STEC CAP Proceedings Document 2014

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2 0 1 4 G ov e r n o r ’ s co n f e r e n c e / S T EC C A P A n n ua l co n f e r e n ce

Ensuring Food Safety: E. coli O157:H7 and other STECs – Progress and Challenges

May 27-29, 2014 Embassy Suites, Lincoln NE


Executive Summary..................................................................................................4 Shiga toxin-producing E. coli...............................................................................4 Presentations.....................................................................................................5 Proceedings........................................................................................................... 10 Governor’s Welcome Dave Heineman................................................................. 10 STEC Ecology in Cattle..................................................................................... 12 MWAS and GWAS Studies to Define Associations between Host Genetics, Microbiome Composition and EHEC Colonization Andrew Benson.................... 12 Seasonal Variation in E. coli O157:H7 Shedding by Cattle: Fecal, Feed and other Factors Thomas E. Besser........................................... 14 Shiga Toxin-producing E. coli in Michigan—from Shedding in Cattle to Human Disease Shannon Manning.............................................. 16 STEC in Produce and the Environment................................................................ 18 The Role of Wildlife in the Dissemination of E. coli O157:H7 and other STECs to Fresh Produce Michele Jay-Russell.................................. 18 Role of Insects in the Ecology of STEC Ludek Zurek......................................20 Dissemination of E. coli O157:H7 from a Cattle Feedlot: Effect of Proximity on Contamination of Leafy Greens, Bioaerosols and Pest Flies Elaine D. Berry....................................................22 The Survival and Growth of STEC in Soil Amendments Used in Agriculture Manan Sharma.............................................................. 24 Produce Industry Needs in Rapid Detection of Clinically Relevant STEC Trevor Suslow.....................................................26 It’s Here, We Found It, Now What Do We Do? An Industry Perspective Will Daniels............................................................28

STEC Prevalence and Intervention Strategies at Slaughter.....................................30 Federal Regulation of STECs and Other Pathogens: Past, Present and Future Bob Hibbert..........................................................30 Quantitative Microbial Risk Assessment: An Update on Objective 4 of the STEC-CAP Grant Michael W. Sanderson.............................32 Recent Research on the Epidemiology of STEC in Beef Production Systems David Renter........................................................34 Recent Research on Antimicrobial Interventions and their Validation to Control STEC in Beef Products Gary R. Acuff.............................36 USDA Food Safety and Inspection Service STEC Update Philip Bronstein........38 Modeling Pathogen Controls in Beef Slaughter Establishments Rachel Johnson-DeRycke.....................................................40 STEC Industry Perspective Brenden McCullough...........................................42 Preventing Outbreaks through Education and Consumers......................................44 STEC Needs and Interventions for Consumer and Retail Audiences Ben Chapman...................................................................44 Reaching the Public with Safe Food Handling Information Christine Bruhn.......46 Translation of Shiga Toxin E. coli: Mitigation to Field Implementation in the Beef Industry Kelly Oliver...................................48 Expanding Food and Nutrition Science Education through Additional, Enhanced Curriculum Development Jason D. Ellis and Dann E. Husmann........50 Consumers and Consumer Advocates Play a Vital Role in Reducing the Public Health Burden Attributed to STEC Illness Vanessa Coffman..................52 Improving STEC Detection.................................................................................54 Rodney Moxley..........................................................................................54 Mick Bosilevac..........................................................................................55 Alina Deshpande........................................................................................56 T. G. Nagaraja...........................................................................................57

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stEc Ensuring Food Safety: Shiga Toxin-Producing E. coli—Progress and Challenges

Proceedings of the 2014 Governor’s STEC CAP Conference

Executive Summary The 2014 Governor’s STEC CAP conference —“Ensuring Food Safety: E. coli O157:H7 and other STECs - Progress and Challenges”— brought together more than 160 national food safety experts to discuss progress and challenges toward protecting the public’s health from the risk of Shiga toxin-producing E. coli, or STEC, in the nation’s food system. Presentations highlighted the latest research on STEC biology and ecology and provided insight from experts in food production, regulation and public policy, the environment and consumer protection. The annual conference stems from a major U.S. endeavor to reduce the occurrence of STEC throughout the beef production chain that continues to threaten public health. Funded by the U.S. Department of Agriculture’s National Institute of Food and Agriculture (USDA-NIFA), this Coordinated Agricultural Project, or CAP, is led by the University of Nebraska-Lincoln and includes 16 participating institutions and 51 collaborators. The long-term goal of this joint public-private venture is to reduce health risks from STECs, while preserving an economically viable and sustainable beef industry.

Shiga toxin-producing E. coli STECs cause more than 265,000 cases of human illnesses in the U.S. each year. Outbreaks occur through consumption of contaminated food and water or through direct contact with infected people, animals or their feces. Cattle and other ruminant animals, such as deer, are natural reservoirs of STEC, which can lead to contamination of meat and milk during harvest and processing. Other foods, such as fresh produce, can also become contaminated with STEC from proximity to animal production facilities and exposure to wild animals. STEC presence in raw and processed foods poses an important human health risk. Thus, the food industry and its regulators have focused substantial resources and efforts toward eliminating STEC from the food supply. E. coli constitute a significant proportion of the bacteria that normally inhabit the intestinal tracts of humans and animals. E. coli don’t typically cause disease. However, the bacterium may acquire certain genes that make it pathogenic. STEC, by definition, are E. coli that produce a potent toxin called Shiga toxin that, in humans, causes blood vessel damage and other injuries that result in hemorrhagic colitis, manifested as bloody diarrhea. Some patients further develop a condition known as hemolytic uremic syndrome, which is characterized in part by kidney failure. The genes that produce Shiga toxins originate from viruses that infect the bacteria, called bacteriophages. After an E. coli bacterium becomes infected with Shiga toxinproducing phages, it is potentially pathogenic to humans. However, Shiga toxin isn’t the only virulence factor required to cause disease. Bacteria must also be able to adhere to human cells and transfer their proteins into the host cell, a process carried out by many other proteins produced by the E. coli bacterium. A subset of STEC produces a collection of proteins that allow them to adhere to intestinal epithelial cells. One of these proteins is intimin, encoded by the gene eae. E. coli that produce, or at least are known to contain, the genes for Shiga toxin and intimin production are known as enterohemorrhagic E. coli (EHEC).


Tests that detect the presence of genes for both Shiga toxin and intimin in the same bacterium confirm the organism to be an EHEC. All EHEC are considered to be pathogens, whereas E. coli identified only as STEC (through detection of Shiga toxin genes and not intimin) are considered potential pathogens. Some STEC do not produce intimin, but adhere to intestinal epithelial cells through production of other protein virulence factors. These STEC are also pathogens, but are not technically EHEC because they do not produce intimin. While hundreds of types of STEC exist, the STEC CAP targets the seven EHEC types most responsible for illness in the U.S. The best known type is O157:H7 (O157), the organism responsible for the 1993 “Jack in the Box” outbreak that led the government to classify it as an adulterant in non-intact beef, such as ground beef. Testing for O157 fundamentally changed beef industry practices. Today, this organism accounts for about 36 percent of STEC infections and is typically the most virulent. However, six other types of EHEC collectively cause almost twice the number of illnesses, and the government now classifies them as adulterants in non-intact beef as well. These EHEC, referred to as “Top 6” non-O157—O26, O45, O103, O111, O121, and O145—are not as well understood and are more difficult to detect. An eighth STEC, O104, was responsible for a European outbreak in 2011. This strain does not produce intimin, so is not an EHEC. However, it produces several other virulence factors in addition to Shiga toxin that make it highly virulent and, for this reason, the USDA-NIFA charged the STEC CAP with testing cattle and beef for this

STEC, by definition,


are E. coli that


produce a potent toxin

Little is known about the ecological niche STEC occupy in the bovine, said UNL’s Andrew Benson. Several researchers described their research in the section, STEC Ecology in Cattle. Benson’s team seeks to identify components of both the gut ecosystem and the host genetic makeup that contribute to STEC colonization and shedding. Benson described his research developing the bioinformatic and statistical tools needed to conduct his studies, the foundational work in mouse models and the data he’s collected in cattle. If he is able to find host genetic traits influencing functions that affect STEC shedding, it could lead to hypotheses to manipulate shedding, Benson said.

called Shiga toxin.

Cattle consistently shed more E. coli O157 in summer than in winter, which corresponds to a higher infection rate in humans during summertime. Thomas Besser, of Washington State University, described his research into understanding the cause of this seasonal shedding variation. His studies suggest that changes in neither the cattle’s gut physiology nor their gastrointestinal ecosystem are responsible. However, a higher population of bacteria-eating protozoa in water troughs during winter months may provide an environmental explanation for lower E. coli shedding rates in the winter, Besser said. Shannon Manning, of Michigan State University, also seeks to identify factors important to STEC shedding. She described research into the prevalence, diversity and persistence of both E. coli strains and the types of bacteriophages that carry

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Executive Summary

virulence factors. Her studies in Michigan cattle herds found much higher rates of non-O157 STEC than O157. Both E. coli strains and bacteriophages were a highly dynamic mix, appearing and disappearing in cattle over time. Manning’s findings suggest the transmission of Shiga toxin-producing phages is critical for the emergence of new STEC strains, particularly O157 in cattle herds. Foodborne disease outbreaks associated with produce are increasing due to proximity to animal operations, wildlife and other environmental factors. Several experts described their work into understanding and preventing STEC in Produce and the Environment. The 2006 spinach outbreak focused attention on wildlife’s contribution to produce contamination, said Michele Jay-Russell of the University of California, Davis. She described her investigation into that outbreak as well as her current research into STEC in wildlife and their potential contribution to produce contamination. Several STEC surveys found low levels of pathogenic E. coli in birds, coyotes, feral pigs and other wildlife. Ludek Zurek, of Kansas State University, is investigating the role flies play in the ecology of O157. His research found large numbers of O157 in feedlot houseflies and that the flies are capable of transmitting the pathogen to cattle, suggesting that houseflies play a prominent role in spreading O157 to produce. Housefly management should be an important part of pre-harvest food safety strategies, Zurek said. California produce growers are encouraged to maintain a 400-foot buffer zone between concentrated animal feeding operations and leafy green fields. Elaine Berry, of the USDA’s U.S. Meat Animal Research Center (USMARC), described her research into contamination of produce fields near a feedlot. Results suggest that 400 feet is insufficient to protect produce and that both flies and wind may contribute to transporting pathogens to fields. The U.S. generates large amounts of manure that provide nutrients to fields, said the USDA’s Manan Sharma. The Food and Drug Administration’s proposed rule would require that any raw manure be applied to produce fields at least 270 days before crops are harvested. Sharma described his research into bacterial survival in manure-amended soils. Preliminary results with O157 suggest that STEC can regrow in composted and nutrient stabilized soils and that the types of manure and soil as well as the method of manure application affect the persistence of STEC. Research is ongoing to better understand the appropriateness of the 270-day interval, Sharma said. As an extension service specialist at UC, Davis, Trevor Suslow described his work with produce growers, sharing their challenges and risks. As animal operations move into areas, they can contaminate nearby fields through water contamination, wildlife movement and sharing fields with cattle, among other factors. Contamination can cause significant economic losses for growers. Growers have asked researchers and public health experts to develop rapid screening tests that differentiate EHEC from the STEC less likely to cause clinical illness in humans, Suslow said. False positive test results occur when genes from Shiga toxin and intimin are detected in test samples, but the genes are contributed by different bacteria, which are therefore not pathogenic. False positives have resulted in needless destruction of product.


Earthbound Farms, the nation’s largest grower, packer and shipper of organic produce, extensively tests its products for O157 and other pathogens, said the company’s Will Daniels. He described its testing programs. The prevalence rate over the past eight years has been less than 0.5 percent. None of the positives represented gross contamination events. The company continues to investigate ways to reduce contamination and improve its production methods. STEC Prevalence and Interventions Strategies at Slaughter are important factors in reducing the risk of STEC. Lawyer Bob Hibbert described the U.S. legal and regulatory system within which animal production facilities must operate. He criticized the current “regulatory creep” taking place and said he believes the current performance standards approach to regulation is flawed and a misuse of finite resources. He suggested solutions be driven by science, a more open policymaking process, comprehensive risk management within industry and effective public communication. A primary goal of the STEC CAP is to develop a quantitative microbial risk assessment (QMRA) to help identify research needs and effective industrial interventions intended to reduce pathogens in beef products. The QMRA is a method to quantify the risks of microbial contamination of a product. KSU’s Michael Sanderson described his team’s efforts to create a model that encompasses risk along the entire beef production chain. The team has made substantial progress in extracting and analyzing existing data through carcass intervention, but lack of data is still a significant challenge. As additional data becomes available, it will be incorporated into the model, Sanderson said. KSU’s David Renter described several studies he and a collaborative team of researchers have conducted to contribute data for the QMRA Sanderson discussed. The studies are helping to better understand when, how much and why STEC occur in beef production. Renter emphasized that many studies refer solely to serogroups and do not confirm the presence of virulence genes. This distinction has important implications for risk assessments and for industry, so care must be taken in defining risks, he said. Validating the effectiveness of interventions to control STEC in beef products is an important part of a successful food safety management system, said Gary Acuff of Texas A&M University. He described several studies he and his collaborators are conducting into intervention effectiveness in both veal and beef productions as well as dry-fermented sausage processors. Results suggest that O157 and the Top 6 non-O157 STEC behave similarly. Philip Bronstein, of the USDA’s Food Safety and Inspection Service (FSIS), said the agency is tasked with balancing the needs of public health, consumers and industry. FSIS tests raw beef products for O157 and the Top 6 non-O157 STEC. Data suggests non-O157 STEC is twice as prevalent in raw ground beef as O157. The prevalence of STEC in veal has raised questions about whether E. coli problems are specific to veal, Bronstein said, adding that although prevalence seems higher in veal, STEC is problematic in both beef and veal. He described the Beef Veal Carcass Baseline Study FSIS is conducting to better understand STEC in beef and veal. He also described the agency’s strain characterization study designed to identify STEC and virulence factors in beef not screened during routine testing. Preliminary results suggest pathogenic STEC other than the current list of FSIS adulterants are present in raw beef.

