.6 2.0 2.1

LA, 2%@55°C 2.5 3.3 1.1 3.1

LB, 1% 5.1 0.7 3.6 0.6

PAA, 0.02% 4.4 1.4 2.7 1.4

ASC, 0.02% 3.9 1.9 2.1 2.0

CPC, 0.5% 1.0 4.8 0.5 3.6

Ransom et al., 2003

Boneless beef short plates & Lean tissue pieces; E. coli O157:H7

Antimicrobial Interventions for Non-intact Beef

Oklahoma State / Ross (2010) study compared 10 different antimicrobials for efficacy on surface inoculated beef subprimals

CytoGuard is a registered trademark of A&B Ingredients

Courtesy: Arun Ramabadran,

Spray Systems Co., Wheaton, IL

Outline


Non-O157 STEC Challenges for the Beef Processor

• Biology and characteristics of the organisms are unknown

• Characteristics of Significance– Thermal resistance– Acid resistance– Dehydration – Aw– Irradiation– Behavior to antimicrobial interventions

traditionally used in beef processing

Characteristics of Significance in Meat Processing – Growth @ 10°C

• Strain O26:H11 survived, but did not grow• LPD - 11.3 h to 39.4 h• Growth rate – 0.032 to 0.045 log CFU/g/h• O145 and O157 had shorter LPD compared to

O45, O103, and O121 (O111 had intermediate LPD)

• After end of LPD, no difference in growth rate

(Chatzikyriakidiu et al., 2011)

Characteristics of Significance in Meat Processing – Growth

(Thippareddi et al., Unpublished)

Characteristics of Significance in Meat Processing – Thermal Resistance

L. monocytogenes Salmonella spp.

Lean Fatty Lean Fatty

125°F 81.3 71.1 61-62 54.3

135°F 2.6 5.8 3.8-4.2 5.43

145°F 0.6 1.2 0.6-0.7 0.54

Z Value 9.3 11.4 10 10

STECs Broth Ground Beef

130°F (54.4°C) 9.39-31.15

140°F (60°C) 0.63-1.28

150°F (65.5°C) 0.08-0.21 1.16-0.76

(Fain et al., 2009; Leong et al., 2011, Vasan et al., 2011)

Characteristics of Significance in Meat Processing – Acid Resistance

• E. coli O157:H7 reductions of >7.70 log CFU/ml was observed in lemon and lime juices* within 72 h at 22°C

• STECs (six serotypes) reduction of >6 log within 72 h at 22°C in lemon and lime juices

(Enache et al., 2009; Kataoka et al., 2011)*Lemon Juice: 6.8° Brix; 2.62 pH; 4.62 TA*Lime Juice: 7.4° Brix; 2.54 pH; 4.82 TA

Non-O157 STECs – Properties of Significance, Acid Resistance

The present study shows that the process used in the manufacture of French sausages results in a complete destruction of NAR STEC strains after 60 days, but it does not have the same effect on the AR STEC strains.

Montet et al., 2009

Outline


Translocation and Thermal Inactivation of Shiga Toxin-Producing E. coli in Blade and Chemically Tenderized Beef

1. Luchansky, J. B., J. E. Call, R. K. Phebus, and H. Thippareddi. 2008. Translocation of surface inoculated Escherichia coli O157:H7 into beef subprimals following blade tenderization. J. Food Prot. 71:2190-2197.

2. Luchansky, J. B., A. C. S. Porto-Fett, B. Shoyer, R. K. Phebus, H. Thippareddi, and J. E. Call. 2009. Thermal inactivation of Escherichia coli O157:H7 in blade tenderized beef steaks cooked on a commercial open-flame gas grill. J. Food Prot. 72:1404-1411.

3. Luchansky, J. B., A. C. S. Porto-Fett, B. A. Shoyer, J. E. Call, W. Schlosser, W. Shaw, N. Bauer, and H. Latimer. 2011. Inactivation of Shiga toxin-producing O157:H7 and non-O157:H7 Shiga toxin-producing Escherichia coli in brine-injected, gas-grilled steaks. J. Food Prot. 74:1054-1064.