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Executive Summary

FSIS’s Rachel Johnson-DeRycke described a risk assessment tool the agency is developing to model pathogen controls in beef slaughter establishments. The model seeks to identify how the number, type and combination of interventions applied at slaughter establishments to remove pathogens affect O157 contamination of beef carcasses. The model may be used to help allocate FSIS resources, as a tool for industry to allocate resources when evaluating interventions and for future rulemaking or guidance documents. National Beef Packing’s Brenden McCullough discussed the slaughter industry’s perspective regarding STEC. He said he would like to see a more targeted approach to STEC interventions from regulators, but acknowledged that the industry was not as proactive as it should have been after the 1993 outbreak and continues to be reluctant to share information. The good news is that O157 prevalence in beef has declined 85 percent since 2000, though improvements are still needed. He applauded the multidisciplinary approach to the CAP process and said he is glad industry was invited to participate. Although much attention is focused on reducing STEC in animal and plant products, Preventing Outbreaks through Education and Consumers is also an important component to public health, said Ben Chapman of North Carolina State University. Restaurants are a primary source of foodborne illness. Chapman described several studies he and a collaborative team are conducting into the messages consumers receive at restaurants and on the Internet regarding food safety. Restaurants provide mixed messages and servers are ineffective risk communicators, he said. In another study, the researchers found that YouTube videos regarding hamburger preparation overwhelmingly model unsafe behavior. Chapman participates in social media and operates the Barf Blog at People serving food in private homes, social and religious groups and for elderly and youth populations also contribute to foodborne illnesses, said Christine Bruhn of UC, Davis. Her research focuses on educating community volunteers and high-risk populations about food safety. She has developed and is testing an online food safety training program based on a previously implemented in-person workshop. Another study looked at the impact of celebrity chefs on consumer behavior. She found that many consumers were unaware of inappropriate safe food handling practices, and believed that the chefs’ behaviors were acceptable. Kelly Oliver described the educational training modules KSU’s Beef Cattle Institute has developed to teach proper control and prevention strategies for industry personnel. The project focuses on both pre- and post-harvest operations, including feedlot operations, packing plants, restaurants and distributors. To train the current and future workforce needed to control STEC, KSU’s Jason Ellis and UNL’s Dann Husmann are developing an educational multi-year curriculum focusing on nutrition science and food production for high school students. The curriculum consists of four courses and is based on the inquiry-based learning method, which encourages developing skills and knowledge so students are able to ask questions and solve problems. To engage students in research, Ellis and Husmann


hope to partner students with researchers. Once the program is finalized and validated, they will expand it beyond the current pilot project in Nebraska and Kansas. Though sometimes overlooked, consumers and consumer advocates play vital roles in reducing STEC illnesses, said Vanessa Coffman of STOP Foodborne Illness. The consumer policy and advocacy organization, founded in 1993, provides a voice for consumers. It engages in numerous policy initiatives and consumer advocacy activities, including congressional visits and participating in social media. Coffman encouraged audience members to engage consumers and consumer advocates, visit the website ( and use the personal stories provided there as context for their work. “If there’s some way that we can work together, I would love to do that for you,” she said. Improving STEC Detection is a critical component to catching pubic health threats before they become outbreaks, to develop economically feasible interventions at meat processing facilities and to aid research. But determining whether Top 6 non-O157 EHEC are present, as well as identifying specific strains, is challenging. UNL’s Rodney Moxley described several studies comparing the effectiveness of different growth and culture plating media and work to develop biomarkers to improve the detection of these organisms. A problem in particular is the lack of a single gene target that can be used to identify the presence of Top 6 non-O157 EHEC in a sample containing a mixture of bacteria. USMARC’s Mick Bosilevac emphasized that PCR methods based on simple detection of stx and eae in such samples do not indicate whether the genes originate from one bacterium, a criterion necessary for confirming the presence of Top 6 non-O157 EHEC in the sample. As such, they are only identified as “potentially positive.” He described several approaches to improve on these classic virulence targets to better detect non-O157 STEC and to help reduce the number of “potentially positive” and false positive samples during STEC monitoring.

mandritoiu /

Alina Deshpande, of the Los Alamos National Laboratory, has developed the first of two PCR-based tests to detect the “Top 8” STEC types (Top 6 non-O157, plus O157 and O104) and three virulence genes. This assay, known as a multiplex oligonucleotide ligated, or MOL-PCR, is different from other traditional PCR assays in that it contains a step that adds to its specificity. In addition, the rapid, highthroughput assay enables large-scale sample screening and is inexpensive. KSU’s T.G. Nagaraja described research comparing three methods for detecting the Top 7 STEC types in cattle feces: traditional PCR, real-time PCR and culture. His studies demonstrate that RT-PCR is the most sensitive method, detecting more potential positives than the other two. However, the culture method sometimes detects non-O157 EHEC in samples that test negative by RT-PCR. Continued research on diagnostic test development to identify Top 6 non-O157 EHEC is needed.

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G ov e r n o r ’ s W e l co m e Food safety and quality are important topics both nationally and globally. As the national


leader in red meat production, Nebraska is particularly concerned with improving beef safety, said Dave Heineman. Food production is Nebraska’s leading economic driver, and its beef production spans the entire spectrum, from cow-calf operations and feedlots to large processing facilities. “One in four hamburgers eaten in the United States starts here in Nebraska,” Heineman said. The state’s beef industry is also important globally. Nebraska is

Dave Heineman

Former Governor of the State of Nebraska

among the top five states in agricultural exports, with livestock and grain products in greatest demand globally. Food production will become even more critical as the world’s population adds another two to three billion people by 2050. “From my perspective, we need to feed the world,” he said. “And that’s why these issues of food safety and all the rest are really critically important as we think about how are we going to feed the world. Food quality and safety are critical to maintaining all of our reputations as the demand for agricultural products continues to grow with the expansion of the global economy.” Heineman thanked those in attendance for their important roles in improving food safety, the University of Nebraska for leading the STEC CAP effort and private industry for its involvement with university research to enhance food safety along the food supply chain. It’s vital work to protect consumers, he said. “They may not know how that food is produced, but they expect the very best food in the world.”


stEc summ ary of co n f e r e n c e p r e s e n tat i o n s

Video of Opening Remarks Video of Closing Comments presentation


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stEc MWAS and GWAS Studies to Define Associations between Host Genetics, Microbiome Composition and EHEC Colonization

Eco l o g y i n C at t l e Little is known about the ecological niche that STEC occupy. To truly understand the ecology of STEC colonization in the bovine, scientists must understand how the gut ecosystem, or microbiome, is created and how it functions, said Andrew Benson. His team seeks to identify components of both the gut microbiome and the host genetic makeup that contribute to STEC colonization and shedding. Each individual—whether human, mouse or cow—has a unique set of gut microorganisms. Some organisms are shared across individuals, but the overall composition is distinctive, like a human fingerprint. This microbiome is randomly determined by such factors as gut disturbance, transmission between mother and child, or exposures to environmental chemicals. Other factors that may affect microbiome composition include the order in which the gut is exposed to organisms and the host’s genetic makeup, diet, metabolism or physiology. Fortunately, knowledge about the bovine genetic makeup is expanding, which will help

Andrew Benson

Professor, Department of Food Science & Technology University of Nebraska

in understanding microbiome composition and influences at the host genetic level. “If we can understand that, it may be possible to incorporate that information back into the breeding programs,” Benson said. “Because if you can use breeding to reduce the levels of shedding of this organism, it would have tremendous impact.”

Developing tools Although Benson’s research has been primarily on a mouse model, that work lays the foundation for cattle microbiome studies. He has been building bioinformatic and statistical tools needed to conduct microbiome studies. Benson described relevant research findings on mouse models to date. Microbes present in the gut can be identified by sequencing their DNA.

Benson’s team has learned that sequencing using metagenomic techniques, a shotgun approach in which all genes from a gut sample are sequenced and used for identification, is as effective as a targeted method of sequencing a specific gene complex present in most microbes, but with variations that allow for microbe identification. He’s also learned that the microbiome behaves as a complex trait, a term

identifying a trait influenced by a combination of environmental factors and genetic predisposition. Benson and his collaborators crossed two mouse lines and monitored the progeny’s gut microbiome as well as the distribution of chromosomal alleles, or genetic variation, from parent to progeny. A study looking for associations between host genetic variation and variations in the microbiome composition revealed 13 different locations in the mouse genome controlling its microbiome, including a variety of different microbial types. Benson’s team next investigated whether genetics controls the microbiome

composition, function or both. Metagenomic studies on the progeny of mice lines bred to resemble the genetic diversity found in human populations revealed that the host controls the function of the gut microbiome, and does so independently of which organism contributes that function. Specifically, they found that the host controls glutamate and butyrate metabolism. Butyrates


provide energy for cells in the colon, while glutamate, a neurotransmitter, may contribute to a connection between the gut and the brain.

STEC study Benson said it’s likely STEC shedding and the local microbiome ecology behave as a complex trait, influenced by both genetic and environmental factors. STEC seem to be transient members of the bovine gut and, typical of complex traits, E. coli O157:H7 (O157) show log-normal distributions in animal populations. Using similar association methods as in the mice studies, Benson and his colleagues are investigating the effects of host genetics, environmental factors and microbiome composition on STEC shedding. Collaborating with the USDA’s U.S. Meat Animal Research Center, the researchers determined the genotypes and the characteristics resulting from genotypes interacting with the environment, or phenotypes, in more than 1,000 animals from 16 common breeds, on four different finishing diets and in three geographic locations. Over four years, a different set of animals from the

same herds were sampled repeatedly and quantified for O157.

...if you can use breeding to reduce the levels of shedding of this

They have now completed the “heroic” effort of gathering the metagenomic sequencing data, Benson said, and have begun to analyze the data to better

organism, it would

understand the associations between the host genome and the microbiome composition and functional traits.

have tremendous

To date, results suggest that diet contributes significantly to microbiome


composition. They found a wide variety of organism types, though Firmicutes were particularly common. The year sampling occurred was a driving factor in the gut microbe composition observed, not surprising given that the diet varied each year. However, composition was unaffected by gender, whether or not the animal shed STEC or the amount of STEC shedding. Now that the enormous task of collecting the data is complete, Benson said his analyses and research are quickly yielding additional results. When asked about his initial hypothesis, Benson said that the population-based information he is gathering will lead him toward hypotheses. In the mouse studies, for example, the data is suggesting a hypothesis that the host shapes butyrate or glutamate metabolism in the microbiome. Likewise, if he is able to find host genetic traits influencing functions that affect STEC shedding, it could lead to hypotheses to manipulate shedding.

Video of Andy Benson presentation

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stEc Seasonal Variation in E. coli O157: H7 Shedding by Cattle: Fecal, Feed and other Factors

Eco l o g y i n C at t l e In northern latitudes, cattle consistently shed more E. coli O157:H7 (O157) in summers than in winters. E. coli infections in humans also peak in summer, so understanding the seasonal variation in cattle is important. While the rise in human infections may be due, at least in part, to the increase in cattle shedding, human and bovine infections may also originate from the same unknown source. “[Seasonal variation] is a very strong phenomenon, and we really don’t have a clue what’s responsible for it,” Besser said. Researchers have identified three possible explanations: first, changes in bovine physiology may better support shedding in summer months, such as hormonal or endocrine factors or changes due to differences in light or temperature; second, changes in the cattle’s gastrointestinal flora, or microbiome, through which E. coli passes, may affect infection rates; or third, cattle are exposed to more O157 in the summer from an unknown environmental source, such as through feed or water.

Thomas E. Besser

Professor, Department of Veterinary Microbiology and Pathology Washington State University

Besser detailed three studies he and his team conducted to test these possibilities.

Physiology and the microbiome Besser’s experiments suggest that neither physiological changes to the cattle themselves nor their gastrointestinal microbiome is responsible for increased shedding in the summer months. His team divided 20 calves into two groups: one group received O157 orally; the others were inoculated intrarectally to bypass the gut microbiome. For two years, each group was inoculated in January and July, and shedding rates were measured for two months. The cattle ate from stockpiled feed to reduce influences from changing environmental conditions and were housed in an open-sided pen to maintain realistic environmental conditions. Shedding rates varied little between summer and winter in either the oral or rectallydosed cattle. In both, shedding rates were high immediately following inoculation and, except for a few high shedders, diminished over time. These results suggest that neither bovine physiology nor the microbes in their guts affect STEC shedding rates between winter and summer. Besser also looked at the shedding rates of individuals and their pen mates. Although he expected to see similar shedding rates in cattle sharing pens and water, he saw no relationship. In one case, the longest and the shortest shedders shared the same pen. The cows were dosed with a 4-strain mixture of O157. Analysis of the different E. coli strains suggests that longtime shedders continue to shed a strain that is common in cattle, but not in humans.


Besser emphasized that this was a small experimental study and that the cattle received higher exposure than they would naturally. What happens at lower levels of exposure is still unknown, he said.

Environmental role Excluding both physiology and the microbiome suggest the environment plays a dominant role in seasonal STEC shedding, Besser said. During the summer months, cattle feed on forage within a few months of harvesting, a feed type that is consistently the most contaminated. Besser’s team tested the role a change in feed source between summer and winter might play in shedding seasonal variation by testing cattle for STEC on multiple farms before and after feed changes in summer and winter, but found no impact. However, studies looking at water troughs suggest troughs may play a role. More O157 are detected in cattle water troughs in the summer than in winter. While that increase may be responsible for higher infection rates during the summer, it may also be a

result of increased shedding.

[Seasonal variation] is a very strong phenomenon, and we really don’t

To investigate further, Besser’s team sampled troughs in winter and summer and

have a clue what’s

found significantly lower numbers of protozoa during the summer, which may be due to higher chlorine concentrations, higher temperatures or decreased sediment depths.

responsible for it.”

The majority of the protozoal species are eating O157, so lower numbers of protozoa in the summer months may account for higher levels of E. coli. Researchers had previously observed that Shiga toxin-producing O157 survive protozoal grazing more effectively than other strains, suggesting that Shiga toxins protect bacteria from predation. The majority of O157 isolated from the environment produce Shiga toxins, which may indicate a selective advantage for its production. What drives that selective pressure is unknown, but protection from environmental protozoa is a good candidate, Besser said. However, unlike the previous study, his team found no protective effects for Shiga toxin-producing O157. “Clearly, a lot fewer bactivorous protozoa are present in the cattle water troughs in the summertime than in the winter, and this is still on the potential list for maybe playing a role in reducing the exposure of O157 in the wintertime,” he said.

Video of Thomas Besser presentation

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stEc Shiga Toxin-producing E. coli in Michigan—from Shedding in Cattle to Human Disease

Shannon Manning

Professor, Department of Microbiology and Molecular Genetics Michigan State University

Eco l o g y i n C at t l e Shannon Manning seeks to identify factors important to STEC shedding by looking at both genotypes and the characteristics that result from genes interacting with the environment, or phenotypes, that facilitate STEC colonization. Her team looked at the prevalence, diversity and persistence of E. coli strains and bacteriophages, the viruses that supply Shiga toxin genes to E. coli, converting them to STEC. She also investigated whether a herd’s diversity of STEC strains was due to transmission of the STEC bacteria or to E. coli acquiring a phage carrying Shiga toxin genes. In a two-year study sampling more than a thousand cattle, Manning found much higher numbers of non-O157 STEC strains than E. coli O157:H7 (O157). Although some STEC likely persist in a herd, overall the strains and phage types were a highly dynamic mix, appearing and disappearing in cattle over time. The STEC population also appears to be more diverse than the Shiga toxin-producing phage population, though the phage population is also diverse. Manning’s findings suggest the transmission of Shiga toxinproducing phage is critical for the emergence of new STEC strains.

STEC prevalence and diversity Manning’s team collected fecal and rectal samples from nearly 1,200 animals on 11 dairy and beef farms over a two-year period, and conducted genetic tests to determine the E. coli strains present as well as the phages carrying the Shiga toxin or intimin virulence genes. They found STEC strains in about 16 percent of the animals, with greater prevalence in beef than in dairy cattle. Manning’s team also found that 70 percent of the animals shed sufficient amounts of STEC to be considered supershedders. However, it’s unclear if the Shiga toxin virulence gene was incorporated into the E. coli bacterium, was part of another bacterium, or came from a free phage. “So I can’t really safely say that 70 percent of animals were super shedding STEC, but we do know that the genes are there,” Manning said. For a more detailed view of the STEC strains present, Manning’s team conducted more thorough testing on a subset of animals from five herds. Surprisingly, O157 was present in only 26 percent, a very low prevalence, she said. In contrast, three-quarters of the animals carried a non-O157 STEC strain. These rates mirror trends in human cases seen in Michigan and nationally. Non-O157 STEC infections are increasing while O157 infections have declined. In Michigan, at least 23 different STEC strains have been found in clinical cases, including 17 percent due to either unknown or undetermined STEC. “I think that it’s really important to state that not just the Top 7 [STEC] cause disease in humans. We’re actually seeing many other serotypes that are associated with clinical infections,” Manning said. Testing also revealed a high diversity of STEC serotypes. For this analysis, she identified distinct STEC profiles, based on the presence or absence of three virulence factors: the Shiga toxin genes, stx1 and stx2c, and the intimin gene, or eae. The researchers found three to eight distinct profiles in each herd and one to four profiles per animal. About 16 percent of the animals had more than one STEC profile, suggesting they’re shedding more than one STEC type.


Although it’s often assumed that the same STEC strain travels through the herd, Manning said she believes that assumption may underestimate diversity.

To further understand diversity and the persistence of strains over time, her team captured detailed snapshots of the STEC strains present in several animals. Over the course of three sampling visits, the researchers found a highly dynamic flow of STEC strains, with animals acquiring new strains and losing others, while other strains remained consistent within the group. “This is just a snapshot of 12 animals over time, and so if STEC dynamics are this complicated, it’s going to be difficult to get a handle on what’s going on at the herd level,” Manning said. To determine if high STEC diversity within a herd is due to transmission of STEC bacteria or to a phage carrying Shiga toxin virulence genes, Manning’s team developed a test to determine phage types while they’re incorporated into the STEC bacterial host genome. For 86 different STEC strains within four herds, they identified 17 distinct phage types in circulation. The dairy herd tested had more phage types than the three beef herds. They also tested for phage types present in a deer herd that shares a pasture with cattle in the dairy herd. They found both herds share a phage type, but it appears to be from STEC, not phage, transmission. It remains unclear if the deer acquired the phage type from the cattle or the cattle from the deer.

To better understand which STEC strains are transmitted within a cattle herd and which represent evidence of phage transmission, they identified the specific phage types and STEC strains in a subset of animals sampled over time. In the first sampling, they identified two STEC strains: one animal carried an O26 strain with phage type 3 and three carried an O6:H34 strain with phage type 4, suggesting phage transmission occurs within a herd.

really important to state that not just the Top 7 [STEC]

In the second sampling, the picture changed considerably: O103:H2 STEC with phage type 2 appeared in several animals, so the STEC strain may have introduced a new phage type. Interestingly, one animal carried three different E. coli strains with two different phages.

cause disease in humans. We’re

In the third sampling, an O26 STEC found in one animal during the first sampling reappeared in many of the animals during the third visit. Interestingly, one animal now carried an O157 strain with two phage types.

actually seeing many other

“We think this suggests that the phage is actually critical for the emergence of these new types, particularly the O157s,” Manning said. “So maybe even looking for these O157s that are currently [Shiga toxin] negative, which we found a ton of, is not such a

serotypes that are associated with

bad idea because you might be able to figure out what predicts phage incorporation.”

Ongoing studies

clinical infections.”

Manning’s studies into STEC and phage prevalence and diversity continue. Because certain STEC characteristics may be associated with its persistence within a herd, Manning’s team is investigating biofilm formation, which allows bacteria to stick together and to surfaces. To date, the researchers have seen significant variation in STEC strains’ ability to form biofilms, and they continue to study the genetic pathways necessary for biofilm formation. Preliminary data suggests that persistent strains are more likely to produce strong biofilms.

Video of Shannon Manning presentation misc/GovFoodConference14/4ShannonManning.html

Additional projects aim to identify the potential gut microbes that may inhibit or promote STEC shedding and to identify an association between cattle immune response and STEC shedding. Manning is also conducting large-scale epidemiological analyses to better identify and understand risk factors for STEC acquisition.

I think that it’s

17 2 0 1 4 STEC C A P P r o c e e d i n g s

stEc The Role of Wildlife in the Dissemination of E. coli O157:H7 and other STECs to Fresh Produce

i n p r o d u ce & t h e e n v i r o n m Domestic and wild animals are potential reservoirs of STEC and enterohemorrhagic E. coli, and may contribute to human disease through undercooked game meat, contamination of food and water supplies and direct contact. Though much work has been done on E. coli in cattle, STEC can be found in nearly every kind of animal species. Michele Jay-Russell’s research looks at how wildlife contributes to fresh produce contamination. Wildlife’s involvement in the spread of STEC is straightforward, she said. Fresh produce is primarily grown in open fields and orchards where animals can forage and defecate. Water sources are also habitat to some animals, such as alligators and turtles living in irrigation ponds. Recent efforts to preserve riparian corridors and other habitats benefit wildlife, but also create difficulties for growers. Wildlife presents risk, Jay-Russell said, but “it’s a manageable risk, from my point of view.” Risk stems, in part, because pathogens can’t be completely washed off raw or

Michele Jay-Russell

Program Manager and Research Microbiologist, Western Center for Food Safety University of California, Davis

minimally processed produce. In addition, many STEC have a low infectious dose, so even a single animal in a field can lead to pathogens entering a processing plant where cross contamination can amplify the risk.

Outbreak research Much research into wildlife’s contribution to produce contamination stem from a 2006 E. coli O157:H7 (O157) outbreak in fresh, prepackaged spinach. Jay-Russell was involved in researching the outbreak, which was determined to originate from a 10,000-acre grass-fed beef cattle herd in San Benito County, California. The ranch had been irrigating the pasture. A large population of feral pigs, a nuisance species in the area, also existed near the operation’s cow-calf area, and nearly 5 percent of the pigs and a quarter of the pig fecal samples tested contained O157. The outbreak strain was also found in pasture soil and some surface water not used for irrigation. How the spinach fields became contaminated remains unknown, but the research established that E. coli outbreaks originate at the field level and not later in the food production process. Research also suggests that even a small amount of pathogenic bacteria entering a processing plant where spinach is bagged can lead to crosscontamination, resulting in a large outbreak. An O157 outbreak in Oregon in 2011 was linked to deer fecal contamination of a local strawberry field. Jay-Russell noted that, as a small farm, that operation would be exempt from the Food and Drug Administration’s proposed new safety rule.

Central coast produce Prior to the 2006 spinach outbreak, smaller outbreaks in spinach and romaine lettuce had been occurring, and water was suspected. Sometimes called the nation’s “salad bowl,” California’s central coast, south of San Francisco, presents unique food safety challenges. Besides produce, the area also has a large number of pasture-based cow-calf operations and a few small feedlots. The area also features wildlife riparian corridors.


ent Jay-Russell presented results from a collaborative 4-year study developing STEC

Best practices

detection methods and a prevalence survey. Working with growers and ranchers, the

Growers are proactive, Jay-Russell

team collected more than 12,000 samples from plants, soil, wildlife and cattle.

said. They conduct environment

The habitat range varied from pastures to residential and riparian areas. It also

assessments, establish setbacks from

included one feedlot.

cattle operations and, if they do find

The researchers found O157 in 2.5 percent of samples from cow-calf ranches,

fecal material, they don’t harvest

but nearly 10 times that amount in the feedlot. Results showed the distinct

within a 5-foot buffer zone.

seasonal prevalence variations typically seen in Midwestern cattle studies.

The FDA’s approach to wildlife and

They found little O157 in samples collected from 18 avian and mammalian species

domestic animals is vague, she added. “They are basically leaving it

across a variety of seasons and locations. However, some positives were detected in

up to the grower’s judgment and have

American crows, brown-headed cowbirds, coyotes and feral pigs. Prevalence primarily

words like ‘reasonable,’ ‘significant.’

ranged from 1 percent to 3 percent, though some ranches had much higher levels.

What’s a reasonable amount of poo

Positives in both the crow and cowbird came entirely from the feedlot and were genetically identical to the cattle, indicating spillover to the birds occurs at that feedlot.

in your salad to me might not be the

The results raise the question of whether a cattle E. coli O157:H7 vaccine could

agency is revisiting its produce safety

reduce the risk of environmental spillover to wildlife, Jay-Russell said. They’ve

rule, it appears likely it will continue

spoken with a vaccine company, but the utility of an approved cattle vaccine for

to leave it to growers to decide.

same as it is for you.” Although the

this purpose has not been tested and remains unknown.

Follow-up studies Jay-Russell described some of the follow-up studies she and her team have conducted. Rodents: Wild rodents are common in California produce fields. In a study

testing more than 1,000 rodents, only one kangaroo rat and a deer mouse were positive for O157. Salmonella rates were also low. Amphibians and reptiles: Collaborating with researchers at the University of

Georgia, Jay-Russell and her team found no O157 in amphibians and reptiles commonly found in Georgia sediment basins and farm ponds. Salmonella was found at the level found in pet store turtles and snakes. Feral dogs: A 2010 E. coli O145 outbreak was traced to romaine lettuce grown

in the southwestern desert near Yuma, Arizona, a major growing region for leafy greens in the winter. The area’s many stray dogs pose a potential food safety risk. In collaboration with the University of Arizona and the Arizona Game and Fish Department, a STEC survey of impounded dogs picked up near produce fields and of coyote scat found no Shiga toxin virulence genes. However, a few contained the intimin virulence gene. It’s unknown if those positives pose a threat to human health, Jay-Russell said. That work continues. Video of Michele Jay-Russell

Cattle and wildlife: The researchers are tagging animals and birds and using


radiotelemetry to investigate buffer zones between feedlots and produce fields.

Preliminary prevalence test results found higher prevalence of non-O157


STEC than O157, nearly 40 to 15 percent. Javelinas and feral pigs had


low prevalence for both types, and rodents contained neither, but did have Cryptosporidium. Radiotelemetry will help them track the movement of birds, and the pathogens they carry, between feedlots and produce fields.

19 2 0 1 4 STEC C A P P r o c e e d i n g s

stEc Role of Insects in the Ecology of STEC

i n p r o d u ce & t h e e n v i r o n m Ludek Zurek’s research focuses on insect microbiology. His investigation into the role flies play in the ecology of E. coli O157:H7 (O157) suggests houseflies may play a prominent role in spreading the pathogen. Housefly characteristics make them a potential vector for O157. They lay their eggs in older manure available in feedlots and dairies, where adult flies pick up the bacteria. Flies evolved closely with bacteria and cannot develop from maggots without a live bacterial community, though the mode of this symbiosis is unknown, Zurek said. Stable flies also lay eggs in older manure but, unlike houseflies, they bite and suck blood. The biting stresses animals, causing drops in weight and dairy production. To avoid economic losses, farmers and ranchers use insecticides and manure management to manage stable fly populations. Houseflies, in contrast, are allowed to proliferate.

Ludek Zurek

Professor, Microbial Ecology, Department of Entomology, Department of Diagnostic Medicine & Pathobiology Kansas State University

Houseflies feed and defecate on fresh-flaked corn, rich in sugar and moisture. Because they can’t feed on solid food, they liquefy it. The food enters a large sac called a crop where it’s stored for later. When the fly is hungry, it moves the food out of the crop and into the midgut for digestion. Bacteria multiply in the crop and can be transmitted from their mouthparts when feeding or through their feces. “So they are a very good potential vector for bacteria that they have in the digestive tract,” Zurek said. Many bacterial types have been found in houseflies, including Campylobacter, Listeria and Salmonella. A study in Japan also suggested houseflies can harbor O157. To determine whether houseflies can transmit O157 between cattle, Zurek and his team first looked at the prevalence of O157 positive houseflies in a Kansas feedlot between June and October. The researchers found 2.9 percent of flies collected from feed bunks and 1.4 percent from corn carried the pathogen. While that may seem low, given the enormous housefly population in a feedlot, even a 1 percent prevalence rate translates into thousands of O157 positive flies, he said. More importantly, they found high concentrations of bacteria. A single fly can carry 150,000 O157 bacteria in its digestive tract. “So that looks like they might actually make a difference in terms of moving these bacteria, at least within the feedlot environment,” Zurek said. In addition, all of the O157 bacteria they tested carried stx2, a Shiga toxin virulence gene. The majority also had stx1 and intimin virulence genes. The average fly also carried about 100,000 fecal coliforms in its digestive tract. Genotyping revealed that the bacteria were closely related, a surprising result given that samples were collected over months. Testing for antibiotic resistance showed the majority of O157 bacteria were resistant to chlortetracycline and oxytetracycline.


ent After establishing that flies carry O157, the researchers investigated the potential for flies to move bacteria from place to place, infecting cattle. They exposed eight calves to flies inoculated with four strains of O157 and collected fecal and water samples for three weeks. Surprisingly, all of the calves were positive after just two days of exposure and remained positive for at least 11 days. Water sample results were more sporadic, with E. coli appearing and disappearing periodically, most likely from calves contaminating it with their saliva. “The houseflies are actually capable of

transmitting E. coli O157 among cattle and likely play a role in the ecology of this pathogen,” Zurek said. The exact mode of transmission is unknown, but flies most likely contaminate feed and water as well as land directly on cattle mouthparts. He added that he believes the higher rate of E. coli infections in the summer is related to houseflies, which are abundant in summer and disappear in winter. Houseflies are also capable of moving bacteria from place to place. They can fly

Houseflies aren’t just a nuisance. They don’t bite, but they can

20 miles, but usually travel no more than six miles on average. They spread from feedlots in midsummer when conditions become crowded. Residential areas are

actually transmit

also attractive to houseflies.

and move all kinds

“So I think housefly management should be an important part of pre-harvest food safety strategies,” Zurek said. “Houseflies aren’t just a nuisance. They don’t bite,

of microbes that

but they can actually transmit and move all kinds of microbes that originate from the manure, including O157:H7.”

originate from the

An additional study found non-O157 in houseflies as well, but all were negative

manure, including

for virulence factors. Those studies continue.