4. Luchansky, J. B., A. C. S. Porto-Fett, B. A. Shoyer, J. E. Call, W. Schlosser, W. Shaw, N. Bauer, and H. Latimer. 2011. Fate of Shiga toxin-producing O157:H7 and non-O157:H7 Shiga toxin-producing Escherichia coli in blade tenderized beef steaks cooked on a commercial open-flame gas grill. J. Food Prot., Submitted 06-02-11.

Escherichia coliEscherichia coli

STEC and ECOH behave similarly!• No discernible differences in translocation between ECOH and

STEC following blade tenderization or chemical injection of beef sub-primals– Majority of cells to top-most 1 cm– More transfer via single pass and lean side inoculation and

tenderization than double pass and fat side inoculation and tenderization (NCBA Study)

Courtesy: Dr. John Luchansky & the ARS Non-Intact Team

STEC and ECOH behave similarly!• No discernible differences in thermal resistance between STEC

and ECOH following cooking of blade tenderized or chemically-injected steaks– Higher temperatures generated greater lethality – No difference in lethality based on steak thickness (1.0 or

1.5 in.)– Subtle differences in thermal resistance between steaks

injected with lactate-containing brine compared to brine without lactate that were cooked at 140F

Courtesy: Dr. John Luchansky & the ARS Non-Intact Team

Outline


Summary

• STECs seem to be similar in characteristics to E. coli O157:H7 based on current research

• Very few interventions eliminate the E. coli O157:H7 and non-O157 STEC risk

• Novel technologies such as carcass irradiation and HPP should be optimized and validated to assure safety of intact as well as non-intact beef products

• The beef industry has made significant strides in reducing risks and costs associated with foodborne pathogens.– However, food safety remains a significant concern due to increasing media and regulatory scrutiny. – E. coli O157 is naturally occurring in animals and the environment, which makes it an ever-present challenge.– The beef industry has a good track record for safety, but there is always room to improve.

• Now there is a tool available to your suppliers that can help reduce E. coli O157 before it enters the packing

plant — in the cattle, at the feedlot — thereby helping reduce E. coli O157 throughout the beef processing chain.– Escherichia Coli Bacterial Extract vaccine* with SRP® technology is the only

conditionally licensed product that can help reduce E. coli O157** carrier state prevalence and shedding at the source — in the cattle.

– Reducing the amount of E. coli O157 coming into the plant can give other interventions in place an opportunity to be as effective as possible.

• The goal for us at Pfizer Animal Health is to help ensure continued confidence in the industry to help keep demand for our products strong.

* This product license is conditional. Efficacy and potency test studies are in progress.** Escherichia coli O157:H7 bacterial strain1Woerner DR, Ransom JR, Sofos JN, Dewell GA, Smith GC, Salman MD, et al. Determining the Prevalence of

Escherichia coli O157 in Cattle and Beef from the Feedlot to the Cooler. J Food Prot 2006;69:2824-2827.

Together, we can reduce E. coli O157.Dr. Brad Morgan, Food Safety SpecialistPfizer Animal Health

E.COLI: Legal Issues and the Big Six

Robert G. HibbertK&L Gates LLP

E.coli 0157:H7 Precedent

– 1994 Policy Statement– Texas Food Industry v. Espy (1994)– Extension to other products – trim, non-intact– Avoidance of rulemaking– Integration with HACCP

E.coli Precedent (continued)

– Commercial response– Technological responses– Exception proves the rule-labeling and product handling

distinctions

Shiga–Toxin Producing E.Coli Serotypes

– Outbreaks– Methodology issues– Marler petition– Pending policy statement

Legal Issues (Regulatory)

– Adulterants under 1(m)(1) vs. 1 (m)(3)

– Policy by Recall

– Abandonment of rulemaking (listeria RTE precedent)

– HACCP and inspection implementation

Legal Issues (Commercial)

– Guarantees and requirements

– Role of testing

– Reliability of testing

– Tort liability

– Insurance concerns

QUESTIONS & ANSWERS

FOR MORE INFORMATIONMohammad Koohmaraie, Ph.D.: [email protected]

Harshavardhan Thippareddi, Ph.D.: [email protected] G. Hibbert: [email protected]

Mike Fielding: [email protected] McDowell: [email protected]

Ecolab: [email protected] Animal Health: [email protected]

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E. coli: Whats next?. HOST Bill McDowell Editorial Director, Meatingplace MODERATOR Mike Fielding...

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