Video of Ludek Zurek presentation

21 2 0 1 4 STEC C A P P r o c e e d i n g s

stEc Dissemination of E. coli O157:H7 from a Cattle Feedlot: Effect of Proximity on Contamination of Leafy Greens, Bioaerosols and Pest Flies Elaine D. Berry

i n p r o d u ce & t h e e n v i r o n m “Historically, foodborne disease outbreaks were commonly associated with animal products,” said Elaine Berry, but now “the dynamic is changing and produce outbreaks have increased.” The increasing numbers are due, in part, to the large E. coli O157:H7 (O157) outbreaks in spinach and lettuce in 2006 and 2010, but also to improved outbreak surveillance systems and microbial detection methods. They may also be due to increasing consumption of riskier fresh-cut prepackaged products. Fresh produce is consumed raw or with minimal processing. Chemical washes and heat treatments are applied to fresh produce, but leafy vegetables are delicate and challenging to treat. “So it’s very important to protect that product in the pre-harvest produce environment, because there are so many potential sources of pathogens and so many different routes of contamination,” Berry said. Some routes are easier to control, such as fences to exclude livestock and wildlife and treating manure to reduce pathogens prior to spreading it on fields. Other routes

Research Microbiologist, U.S. Meat Animal Research Center

are more difficult, such as birds, flies and airborne transmissions from livestock

USDA-Agricultural Research Service

After the 2006 outbreaks, California growers created the Leafy Green Products


Marketing Agreement in collaboration with scientists, food safety experts, processors and sellers to provide guidelines for the safe production and harvest of leafy greens. The agreement suggests a 400-foot buffer zone between concentrated animal feeding operations and leafy green fields, but that distance lacks scientific confirmation. “That turns out to be one of the very critical questions that needs to be answered for the produce industry: What are the appropriate setback distances or buffer zones that we can place in between animal production and leafy green fields that’s going to reduce the risk of contamination with pathogens?” Berry asked.

Feedlot proximity Berry and her team conducted a two-year study to determine the impacts of environmental conditions and proximity to a cattle feedlot on the prevalence of O157 in a leafy green produce crop. Their results suggest that O157 can be transported from a cattle feedlot to nearby fields. Although pathogen levels decreased as distance from the cattle increased, they found a 400-foot buffer zone to be inadequate to prevent field contamination. At the USDA’s U.S. Meat Animal Research Center in Nebraska, they grew leafy greens 200 feet, 400 feet and 600 feet downwind of a feedlot. Throughout two growing seasons, they collected samples from the leafy greens, feedlot surface manure, air and pest flies. They also recorded weather and feedlot pen conditions. Not surprisingly, the results found high levels of O157 on the feedlot surface, with both years averaging 72.5 percent positive samples. In the fields, generally, O157 decreased as distance from the feedlot increased and, at times, the pathogen wasn’t found on leafy greens at 600 feet. Overall, for both years, 3.5 percent of the samples tested positive for O157 at 200 feet, 2.2 percent at 400 feet and 1.8 percent at 600 feet, though numbers were higher the second year.


ent The higher 2012 numbers were due to a single sampling date. A few days prior, the feedlot operation replaced 300 head of cattle under particularly dry, hot and windy conditions. Because the cattle were moving and kicking up dust, the wind probably carried the bacteria off the feedlot surface and into the crops, Berry said.

Wind and flies The researchers also investigated whether pest flies or dust can transfer the pathogen from a cattle feedlot. They found E. coli present in both air samples and flies. Berry and her team collected air samples and

tested them for O157 and for total E. coli. They detected E. coli in the samples, suggesting that pathogenic strains can be transmitted by air. They failed to detect O157, however, perhaps because O157 is present in much lower numbers than general E. coli. E. coli prevalence in air samples decreased as distance from the feedlots increased, and remained the same even when pen conditions were muddy. Minimal E. coli was

...current buffer zone distance guidelines of 400 feet aren’t adequate

found in the air after the pen surface had been scraped clean and before new cattle were brought in. However, high levels of E. coli were detected on a dry, dusty day a

to reduce the

month later. The cooler temperatures that day also encouraged playful activity in the cows, which kicked up dust.

risk of produce

Flies may also be a source of pathogen contamination in produce. Livestock manure

contamination from

is an important developmental site for flies. Many pest fly species also spend time resting in vegetation, and some feed on plant nectar, pollen or the honeydew secreted

STEC O157 when

by other insects. So the potential exists for flies to transport pathogens from manure to produce.

it’s grown near

Berry and her team collected flies during the growing season and tested them for


O157. Overall, the researchers found high prevalence of positive flies: nearly 19 percent at the feedlot and about 10 percent at each field. Genetic testing of the O157 types sampled in the fields, flies and the manure didn’t reveal clear sources for the field contamination.

Conclusion “What does that mean for produce growers?” Berry asked. “What it suggests is that current buffer zone distance guidelines of 400 feet aren’t adequate to reduce the risk of produce contamination from STEC O157 when it’s grown near feedlots.”

Video of Elaine Berry presentation

More research is needed to determine adequate distances to reduce the risk. misc/GovFoodConference14/18-

When cattle pens are dry and dusty, an increased risk of airborne transportation


of E. coli exists. This risk is exacerbated when cattle movement is generating airborne dust. In addition, cattle pest flies carry O157 and may also be a source of produce contamination.

23 2 0 1 4 STEC C A P P r o c e e d i n g s

stEc The Survival and Growth of STEC in Soil Amendments Used in Agriculture

Manan Sharma

Research Microbiologist, Environmental Microbial and Food Safety Laboratory USDA-Agricultural Research Service

i n p r o d u ce & t h e e n v i r o n m The U.S. generates large quantities of manure, so putting it on fields to provide nutrients is a good use for it, said Manan Sharma. “We want to be able to do it in a way that doesn’t contaminate produce with the transfer of pathogens from the manure to the soil.” Manure comes in many forms and contains numerous pathogen types. Agencies, including the Food and Drug Administration, have developed rules and handling guidelines to prevent contaminating produce. The FDA’s proposed rule, called the Standards for the Growing, Harvesting, Packing, and Holding for Human Consumption, would require that any raw manure be applied to a produce field at least 270 days before crops are harvested. Crops are grown in manure-amended soils, but current regulations are based on reports involving the survivability of E. coli O157:H7 (O157) in manure, Sharma said. Amended soils change the environment in which bacteria live and could potentially grow. So research must assess pathogen survival in different manure and soil types, manure application methods, and geographic and climatic conditions, such as rainy versus hot, dusty regions. Sharma and his team are investigating the length of time that STEC and other bacterial pathogens can survive in soil amendments. Preliminary results suggest that the types of manure and soil as well as the method of manure application affect the persistence of E. coli. In addition, STEC can regrow in composted and nutrient stabilized soils and in compost teas supplemented with certain nutrients.

STEC survival To determine if the 270-day waiting period the FDA proposed is sufficient to prevent transferring pathogens to produce crops, Sharma and his colleagues inoculated manure spread on conventional and organic soils with E. coli strains in low and high concentrations, then studied the survival of O157 and nonpathogenic E. coli. The 3-year study is a collaborative effort with the FDA and the University of Maryland Eastern Shore. To date, the study is at 212 days, and the team is still able to detect E. coli in research plots. Sharma described summer 2012 results from a single field, which represent a common pattern across all study sites. E. coli immediately grew following application to manure-amended soils, followed by a rapid die-off. By day 7, populations were very low. Then, in some manure types, the E. coli resuscitated, growing again before dying back once more. This trend is more pronounced in nonpathogenic E. coli applied at high populations, likely due to a greater number of bacteria able to recover from stress. Although the data is still incomplete, the growth spikes coordinate with rain events, and Sharma said rain likely provides the moisture that stressed pathogens need to regrow, depending on manure type. In one example, nonpathogenic E. coli populations resuscitated most vigorously in poultry litter, though growth spikes also occurred in dairy liquid and horse manure.


Sharma said relationships among the time of bacterial application, nutrients present and bacterial stress probably play roles in bacterial growth. Introducing something that aids the bacteria, such as rainwater, helps them recover. He hypothesizes, however, that the longer the bacteria are in the field, as the stress increases, the bacteria are unable to recover.

ent For the O157 strains, the second resuscitation event due to rain seen in other populations didn’t occur even at higher population levels. This result was possibly due to the bacterial strains used in the experiment, which didn’t come from agricultural settings and may have been more susceptible to stress. The researchers also found differences in growth responses when manure was tilled into the ground versus applied to the surface. The tilled application saw the initial growth spike, but no growth following the rain events. That difference may be due to bacteria being more or less stressed by the soil environment following tilling or not having as much access to moisture from the rain.

Compost Composting manure is an important biological process that stabilizes nutrients, reduces pathogens and minimizes odor and vectors. Compost piles must reach 55°C to generate enough heat to kill pathogens. In addition to manure, compost can be made from other feedstocks, such as yard and food waste.

The FDA’s proposed rule would require compost producers to document that they achieved 55°C temperature, and compost must be applied at least 45 days prior to harvest. An informal survey found that most compost producers use one of two methods to test compost: the EPA’s 1680 test, used to recover fecal coliforms from biosolids, and the Test Method for the Examination of Composting and Compost, or TMECC, recommended by the U.S. Composting Council.

STEC can regrow in composted and nutrient stabilized soils and in compost

Sharma and his team compared the tests’ ability to detect E. coli and fecal coliforms in compost. They performed the EPA and TMECC procedure on compost samples taken from a variety of feedstocks in 19 states and inoculated with both nonpathogenic E. coli and O157 strains. The EPA method recovered a statistically significant greater percentage of E. coli compared to TMECC, 69 percent to 48 percent respectively. Because the EPA method tests 5.6 grams of compost compared to 0.33 grams in the TMECC method, it yields more E. coli.

teas supplemented with certain nutrients.”

Another study sought to determine whether certain physical factors, such as pH or moisture levels, could accurately determine the regrowth potential of O157 or Salmonella in finished commercial compost. Analyses using a sophisticated statistical model they developed showed that three factors contribute most to regrowth: percent moisture, carbon-nitrogen ratio and the total organic carbon levels. However, no single factor or feedstock type predicted the regrowth of O157 in compost. Sharma noted that, like most commercial compost, they bagged the compost, which may have affected moisture levels. Compost stored outside may behave differently.

Compost teas Video of Manan Sharma presentation

Some growers apply compost teas to control for certain diseases or to improve plant health. Teas are finished compost steeped in water and are sometimes supplemented with additives or nutrients. The FDA’s proposed rule states that unaltered compost teas can be used within one hour of preparation without restriction. However, previous studies have shown that adding certain nutrients to compost teas encourages pathogen growth, increasing the risk to produce. So teas with additives are considered raw manure and must be applied at least 270 days prior to harvest. misc/GovFoodConference14/19MananSharma.html

25 2 0 1 4 STEC C A P P r o c e e d i n g s

stEc Produce Industry Needs in Rapid Detection of Clinically Relevant STEC

i n p r o d u ce & t h e e n v i r o n m Proximity to animal production clearly risks contaminating edible crops with pathogens, and in many places animal and horticulture are closer than ever, said Trevor Suslow. As an extension research specialist, he works with growers, but says he sees the need to work with animal producers as well to manage this key source of pathogenic contamination. Suslow shared the challenges and risks growers, handlers and processors face. Pressure to expand pathogen testing is mounting, but that will be problematic, he said, especially for resolving STEC risk. More discriminatory STEC testing that focuses on clinically relevant pathogens and that is rapid, sensitive and affordable is needed for better decision-making.

Product contamination A few months after the well-known 2006 spinach outbreak, another E. coli O157:H7 (O157) outbreak stemming from shredded lettuce at Taco John outlets occurred.

Trevor Suslow

Extension Service Specialist, Department of Plant Sciences University of California, Davis

That outbreak was traced to dairy wastewater applied to nearby silage crops and other crops destined for processing. Lettuce fields designated for minimal processing were close by. Suslow contributes the outbreak to lack of awareness or concern for the multiple vectors of enterohemorrhagic E. coli (EHEC) and other STECs in close proximity to lettuce fields. “So one might ask: Weren’t there sufficient hazards present to trigger what we now view as a pre-plant red flag?” he asked. “Sadly, even with all the information that’s out there, even with all of our effort, we see isolated examples where lack of awareness or lack of understanding of the potential risk is still happening.” Many sources contribute to microbial pathogen risks, including runoff, dust, flies, rodents, birds, domestic animals and agricultural traffic. In California, animal production operations are moving in next to crops, some of them long established perennial orchards. Even a cow-calf operation using a pasture for grazing in the winter can contaminate fields planted in the summer when dried manure becomes airborne or animals move through both fields, spreading pathogens. In other cases, water conveyances can transport pathogens from an animal operation to nearby crops. And grazing animals on crop residue has become a trend in California, but the activity is a potential source of contamination for STECs and non-O157 EHECs. Suslow described a case in which a dairy and nearby romaine lettuce production shared a cross connection to the water supply, a deep aquifer pumped to a reservoir for irrigation. An event raised concern that an elevated level of indicator E. coli could occur, triggering a deeper look for pathogens. Testing the source water indicated high levels of E. coli, including O157 and other STEC, as well as Salmonella. The overhead irrigation emitters also tested positive. Irrigation was stopped, and the crop destroyed. However, tests for indicator E. coli on 60 romaine head samples taken eight days after irrigation found no evidence of pathogens. A direct connection between the contaminated water and pathogen survival on irrigated crops isn’t straightforward, but the pathogen seems to die off fairly rapidly, Suslow said. Crops other than leafy greens also risk pathogen contamination. Zipper-fruited citrus, for example, are at greater risk. Because the rinds are more delicate for easy peeling,


they don’t go through a wash system, and contaminated surface material can accumulate on the fruit.

ent “As far as we know, these are extraordinarily rare events, but clearly they happen,” Suslow said of E. coli outbreaks. But he added that, in some cases, the close proximity to animal production is a strong possibility for contamination events. How close is too close? he asked. In one example, Suslow’s team sampled 430 cantaloupes in a field next to a small dairy and found hotspots for indicator E. coli, a common finding in riskbased investigative sampling whether due to flooding or to close proximity to an animal facility. However, these hotspots were surrounded by low levels of E. coli, suggesting that something other than proximity was responsible for the contamination. In this case, the grower had arranged with the dairy operator

to spread the manure collected after scraping the waste lagoon directly on the field. Circumstances vary, and situations are not often straightforward, Suslow said.

STEC testing STEC are diverse and their relevance to human infection is uncertain. “Current evidence from molecular and cultural confirmation of STEC on raw and finished product by those who do test routinely—surveillance testing of their product—strongly

Food safety is critically important, but environmental protection also has

suggest that consumers are regularly exposed to viable contamination at low levels, but it’s there on product marketed by those who do and do not test,” Suslow said.

some very strong

“Should this knowledge impact testing practices and policies? Clearly, there doesn’t seem to be a direct correlation with sporadic illness, certainly not with outbreaks. What criteria might guide these policies?”

societal benefits,

Pre-harvest testing with positive results for EHEC can force the destruction of entire

and we need to

fields because the produce is deemed adulterated. However, the genes being detected

figure out a way

by these tests in some cases may have come from a combination of more than one type of bacteria, none of which is pathogenic. This constitutes a false positive test

to achieve both

result. Suslow also expressed concern that false negative results can occur when

of them.”

EHEC lose the Shiga toxin virulence gene due to stresses from testing procedures. To lessen the economic hit from crop destruction, growers have asked researchers and public health experts to simplify the complexity of STEC diversity and testing. Suslow’s group organized a colloquium with the Center for Produce Safety and other associations to challenge the Center for Produce Safety and its affiliates on issues surrounding STEC test outcomes. Colloquium participants agreed that rapid screening tests that differentiate EHEC from other STEC less likely to cause clinical illness in humans are needed. Objective criteria are needed to resolve the role of these diverse organisms, Suslow said.

Video of Trevor Suslow presentation

Healthy diverse ecosystems help to keep pathogens in check, he added. “It really is


an important issue, and one that’s really certainly near and dear to my heart. Food


safety is critically important, but environmental protection also has some very strong societal benefits, and we need to figure out a way to achieve both of them.”

27 2 0 1 4 STEC C A P P r o c e e d i n g s

stEc It’s Here, We Found It, Now What Do We Do? An Industry Perspective

Will Daniels

Chief Food Integrity Officer Earthbound Farms

i n p r o d u ce & t h e e n v i r o n m Earthbound Farms, the nation’s largest grower, packer and shipper of organic produce, extensively tests its products for E. coli O157:H7 (O157), enterohemorrhagic E. coli (EHEC) and Salmonella, said Will Daniels. The company tests inputs in the field, including water, fertilizer and transplants. Because fertilizers, composts, liquid teas and pelletized products are particularly risky, it works with manufacturers to ensure their products’ safety. The company also used to test seed, but after five years of negative test results, it eliminated seed as a major risk and stopped testing. Earthbound conducts tissue testing in the field on crops destined for both field packing and the manufacturing facility. These dual-purpose fields are tested prior to harvest so as not to implicate the field-packed products in case a positive sample is later found at the manufacturing facility. After harvesting, products go to the facility where testing continues during processing and as finished goods. The challenge is that contamination is not uniform. It’s sporadic and difficult to find, making a focused, frequent sampling system important. “The key here is that it’s a comprehensive program,” Daniels said. “This is not our answer to food safety; it’s just one hurdle in a multi-hurdle approach to food safety in a process that doesn’t have a kill step.”

Testing program Field Sampling Plan: Designed for field-packed products, such as head lettuce, the

field sampling program tests 60 samples from 1-acre blocks three to five days prior to harvest. The small acreage size improves the ability to find pathogens and, if a positive is found, limits the area requiring destruction. Although field testing is important, Daniels said the company is lucky to find a positive in the field. It has a better chance of finding contamination in sampling raw product at the facility and, therefore, puts more emphasis into sampling harvested raw products. Wash system: Earthbound uses a triple-wash system that washes twice in lightly

chlorinated water followed by a freshwater rinse. While this system has been shown to reduce microbes, it was designed to prevent cross contamination from one batch of lettuce to the next. Sterilizing lettuce would also pose hazards, he said. Organisms that cause spoilage can be beneficial because they indicate microbial growth is occurring, which is particularly important if pathogens are present. Raw product sampling plan: Leafy greens are harvested by mowers, picked up

by belts and transferred via refrigerated truck to an Earthbound facility. The raw sampling program encompasses this entire process, including the containers. Each field is broken into four pallet sampling units, about 1,500 pounds total, and 60 samples from each pallet are tested. Negative results are available in 12 hours; positive results in 16 hours.


When positives occur, the pallet is destroyed and a team returns to the field to identify the source of contamination. Finding the source is rare, but when identified, the company takes steps to prevent further contamination. In 2008, for example, the team investigated a ranch with multiple positives. It found residual water in the bottom of an unused overflow reservoir. A sample tested positive for the strain found in the product. Further investigation determined that the overflow reservoir was connected to the irrigation system watering the entire 270-acre field.

ent Earthbound removed the ranch from its supply base, a significant hit to the company’s production. The grower severed the cross-connected water source, retested the water and was readmitted. The company found no further contamination from that supplier, though other suppliers in the same area still tested positive. The contaminated water source wasn’t a large risk when it was found, but may have escalated into an outbreak later in the season without intervention. Finished goods sampling plan: Finished goods are broken into 2-hour manufacturing

units. Throughout the 2-hour window, 60 samples are tested. At the beginning and end of each 2-hour unit, water samples are collected and stored. If a sample is positive, the water is tested. If the water is negative, they assume the contamination is contained within that 2-hour unit and that product destroyed. A positive water sample would expand the scope of water testing, resulting in destroying more product or possibly a recall. However, to date, no water samples have tested positive. That’s encouraging, Daniels said, and demonstrates the wash system’s effectiveness in preventing cross contamination.

Test and hold data

Earthbound is participating in studies to improve the wash system. To date, citrus-based washes and high-powered ultrasound have not improved washes. Other changes are also being studied, including increasing agitation or contact time with washes, different temperatures or gaseous intervention either pre- or post-wash.

Overall, the prevalence rate over the past eight years has been less than 0.5 percent. None of the positives indicated gross contamination events, suggesting that either the company has gotten lucky or the sampling program is appropriate to catch even small contamination events, Daniels said. Pathogen prevalence varies considerably year to year, and no patterns have emerged. More positives appear in the raw product phase, but still appear in finished goods as well. Although the company investigated potential cross contamination within the facility, testing suggests the contamination gets through raw material testing and isn’t completely removed by the wash system.

When asked about irradiation, Daniels said that, although the federal government approved irradiation for romaine lettuce and spinach several years ago, consumers have overwhelmingly rejected it. If preliminary studies suggest it’s beneficial, he said he would like to

Testing data suggests a correlation exists between positive results and a spike in the heat index. To better understand this connection, Earthbound is working with the Food and Drug Administration’s Risk Modeling Team and NASA to develop a riskmodeling tool to help predict proliferation events.

Future study Daniels said he believes many vectors are responsible for EHEC contamination of leafy greens and that controlling them all is impossible. For example, he questioned whether, if feral pigs are shown to be a source, their elimination would affect contamination rates. A Salinas, California, grower installed a 7-foot-tall, 3-foot-deep fence to keep out pigs and deer to prevent contamination, but positives still occur.

see it developed.

He said he also believes bird activity contributes to positives. He encouraged researchers to take samples on the outside of the bird because that may be a source of gross contamination. Starlings and blackbirds flock in pastureland and at water troughs, then go to crop fields looking for insects. Feral pigs damage fields, so their presence is obvious. Birds, however, leave no trace.

Video of Will Daniels presentation

Daniels would also like to see more rapid and accurate testing at lower costs, which would encourage more testing and reduce the impact of positive results. Real-time monitoring of irrigation system water, for example, could shut off or divert water before it contaminates a field.

GovFoodConference14/21-WillDaniels. html

29 2 0 1 4 STEC C A P P r o c e e d i n g s

stEc Federal Regulation of STECs and Other Pathogens: Past, Present and Future

Bob Hibbert

Partner, Food and Drug Practice Morgan, Lewis and Bockius

p re va l en ce & i n t erv en t io n Before scientific advances can be applied to the meat industry, they must first be filtered through a legal and regulatory system, said Bob Hibbert. Three statutes cover products regulated by the USDA: the Federal Meat Inspection Act, Poultry Products Inspection Act and Egg Products Inspection Act. The Food and Drug Administration’s Food, Drug, and Cosmetic Act covers all other foods, including produce. Hibbert said he believes the current performance standards approach to regulation is flawed and a misuse of finite resources.

A complicated law The Federal Meat Inspection Act affirmatively applies the U.S. mark of inspection, which indicates the product was produced in compliance with federal regulations, if carcasses and parts are found to be “not adulterated.” That language allows the government to withhold the mark without first establishing something is wrong with the product. The USDA can withhold the mark until they are satisfied the product hasn’t been adulterated. That language is the fundamental difference between the USDA’s Food Safety and Inspection Service (FSIS) system and other food regulations. The concept of adulteration is key, Hibbert said. In simplified terms it means wholesomeness. Food that is not adulterated can be sold in commerce; food that is considered adulterated cannot. All federal and state statutes contain multiple definitions that categorize a product as adulterated or not. Pathogens are a critical factor. In the Adulteration Proviso of the Federal Meat Inspection Act, a product is considered adulterated if it bears or contains any poisonous or deleterious substance harmful to health, unless it’s not an “added substance.” In other words, a naturally occurring pathogen doesn’t adulterate the product if it is present in an amount that doesn’t ordinarily sicken people. With the exception of E. coli O157:H7 (O157) and other STEC, the default assumption is that raw meat and poultry containing pathogens are not considered adulterated, including raw poultry containing Salmonella, because the implicit assumption is that proper handling, such as cooking, will kill the pathogen. The proviso, however, contains two escape clauses. A product can be considered adulterated for any reason, if it’s found to be unsound, unhealthful or otherwise unfit or if the product is prepared, packed or held under insanitary conditions. This language allows the government to condemn goods without proving anything’s wrong, for example, if they discover it in a filthy or infested warehouse, Hibbert said.

E. coli 0157:H7 crisis In the 1970s, the American Public Health Association sued the government to require Salmonella warning labels on poultry products. They lost. The court determined that Salmonella does not constitute an adulterant because American housewives know how to prepare food. This case helped secure the view that pathogens are not adulterants. “That changed in the wake of the O157:H7 crisis that stemmed from the terrible tragedy in the upper Northwest in the early ‘90s,” Hibbert said.


Following the 1993 Jack in the Box outbreak, the USDA announced it now considers O157 in ground beef an adulterant because pathogens in the middle of the hamburger patty may not be cooked sufficiently. It implemented testing and enforcement measures, and the beef industry sued. There were two dimensions to the case: first, the determination that O157 is an adulterant and second, the process by which that determination was reached. That O157 should not be in the ground beef supply is

s t r at egi es at s l augh t er hard to oppose, Hibbert said, but the announcement violated the Administrative Procedure Act, which requires the government to request public comment before making a final decision. However, the judge ruled in favor of the government. Today, O157 and six other EHEC are considered adulterants. But Hibbert said he believes the government presumed, overbroadly, that when it comes to pathogens, it doesn’t have to follow rule-making procedures. At the same time, the food industry understood that fighting to keep unsafe food in the food supply was bad public relations and became gun-shy about challenging the government’s determinations. The result is that the government began regulating by bureaucratic fiat, he said. In 1996, FSIS implemented the so-called “mega-reg” that established the pathogen reduction performance standards system in place today. In 1999, it expanded E. coli testing to include non-intact meat and trimmings. It continued announcing expansions, including the additional non-O157 STEC in September 2011. At the same time, FSIS began withholding the mark of inspection if it was unsatisfied that regulatory procedures were followed, regardless of pathogen levels. Today, FSIS makes announcements, then requests comments. Hibbert likened it to planning and booking a family vacation, then asking your family where they want to go. “In this area that’s how FSIS rolls these days and that’s how they’re going to continue to roll…What you see now is this sort of regulatory creep,” he said, including pushing

Conclusion Today, two streams of regulation exist: adulteration and performance standards. Adulteration is strain specific. “Are we looking at 100 strains in 10 years? Is that a feasible system and is it practical to do brickby-brick regulation in that fashion?” he asked. But he argued that the performance standard approach is both legally and scientifically suspect, can’t be enforced and misuses finite public and private resources.

to consider primal cuts adulterated and using ambiguous concepts, such as “hot days.”

Salmonella In 2001, Supreme Beef sued FSIS for suspending the grinder company’s operations due to high Salmonella numbers, a supposed indicator of improper process control under the “mega-reg” performance standards. The court sided with Supreme Beef, determining that the government needed a clear legal basis, not one based on regulations. Although the government asserted insanitary conditions, the problem was incoming raw materials, Hibbert said. The ruling means the performance standards can no longer be enforced. Instead, FSIS now conducts the tests and looks for reasons other than the failure to suspend operations.

He suggested that solutions be driven by science, a more open policymaking process driven by comment and rule-making, comprehensive risk management within industry, and effective public communication. The ultimate goal is to maximize everyone’s efforts to protect the public health, he said.

“So you have the somewhat awkward dance that goes on where it’s really the performance standard failure that’s getting you into the regulatory box, but them saying that it isn’t, which raises the question: What is all of this performance standard testing about, particularly when the government is hell-bent on expanding them?” Hibbert said. “You have finite resources, and it seems to me all of that testing, all of that focusing on so-called standards that are not enforceable, that they could get more bang for the buck elsewhere.” Today, focus is shifting toward Salmonella. Although the pathogen isn’t considered an adulterant, FSIS has used the unsafe escape clause in the law. When a Salmonella outbreak occurs, they issue a recall. In some cases that’s understandable. In others, the line is unclear, he said.

Video of Robert Hibbert presentation

Some groups, such as the Center for Science in the Public Interest, are pushing


the government to consider some Salmonella strains adulterants. The petition is pending, and Hibbert said he believes that if the USDA one day agrees, it will make a declaration without prior public comment.


“Is that feasible?” he asked. “Scientifically, economically and otherwise. And what


defense is a company going to have, if we get to that point?”

2 0 1 4 STEC C A P P r o c e e d i n g s

stEc Quantitative Microbial Risk Assessment: An Update on Objective 4 of the STEC-CAP Grant

p re va l en ce & i n t erv en t io n The dynamic nature and enormous complexity of the beef production system creates challenges to improving food safety, said Mike Sanderson. As aspects of the system change, the data may no longer be relevant. As systems change, some relationships within the system are difficult to observe directly and must be inferred, and some important outcomes are driven by rare events, which make them difficult to study. “At the end of the day, we still have to make decisions, and we need to make decisions that are the best that they can be for food safety,� he said. A primary goal of the STEC CAP is to develop a quantitative microbial risk assessment to help identify research needs and effective industrial interventions to reduce pathogens. The risk assessment is a method to quantify the risks of microbial contamination of a product. Individual studies provide pieces of data throughout the production system that can be tied together to develop a coherent model representing the real system, which can help guide decisions.

Michael W. Sanderson

Sanderson described efforts to develop a risk assessment tool that focuses on

Professor, Diagnostic Medicine and Pathobiology

analyzing exposure to pathogens. The model encompasses risk along the entire beef

Kansas State University

address such aspects as illness and mortality rates, which are important variables in

production chain, from the farm to consumers. Hazard and risk characterizations then understanding the process and research needs. Developing the model requires a tremendous amount of data from throughout the production process. One task involves defining prevalences and concentrations for the Top 7 EHEC at every step, including frequency of prevalence, for example, how often prevalence is 20 percent or 50 percent. That level of specificity is needed for different classes of cattle, seasons, production plant sizes and products. All data must then be linked together to understand, for example, how hide contamination levels relate to carcass contamination levels. To illustrate the complexity, Sanderson described capturing prevalence and concentration data at each step in the pre-harvest level, including different classes of cattle, in different seasons, perhaps with one or more pathogen-reducing interventions, followed by transporting the cattle to processing. The team collects the data from published literature, then validates the studies and decides which data to use and how to put it in a comparable form to combine it.

Limited data After compiling the data, Sanderson and his team look for statistical distributions that represent the real world. Distributions that fit well can be used in the model. He said he’s hampered by lack of data. For example, while distributions of summer and yearround data of fecal STEC prevalence in feedlot cattle fit observed data well, winter data do not. STEC fecal concentrations, a necessary model component, also lack data. At slaughter, hide STEC prevalence is important. Hide prevalence at feedlots and at the slaughter plant are similar, but most of the limited data is specific for E. coli O157:H7 (O157), and enumerable rates vary greatly. Enumerability indicates more than 40 colony-forming units per 100 square centimeters. At the feedlot, the enumerable rate is about 4 percent, indicating that many positives are at levels too


low to count. At the plant, the percentage jumps to 19 percent.

s t r at egi es at s l augh t er “So while overall prevalence didn’t change, the proportion of those that were higher changed pretty dramatically,” Sanderson said. “We’d like a better estimate than just what proportion are above some certain level. We’d actually like to know the counts for everybody and be able to build a distribution that represents what are the counts of O157 or O26 on hides when they enter the plant.” Data scarcity, again, is to blame for an ill-fitting hide concentration distribution. In addition, while most samples fall into a common range, one sample occasionally is much higher. This data set may be too complex to develop a good distribution and may require a mixture distribution, Sanderson said. Hide to carcass STEC transfer is another critical point that requires a quantitative connection between hide STEC concentrations and the risk to carcass concentrations. But the required data is limited. “This is a key point to get into the actual product realm: How much of that carcass is contaminated?” Sanderson asked. Some carcass prevalence data is available. About 15 percent of pre-evisceration carcasses are contaminated. Post-evisceration prevalence levels drop considerably

following interventions and are quite low by the time carcasses are chilled. The low levels make it challenging to model because it is difficult to estimate the impacts of interventions. At this point, outbreaks are rare events.

Conclusion “We’ve made substantial progress in extracting and analyzing the existing data through carcass intervention,” Sanderson said. His team continues to collect data

We’ve made substantial progress in extracting and

further along the production chain.

analyzing the

The model’s draft code is written. It’s a full 2nd-order simultaneous STEC-8 model, which means it handles variability and uncertainty separately and can help sort out

existing data

where more data would be helpful. They continue to refine the code for smoother and more flexible operation and implement easier methods to enter data and make

through carcass

parameter changes. They will also calibrate the model results and test it to ensure


results are realistic. Data gaps are problematic, Sanderson emphasized. “We don’t have near the concentration data that we need in order to be able to quantitatively assess and predict what risk is.” Relationships and correlations are required to link farm to consumption steps and quantitatively describe how they link. Additional non-O157 STEC data, including non-fed beef data from culled beef and culled dairy, are also needed. When asked about the model’s potential uses, Sanderson said the model will help target research by pointing out where more data is needed and where it would be

Video of Michael Sanderson

most valuable. It could also be used to test the impact of slaughter interventions on


public health, and perhaps provide ideas for effective practices. Economists could

also use the model to better understand the most cost effective use of resources to


improve public health. As additional data becomes available, it will be incorporated into the model, he added. “We look forward to wrapping all that up into a nice package.”


33 2 0 1 4 STEC C A P P r o c e e d i n g s

stEc Recent Research on the Epidemiology of STEC in Beef Production Systems

p re va l en ce & i n t erv en t io n Quantitative risk assessments developed to improve beef safety require data on STEC prevalence in beef production systems. The data exists, but is limited, especially for non-O157 STEC, said David Renter. He described several studies he and a collaborative team of researchers have conducted to better understand when, how much and why STEC occur in beef production. The data they generate contributes to the quantitative risk assessment being developed under the STEC CAP. In an early study the team looked at the prevalence of E. coli O157:H7 (O157)

and non-O157 O groups in feedlot fecal samples. Because E. coli detection methods are still improving, the researchers tested the samples via culture and PCR, a genetic diagnostic tool. They learned that, while they found many O groups in the samples, the bacteria didn’t contain the virulence genes that would classify them as either a STEC or

David Renter

Professor, Epidemiology, College of Veterinary Medicine Kansas State University

enterohemorrhagic E. coli (EHEC). They also found little agreement between the culture and PCR prevalence results, which highlights the importance of further refining detection methods, Renter said. “We’re trying to determine prevalence as accurately as we can. We need to have detection methods that do that and do that well.” Another study focused specifically on E. coli O26 prevalence. Surprisingly, the

culture method detected more O26 than did the PCR method. Similar to the previous study, few O26 bacteria contained the Shiga toxin virulence genes, so most of the O26 recovered from feces in the feedlot population were not STEC. The researchers also found that neither the O157:H7 SRP vaccine nor a directfed microbial fed to cattle affected fecal shedding of O26 serogroups. To determine if E. coli O104:H4, the cause of a German outbreak, could be

found in the beef system, the researchers developed diagnostic methods to detect it. They sampled eight feedlots and found about 20 percent of the fecal samples contained E. coli O104. However, none of these were O104:H4, nor contained any of the virulence genes found in that organism. A larger study of 29 feedlots found a similar prevalence of E. coli O104 that were not O104:H4 and did not contain the virulence genes found in the German strain. Other studies have confirmed these results. “So there’s some pretty good indication that cattle are not the reservoir for this particular strain,” Renter said. A study looking at the effect of the O157:H7 SRP vaccine and a low-dose

direct-fed microbial on the top six STEC O groups found no significant effect on fecal shedding in the feedlot population. This study also provided longitudinal samples from multiple pens over time. Researchers have documented the tremendous variability in O157 prevalence from pen to pen and over time, but little research has been done on non-O157 STEC variability. This study found extreme variability from pen to pen and over time. In fact, the prevalence can be very high and low in a pen on the same day or in pens across time.


s t r at egi es at s l augh t er In contrast to previous studies, Renter and his team also found significant associations between O groups. For example, a sample positive for O157 was three to nine times as likely to contain O26, O45, O103 and O111 serogroups. Because of the inconsistency with previous results, Renter said more work is needed to understand potential associations between O groups and epidemiological risk factors. This example highlights the opportunity to obtain much more information from one study or sample set available through the collaborative nature of the CAP grant, he added. The final study—the 2013-2014 Feedlot Cattle Study—was an enormous

collaborative effort and continues to generate numerous results, Renter said. To determine the prevalence of STEC serogroups and Shiga toxin and intimin virulence genes, the researchers collected fecal samples from feedlot cattle immediately prior to harvest and hide and carcass samples from the same animals in the process facility. They sampled in summer and winter. O-group prevalence varied greatly. They detected some O groups in small amounts,

while nearly 60 percent of the fecal samples contained O103. However, in most cases, less than 1 percent contained the Shiga toxin and intimin genes. So STEC and, more importantly the intimin-carrying EHEC, were present in much smaller numbers than the E. coli O groups of interest. The exception was O157. Nearly 43 percent of the fecal samples contained O157, all of which contained both Shiga toxin and intimin genes. Many studies refer solely to serogroups, not virulence factors, Renter said. But many of the O-group isolates don’t contain Shiga toxin or intimin genes, so researchers

We’re trying to determine prevalence as accurately as we

must think past serogroup results. This distinction has important implications for risk

can. We need to

assessments and for industry. “It’s really important that we emphasize, when we’re looking at potential risks, that we are careful on what we use as a definition.”

have detection

Additional preliminary results comparing summer and winter demonstrate that the number of positive non-O157 O groups drop dramatically in winter, the same seasonal effect that has been well described for O157.

methods that

Renter and his team are expanding their work to include more feedlots in multiple

do that and

states, as well as including more plant interventions and adding culled beef and

do that well.”

culled dairy cows. Additional collaborations are studying other aspects, such as the role of gut organisms and risk modeling. Another aspect of Renter’s research investigates risk factors from the macro-level, such as seasonal variation and management factors, to the micro-level, looking at microbiological factors that may influence STEC in the beef system.

Video of David Renter presentation

35 2 0 1 4 STEC C A P P r o c e e d i n g s

stEc Recent Research on Antimicrobial Interventions and their Validation to Control STEC in Beef Products

Gary R. Acuff

Director, Texas A&M Center for Food Safety Professor and AgriLife Research Faculty

Texas A&M University

p re va l en ce & i n t erv en t io n Validating the effectiveness of interventions applied at slaughter establishments to control STEC in beef products is an important part of a successful food safety management system, said Gary Acuff. Hazard Analysis and Critical Control Points is a food safety management system designed to prevent hazards in food production. The Food and Drug Administration and the USDA regulate products through mandatory HACCP programs. “At the heart of HACCP really is validation. If you don’t have validated control, or validated process control, then you really don’t have a successful HACCP program. It’s the thing that stands between success and failure, I think, with HACCP,” Acuff said. HACCP codex documents state that “validation involves obtaining evidence that control measures, if properly implemented, are capable of controlling the identified hazards.” Obtaining evidence involves multiple approaches, including using predictive models, collecting scientific literature and conducting laboratory or plant facility studies. Safe harbors identifying conduct that constitute compliance with a rule have also been established. All of these approaches provide data to validate process control and to understand how much process control exists. Acuff and his colleagues focus on microbiological studies at a laboratory scale as well as on some studies in pilot plants or commercial facilities. “We’re working very hard to supplement the information that’s available out there in terms of validating these process controls,” he said. The ideal validation approach would measure the pathogen before and after a critical control point during processing to determine the intervention’s effectiveness at reducing pathogen levels. However, pathogens often aren’t present in sufficient numbers or consistently enough to measure. Indicator organisms, such as coliforms, Enterobacteriaceae and E. coli, correlate closely to Salmonella and STEC, but these populations are insufficient or too inconsistent to work well for validation. Surrogate bacteria, however, have proven a more consistent alternative, Acuff said. These nonpathogenic bacteria can be applied in large and consistent numbers as substitutes for pathogens in order to measure reductions related to an intervention. The USDA, Acuff and other researchers working on intervention validation have teamed to work collaboratively to implement validation guidance and assist plants that need validation help. This Consortium of Food Process Validation Experts has published numerous papers. Acuff described several studies consortium researchers are conducting. Veal has received attention lately because it may be more highly contaminated

and therefore presents greater risk than beef. A collaborative study examined methods to decontaminate veal. Looking first at hide-on bob veal, the researchers applied surrogate bacteria to hides and measured populations following several interventions. Scalding consistently reduced bacterial populations, and following scalding with a 180°F wash caused a 5-log reduction. A final lactic acid spray almost completely eliminated bacteria.


s t r at egi es at s l augh t er Treating dressed veal requires more finesse because carcasses are more sensitive to color changes and other issues than hides. As with hide-on veal, the researchers applied surrogate bacteria and measured populations following interventions. After a final wash at about 125°F, all samples had reduced populations. They found little difference among concentrated lactic acid, and the antimicrobial sprays Beefxide and Citrilow, about a half log to a log reduction in each. “Not really what we were hoping to see in terms of reduction for acids, but some reduction, and it’s certainly significant and another option for veal processors in terms of a way to actually reduce the presence of pathogens on their products,” Acuff said. Another study investigated the effect of the antimicrobial lauric arginate

(LAE) on E. coli O157:H7 (O157) and non-O157 STEC. On vacuum-packaged subprimals inoculated with pathogens, the researchers compared two LAE brands as well as two mixes, one using tap water and another deionized water. They found no difference between brands or water mixes. A significant reduction occurred when inoculation levels reached 100 parts per million for O157 and 200 parts per million for non-O157 STEC. The interventions were similar and effective, but didn’t result in huge reductions, Acuff said.

Other studies are ongoing. One

is investigating the validity of research methods to inoculate products with pathogens or surrogates to accurately represent real-world conditions, such as inoculating cold versus warm products. Other studies are providing data to include in risk assessment tools. Such data includes growth models establishing log growth rates, lag times and other parameters to understand STEC growth rates as a function of temperatures. Preliminary results suggest that O157 and non-O157 STEC

Results of a study investigating treatments to reduce STEC on non-intact

products determined that STEC and O157 behave similarly. The catchphrase: If it works for O157:H7, it will work for the Big Six pathogenic E. coli strains. Industry provided input for this study. The researchers used pathogenic E. coli strains, pilot-scale commercial food processing equipment and entire subprimals. The study investigated antimicrobials delivered by an air-assisted electrostatic spraying system, a unique system that requires minimal volume but provides almost complete coverage of the product. The study included a variety of products, including ground patties and veal cutlets, as well as processes, such as blade tenderization, cooking and electrostatic spray. While most O157 and other STEC remained within a centimeter of the surface in blade tenderized or chemically injected beef subprimals, some pathogens became distributed throughout the steak. Additionally, the researchers found no difference between O157 and non-O157 STEC levels after cooking various non-intact products, though not surprisingly high temperatures provided greater reductions. They also found that tenderized beef results in greater risk of illness than intact steaks, a 2-fold increase for blade tenderized and 4-fold for chemically injected products.

behave similarly.

Another study provides dry-fermented sausage processors with validated processes

to achieve the USDA’s Food Safety and Inspection Service’s 2- or 5-log reduction requirement and to meet the NCBA Blue Ribbon Task Force Requirements. The study measured STEC levels following interventions of relative humidity at lower temperatures, different pH levels and a variety of heating temperatures following fermentation. The researchers found that higher temperatures with lower pH result in greater STEC reductions. Fermentation delivers reductions of about 0.7-log to 1.6-log, regardless of the target end-point pH. Additional reductions in pathogen levels were also achieved during the come-up time (CUT), the time required after fermentation to heat the product to the target internal cooking temperatures of 110°F to 130°F. In general, fermentation to pH 4.6 and post-fermentation heating achieved a 5-log reduction in less time than fermentation and post-fermentation heating to pH 5.2.

Video of Gary Acuff presentation GovFoodConference14/7-GaryAcuff. html

37 2 0 1 4 STEC C A P P r o c e e d i n g s

stEc USDA Food Safety and Inspection Service STEC Update

p re va l en ce & i n t erv en t io n “As regulators we walk thin lines,” said Philip Bronstein. “On one hand, we need to be able to have enough food both cheap enough and abundant enough to feed the nation and the world…but we also need to make the food supply as safe as possible.” The Food Safety and Inspection Service (FSIS) is a public health agency as well as a regulatory agency and must balance the needs of public health, consumers and industry. Bronstein described several studies FSIS is conducting to improve beef safety and regulatory testing.

FSIS STEC sampling

Philip Bronstein

Senior Microbiologist, Office of Public Health Science USDA-Food Safety and Inspection Service

In 1994, FSIS began testing raw beef products for E. coli O157:H7 (O157) and several years later declared any non-intact beef product containing the pathogen as adulterated. FSIS uses five key criteria in its risk assessments: the hazard is present; it is associated with severe illnesses; it has a low infectious dose; person-to-person transmission can occur; and, finally, that typical cooking doesn’t kill the pathogen. Americans commonly eat hamburgers medium or medium rare, which may not kill all O157 present. In June 2012, FSIS expanded testing to include the Top 6 pathogenic non-O157 EHEC. Their presence, confirmed by the presence of isolates containing both the Shiga toxin and intimin virulence genes, renders a product adulterated. “I’m actually very proud of the agency,” Bronstein said of the virulence gene testing. “We’re trying to become more refined in our regulatory definitions.” The Top 6 non-O157 EHEC are important, he added, causing more than half of the STEC foodborne illnesses from 2000 to 2008. O157 caused about 35 percent of cases. The commodity linked to STEC illnesses is also an important consideration. Nearly 30 percent of cases are linked to beef, but grains, beans and leafy greens are increasingly associated with STEC illness. Evidence suggests that cattle manure used to fertilize leafy green fields could be a contributing factor. “I think that’s something else that we all have to be cognizant of when we think of STEC is that it’s not just beef, it spans our whole food spectrum,” Bronstein said. Data from recent FSIS routine testing of raw ground beef components taken in a nearly two-year period indicate that non-O157 STEC appear almost twice as often as O157, 0.7 percent and 0.42 percent respectively. The difference in prevalence between beef and veal has become a recent issue, he said. Some companies that slaughter only beef believe E. coli problems are specific to veal. Testing found 0.37 percent O157 and 0.58 percent total non-O157 STEC in raw beef trim, while in veal the prevalence was 3.57 percent for O157 and 7.89 percent for non-O157 STEC. While E. coli prevalence for STEC is higher in veal than in beef, the data is skewed somewhat because the percentages represent thousands of beef samples and fewer than a hundred veal. “I think it’s safe to say there are problems in both,” Bronstein said.

Beef Veal Carcass Baseline Study 38

To better understand the effectiveness of interventions intended to reduce pathogens in both beef and veal production, FSIS is conducting a Beef Veal Carcass Baseline Study in collaboration with the USDA’s U.S. Meat Animal Research Center (USMARC). The study will provide process control criteria, estimate the prevalence

s t r at egi es at s l augh t er and quantitative pathogenic organism levels at two steps of the slaughter process, and estimate the presence and quantitative levels of indicator organisms.

The analysis continues, and Bronstein noted that the results could lead to changes in the way FSIS approaches analyzing adulterant STEC in beef or to refinement of FSIS adulterant STEC definitions. If these STEC are associated with severe human illness, then FSIS may consider expanding its list of adulterant STEC, he said, adding that he doesn’t believe that will happen soon.

For the study, the agency is collecting about 4,000 samples from two points on the carcass at 133 beef and veal slaughter establishments. The samples will include all veal subclasses. The samples are collected during post-hide removal before any interventions are applied and during the later pre-chill step following the last intervention. In preliminary studies, the “shakedown stage,” FSIS learned that about 55 percent of the establishments apply interventions during the de-hiding process, so data from those locations will be analyzed separately. The study targets O157, the Top 6 non-O157 EHEC and Salmonella. It also looks at indicator organisms generic E. coli, total aerobic bacteria, Enterobacteriaceae and coliforms.

FSIS lists research priorities on their website, many of them related to detection methodology, screening, enumeration characterization and ways to obtain cheaper, faster and more information.

Preliminary shakedown data found nearly 20 percent of samples were positive for Salmonella after de-hiding, and 3.22 percent positive at the pre-chill stage. Three samples had the same Salmonella serotype in both locations, which might indicate that the establishment was unable to remove the pathogen from the carcass during processing. A detailed breakdown of positive results by pathogen demonstrated that post-hide samples contained about 1 percent O157 and 8 percent non-O157 EHEC. Pre-chill samples found no O157 and about 3 percent non-O157 EHEC.

“We really want to work with industry to try to reduce the risk of STEC in our food supply, Bronstein said. “And I think these collaborations through academics and [the USDAAgricultural Research Service] is

Bronstein said that some in the industry believe testing for non-O157 EHEC is unnecessary because O157 results can be used as an indicator instead. However, these results suggest that O157 is not always present alongside non-O157 EHEC. The FSIS is collecting data to support that position.

what we really have to do.”

When that conclusion was questioned because the positive numbers are comparing O157, a single organism type, to a pool of six non-O157 EHEC, Bronstein replied that, as an indicator of process control, it’s preferable to look for a larger collection of pathogens and apply it to a smaller collection. “I think you can make a really strong argument for casting a wider net,” he said, though FSIS is mindful that testing for non-O157 EHEC increases the expense for industry. The results show a higher percentage of pathogens at post-hide removal in veal than in beef products. With veal, the pathogen more often remains at pre-chill, indicating that veal producers have a harder time combating these pathogens than beef producers. The preliminary 3-month shakedown study is complete, and the yearlong study begins July 2014.

Strain characterization study In another collaboration with USMARC, FSIS is conducting a study to identify STEC and virulence factors in beef not screened during routine FSIS testing. The researchers test samples for FSIS adulterant STEC and for Shiga toxin and intimin virulence genes. They then analyze positive samples to determine the O group and any Shiga toxin genes, intimin gene or other potential virulence factors. To date, 18 percent of samples have been positive for O157, lower than the original prevalence found during routine FSIS screening because study broths were frozen and thawed. Two samples contained a non-O157 EHEC and two samples contained one STEC not classified as an adulterant; each contained the Shiga toxin and intimin virulence genes. These results suggest that potentially pathogenic STEC other than the current list of FSIS adulterant STEC are present in raw beef.

Video of Philip Bronstein presentation misc/GovFoodConference14/9PhilipBronstein.html

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stEc Modeling Pathogen Controls in Beef Slaughter

p re va l en ce & i n t erv en t io n The USDA’s Food Safety and Inspection Service (FSIS) is tasked with ensuring the safety of meat, poultry and egg products. To help fulfill that mission, FSIS developed a risk assessment tool to model pathogen controls in beef slaughter establishments. The model seeks to identify how the number, type and combination of interventions applied at slaughter establishments to remove pathogens affect E. coli O157:H7 (O157) contamination of beef carcasses, said Rachel Johnson-DeRycke.


The model encompasses the entire slaughter process, beginning with incoming contamination and its transfer from the hide and gastrointestinal system to carcasses, followed by three stages of interventions and, finally, converting carcasses to ground beef.

Rachel Johnson-DeRycke

The data used to build the model came from peer-reviewed literature and a 2007 survey of all 632 U.S. slaughter establishments regarding their practices. The survey provided a snapshot on the use of 11 different interventions. Establishments use more than 11 interventions, and a recent survey has captured additional information that will be included in future models. Peer-reviewed literature provided data on log reductions of pathogens expected from each intervention, as well as O157 prevalence and concentrations on cattle hides and in the gastrointestinal systems. “There are some pretty significant data gaps,” Johnson-DeRycke said, adding that ongoing research will help improve the model.

Public Health Specialist, Office of Public Health Science

USDA-Food Safety and Inspection Service

Because of limited data, FSIS included any study deemed relevant. Data regarding pathogen prevalence and contamination levels at slaughter were limited to the U.S. However, intervention studies, regardless of the country studied, were used if the practice investigated is equivalent to U.S. interventions. The number of establishments using each intervention and the percentage of carcasses slaughtered by that intervention are inputs to the model. Fewer than 20 establishments slaughter as much as 90 percent of the cattle, and therefore have the largest impact on overall production. FSIS next identified the effectiveness of each intervention as reported in the published literature. Johnson-DeRycke said that because many studies are conducted in a laboratory on cuts of meat as opposed to on a carcass moving through a slaughter establishment, the results most likely represent the best-case scenario. That figure is represented as a “high” value log reduction. The model also includes “default” and “low” reductions that may better represent actual values. The model is combinatorial, simulating the production of 1 million grinder lots of ground beef. Because all 632 establishments are included in the survey data, all are represented in the model, which includes production volumes. In addition, although 2,048 intervention combinations are possible, in reality only 75 combinations exist among the different slaughter establishments. Interventions are divided into three stages: hide, pre-evisceration and postevisceration. Hide interventions include whole animal hide washes, head washes, chemical dehairing and hot and cold water washes. Pre-evisceration interventions include steam vacuuming, organic acid washes and additional hot and cold water washes. Post-evisceration interventions include organic acid washes, water washes and steam vacuuming.


s t r at egi es at s l augh t er Establishments use different intervention techniques, which affect anticipated reductions. The 2007 survey didn’t include specific information regarding techniques, such as water temperature or hand sprayers versus cabinet washes. “We really had to make some assumptions there in terms of translating what those log reductions from the literature would be when applying them to the model and that was definitely one of the biggest challenges for us,” Johnson-DeRycke said. Two steps in the slaughter process provide opportunities for cross contamination: dehiding, because the carcass can become contaminated as the hide is removed, and evisceration. Although nicking the viscera is rare, it causes significant contamination. For both contamination events, the model simulates different possibilities regarding how often and how much contamination is expected. Finally, the model simulates how many carcasses end up in meat trimming combination bins and grinder lots. Large establishments can have up to 10,000-pound grinder lots. The model also includes decay values. Interventions within each stage don’t reach the same reductions in combination as they do individually. The first intervention removes most of the gross contamination, and subsequent interventions are less effective, removing pathogens resistant to other treatments or in hard-to-reach places.

The model may be used to help allocate FSIS resources, as a tool for industry to allocate resources when evaluating interventions, and for future rule-making or guidance documents.

“We really had a hard time finding any type of research that had been done or anything published on this concept of just how much of a decrease we would expect,” Johnson-DeRycke said. So the model allows the user to select a “decay value,” which calculates slight reductions to the second and subsequent interventions. Each intervention stage is modeled independently to allow for the additional contamination that occurs during dehiding and at evisceration, so decay values are reset at each stage.

“This was built as an exploratory tool to use the survey to see what kind of differences we could start to parse out among the interventions…When we build models of a process, we can adapt them to a lot of different things, and I think this model in particular will be one that we end up using for a lot of different things,” JohnsonDeRycke said.

Because the order of each intervention within an establishment is unknown, the model uses the most effective intervention first, and follows in decreasing order. Those presets can be manually rearranged to accommodate a specific establishment. The model begins with the baseline scenario of what establishments already do, then adds production volume to the intervention. For example, to see the effect of adding a carcass steam vacuum treatment to all establishments, all non-vacuuming establishments are moved to the vacuuming category. The decay value from other interventions and the establishments’ proportion of beef production are reflected. Rerunning the model shows the new contamination levels in grinder lots.

Some results are available online.

The model demonstrates that the overall effect of adding interventions is dependent on the effectiveness of that particular intervention, the current production level under a given intervention and the number of other interventions already in use at a given establishment. Adding post-slaughter dehairing would have the highest reduction, however post-evisceration interventions have a greater effect overall, a reflection that contamination lingers after dehiding and evisceration. Video of Rachel Johnson-DeRycke

FSIS is collecting new data to use in the model. A 2013 survey asked questions regarding additional intervention applications. Information gathered from an FSIS Beef Veal Carcass Baseline Study will also be incorporated. But the agency would like additional information regarding decay values, dependence within slaughter areas and independence between slaughter areas, and the economics of interventions.

presentation misc/GovFoodConference14/25RachelDeRycke.html

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stEc STEC Industry Perspective

p re va l en ce & i n t erv en t io n Brenden McCullough described the slaughter industry’s perspective regarding STEC intervention strategies at slaughter. “It’s about risk assessment for us. It’s about looking at the overall picture and trying to figure out what works, what doesn’t, what gives us the best opportunity to never make anybody sick and that’s what it’s all about,” he said. McCullough applauded the multidisciplinary approach of CAP. Industry has struggled for years, using study results to implement changes only to find 90 percent of them do not work as shown in the laboratory, he said. Industry then struggles to justify what it did. He used steam vacuuming as an example of an intervention that may get good results in the laboratory and that a manufacturer may say achieves excellent reductions, but in the plant does not. In some cases, he said, interventions can cause greater contamination.

Brenden McCullough

Vice President Technical Services National Beef Packing

But he also admitted that he’s nervous about the data that will come out of the CAP process. Will the industry be able to use the data to not only improve the process, but also “stand up and be whole in the USDA’s eyes when you deal with these things?” he asked. Large customers, such as Costco, may also see the data and request changes. Yet industry must change, he acknowledged. Industry buried its head after the 1993 Jack in the Box outbreak and was not as proactive as it should have been. Lack of knowledge played a role, but so, too, was the unwillingness of some industry leaders to change. Attitudes are different today. “I would say that most of us, if not all, understand that if we do not embrace and seek to gain the knowledge that we can get from the folks here and the research and the studies and be a part of that process, we’ll rue the day because this thing’s not going to go away, and it shouldn’t. There’s not a one of us that wants to produce a product that’s going to make somebody ill,” McCullough said. Industry’s goal is to eliminate pathogens in meat, lofty and perhaps unobtainable, but that goal must drive the process, he said. In the plant, they test frequently and continually analyze the results to monitor the process and react appropriately. Testing doesn’t guarantee food safety, but ongoing assessment of the process allows the plant to take action when abnormal results begin appearing. When contamination appears, product within that timeframe is removed from the non-intact product realm. But he said he’s also concerned that regulators “paint everyone with the same brush.” An issue with one plant, for example, is an isolated incident. That plant should be held accountable, but the incident shouldn’t force changes industry-wide. In addition, when looking at interventions and types of pathogens, the data may show that a more targeted approach is more effective than across the board changes, he said. It appears not all STEC are found in beef, he added, referring to the USDA’s Beef Veal Carcass Baseline Study shakedown data. If some STEC are proven to not be a food safety risk in beef, he’d like to see that acknowledged and a more focused approach applied. It’s frustrating, he added, to hear presenters say STEC are on the rise, without providing context. Beef, veal and dairy cows are different animals, processed in different ways and in different environments. “We have to understand that we can’t treat everything the same, and expect to get a good result. We need a target,” he said.


Industry has challenges ahead. It’s been reluctant to open its doors for testing and research because of the repercussions the data could cause with regulatory agencies.

s t r at egi es at s l augh t er Twenty years ago, when industry first began working on reducing E. coli O157:H7 (O157), it tried many interventions, some that worked and others that didn’t. The data from that time now carries a “heavy weight” with regulators, so industry is treading carefully. But talks with academia are happening, and industry is committed to improving the process. “We know some of that data’s wrong,” he said of the USDA’s studies. “Why doesn’t the agency know it? We don’t share it with them. We need to do a better job of that.” The good news, however, is that O157 prevalence in beef has declined 85 percent since 2000. Levels do rise in hot weather and never entirely disappear, so improvements are still needed. But what industry has learned from O157 should help control the entire STEC population, he said. The USDA’s Healthy People 2020 includes goals for foodborne illnesses from several pathogens. The industry already meets Listeria goals and is close with STEC, but Salmonella incidence rates are still high. USDA is committed to helping industry drive down incidence rates, McCullough said.

When asked about what industry can do about contamination before cattle arrive at the plant, McCullough said that pre-harvest interventions are important, but difficult. Without solid data that such interventions drive down contamination, ranchers are reluctant to invest money in them. Unlike in the poultry industry, ranchers and slaughter establishments operate independently of each other.

It’s about looking at the overall picture and trying to figure out what works,

McCullough was also asked about the use of internationally sourced meat in the U.S. industry’s grinder products. He said that any raw product is a concern, and foreign

what doesn’t, what

meat should be monitored equally. Lot sizes vary considerably in other countries, and some are huge. It’s easy to get caught up in recalls, for example, if you share grinder lots with a company undergoing a recall.

gives us the best

McCullough emphasized that he doesn’t speak for all industry, but he said that he’s

opportunity to never

glad industry was invited to be part of the STEC CAP process. Industry is looking for

make anybody

new interventions that are economically feasible and it wants to share information. It also wants to help guide sample testing—not to drive outcomes—but to help


create an effective process that can be implemented. “This partnership, the different aspects that everybody covers, is just amazing,” he said.

Video of Brenden McCullough presentation

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stEc STEC Needs and Interventions for Consumer and Retail Audiences

preventing outbreaks thro The recent and ongoing outbreak of undercooked hamburger linked to more than 15 different restaurants highlights the importance of intervening at the consumer and retail level to prevent foodborne outbreaks, the focus of Ben Chapman’s research. The risk doesn’t come just from ordering a hamburger at a restaurant, but from eating at restaurants that serve beef due to cross contamination from beef to other products, Chapman said. His work is based on behavioral theory. The theory of planned behavior is most applicable to food safety behavior. When people make choices, they are influenced by their attitude toward the behavior, such as whether they believe it’s risky or not. They’re also influenced by what other people are doing, so someone on television modeling a behavior confirms that behavior’s acceptability. The final influence on behavior is whether they think they have control over the outcome. Those three influences lead to intention to do that behavior, followed by doing the behavior.

Ben Chapman

Extension Food Safety Specialist, Professor North Carolina State University

In the current hamburger outbreak, the perceived behavioral control in the restaurant is ordering a cooked burger. The restaurant follows through by bringing the prepared burger. The Food and Drug Administration’s food code states that a restaurant serving undercooked meat must disclose the risk associated with consuming it and must remind consumers of the risk when they order it. Restaurants often put that notice on the menu, or they leave it to servers to communicate the risk. Chapman and his team conducted a project focused on menus and servers as risk communicators. “I think now that servers are not good risk communicators, and I don’t think that we should expect them to be good risk communicators. But the law says that that’s how it should work,” Chapman said. During the recent outbreak, the USDA’s blog stated: For consumers, we advise all consumers to safely prepare raw ground beef products by cooking them to 160°F. When dining out, this is equivalent to ordering your burger well done. “It is not at all,” Chapman countered. “There is no definition for ‘well done’ at a restaurant.” He led a collaborative team that visited 220 restaurants nationwide and ordered well-done and medium-rare hamburgers. The variety of colors among the hamburgers suggests a single meaning for “doneness” doesn’t exist. Color doesn’t definitively identify what has been cooked to 160°F, the study concluded. The researchers also looked at menus and found numerous messages. The study demonstrated that restaurants provide mixed messages. One menu, for example, suggested ordering their burgers rare because the beef is ground on site, followed by the food safety warning regarding consuming undercooked foods. In addition, they found that servers are expected to be risk communicators at the same time they are suggesting a riskier burger for quality reasons. “It’s kind of a shame, and it’s a little bit of a problem,” Chapman said. Researchers also asked servers about the method of doneness. About 67 percent


of the information they provided was incorrect based on food code criteria. Nearly 70 percent cited color, while others mentioned touch or time. Temperature was

ugh education & consumers mentioned, but was outweighed by incorrect messages. Server messages ranged widely. One server, for example, said the USDA had outlawed E. coli in beef, so you could order a burger medium rare and be safe. Another said he wouldn’t let his pregnant sister eat a burger medium rare, so he didn’t recommend it to patrons either. When questioned about which hamburgers were safe to eat from a food safety standpoint, 30 percent of servers said rare or medium rare. Chain restaurants were more conservative in their responses than independent restaurants. Interestingly, medium hamburgers are much more acceptable at independent restaurants compared to chains, while medium-well to well-done hamburgers are recommended more at chains. This result suggests something in the corporate culture affects servers. The research is ongoing and will look at demographic and geographic as well as urban versus rural differences. This data is powerful, Chapman said. He plans to make recommendations to improve the USDA food code so that appropriate information will allow people to

make better decisions. A second project looked at YouTube videos and the information modeled for consumers. Chapman and his team reviewed 87 videos on how to make a hamburger or cheeseburger with 12 million total views, and coded each for positive and negative behaviors. Negative behaviors far outweighed the positive. Cognitive behavioral theory states that a modeled behavior is more likely to be emulated. Only one video out of

There is no definition for ‘well done’ at a restaurant.”

87 used a thermometer to check doneness. The others used different methods, such as pushing on the burger to check for firmness. In a third study, the researchers investigated food safety at food pantries, part of a commitment to deliver food safety material through 4-H clubs. They visited 97 North Carolina pantries. Though food normally isn’t prepared, large cuts of meat or ground beef are routinely subdivided into smaller bags for refreezing or for consumers. About 20 percent of people living in the U.S. in the past five years accessed a food pantry. Beef handling is prevalent, yet most states, including North Carolina, don’t regulate pantries. The study is ongoing. Chapman participates in social media activities, including producing the Barf Blog ( He and a colleague wrote 620 posts in the first half of 2014, 88 of them specific to STEC, and now have more than 23,000 subscribers. He and his team have also outlined approaches for food safety messaging in social media in a paper soon to be published. Social media provides researchers with access to real people to find out what they’re talking about and doing.

Video of Ben Chapman presentation

To communicate within social media, it’s important to provide evidence, not just

dictate prescriptive information. “As educators we need to tell people the why,”


Chapman said. Social media is also about engaging people and having dialogues, not


just giving them information. Social media is difficult and can be overwhelming, he added. “So if you’re going to go down the road into social media be sure you have time to invest into it.”

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stEc Reaching the Public with Safe Food Handling Information

preventing outbreaks thro Most foodborne outbreaks stem from restaurants, but people serving food in private homes, social and religious groups and for elderly and youth populations also play important roles, Christine Bruhn said, adding that no formal outreach program exists in these food safety areas. Her research focuses on educating community volunteers about food safety by identifying the food safety guidelines people need and providing information to them.

Online versus in-person training Previous work demonstrated that 3-hour, hands-on workshops given by community service volunteers effectively increased participants’ knowledge, motivation and reported follow-up behavior. But workshops are a significant commitment for volunteers, so Bruhn and her team have developed and are testing an online version.

Christine Bruhn

Director, Center for Consumer Research

University of California, Davis

The researchers converted the workshop program into several online modules based on USDA guidelines for food safety, focusing on cook, clean, chill and separate principles. The online information is conveyed through text, video and slides. It was also translated into Spanish. Participants are presented with an introduction, then take a pre-test. After completing the modules and reviewing a summary, a post-test measures the knowledge gained. The in-person workshop has been completed by 174 people, the online version by 89 people. Both groups increased their knowledge of food safety, Bruhn said. Some questions demonstrated that many already knew the information, and overall, those that completed the online version had more prior food safety knowledge although they, too, improved. To date, no one has taken the online version in Spanish. In the workshop, participants’ knowledge increased from about 65 percent correct answers to more than 80 percent. Those who indicated they had prior food safety training, from a high school class to food safety certificates, scored better initially, but also increased their knowledge. The participants cook for service groups, such as Kiwanis or Rotary clubs; youth groups, such as 4-H clubs or Little League teams; faith groups, such as church potlucks; and senior centers. “Senior centers and no food safety knowledge? Yes!” Bruhn said. “So it’s very important to be able to reach these people because they were reaching a high-risk audience.” One test question asked about awareness of high-risk individuals. Children, older adults, diabetics and pregnant women are all at increased risk, but knowledge of that risk was low, particularly among workshop participants. In all cases, in both workshops and online, participants increased their knowledge regarding these risky populations. Additional examples of handling practice questions, including storing leftovers in shallow containers and using a thermometer to test doneness, demonstrated that participants increased knowledge after both the workshop and the online training. The in-person workshops included visual aids. For example, when discussing the


importance of temperature control, the trainer put a jelly bean in a jar. To simulate bacterial growth, the jelly beans “doubled” every 20 minutes until the jar was full.

ugh education & consumers “That visual impact of the importance of temperature control is very powerful in realizing … that they have to make sure their food is cold,” Bruhn said. “I was concerned that this wouldn’t translate online, but it did. We were able to do visual things online.” The team is continuing to share the system, particularly with Spanish participants.

Celebrity chefs Bruhn and her team also investigated the impact of celebrity chefs on consumer behavior. They evaluated 60 cooking shows and showed clips to study participants of Rachael Ray, Paula Deen, Bobby Flay and Guy Fieri modeling negative behavior, such as licking fingers, chopping cilantro on top of raw fish juice and not testing for doneness. Some consumers recognized the errors; others didn’t. Those who didn’t tend to believe it’s fine to do those behaviors at home. Those that do recognize the errors were either disgusted at the chef or excused the chefs’ behaviors.

Culinary students lost respect for the chefs and felt it was their moral obligation to model appropriate behavior. The team is still analyzing the results, but Bruhn said she hopes to present the findings to the Food Network to show that, not only do people imitate dangerous behavior, but also lose respect for celebrity chefs. Is the Food Network responsible for

Senior centers and no food safety knowledge? Yes!”

foodborne illness? Bruhn asked.

Reaching high-risk populations Pregnant women are at increased risk of foodborne illness because their weakened immune systems allow bacteria to overpower them. Although E. coli doesn’t cross the placental barrier, pathogens can make women, the fetus and newborn babies ill. Diabetics are also at higher risk because of reduced gastric acid production and decreased bowel motility. Educational programs for both groups fail to include food safety, and health professionals have acknowledged incompetence in this area. Bruhn and her team are comparing three methods to change high-risk individuals’ behavior: an in-person, 3-hour interactive program; providing USDA educational materials; and reading those educational materials along with telling personal stories. They use pre- and post-surveys to identify attitude changes and follow-up phone calls to determine behavioral changes. “We find that people were excited, especially the diabetic people were terribly excited to have the information,” Bruhn said. They were given refrigerator and cooking thermometers and asked to record temperatures. The activity helped commit them to the training. Video of Christine Bruhn presentation

“Our summary was that educational changes are making a difference, at least in

knowledge,” Bruhn said. The personal interactions, in particular, were positive and


seemed to lead to behavioral changes, though follow-up surveys are ongoing.


“It’s amazing that these high risk individuals, especially those with diabetes, are neglected as far as food safety information is concerned,” Bruhn said. The USDA has information for diabetics, but no one is delivering the information to these groups.

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stEc Translation of Shiga Toxin E. coli: Mitigation to Field Implementation in the Beef Industry

preventing outbreaks thro The Beef Cattle Institute (BCI) of Kansas State University is the national online training center for beef quality assurance. To date, the institute has reached 30,000 people, said Kelly Oliver. To help reduce E. coli contamination in the food supply, BCI has produced educational training modules to teach proper control and prevention strategies for industry personnel. The project focuses on both pre- and post-harvest operations, including feedlot, cow-calf, veal and cull dairy cow operations, packing plants, restaurants and distributors. To create the modules, BCI interns sorted through all available research and created scripts. A panel of experts reviewed the scripts, and a final narration was developed. BCI is now working with food producers to obtain video footage, photos and other supporting material to produce the final training modules. It’s also conducting videoed interviews with experts to include in the modules.

Kelly Oliver

Project Coordinator, Beef Cattle Institute Kansas State University

The website contains hundreds of training modules for producers. They can view the modules online, take an online quiz and receive a certificate of completion. All modules are available in English and Spanish. The participant cost is $25. To date, the modules, though not yet live, have reached several thousand people.


Video of Kelly Oliver presentation

ugh education & consumers

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stEc Expanding Food and Nutrition Science Education through Additional, Enhanced Curriculum Development

preventing outbreaks thro The overall goal of Jason Ellis and Dann Husmann’s educational project is to train the current and future workforce needed to control STEC, from researchers to production agriculturalists and even future consumers, Jason Ellis said, speaking for the duo. They are developing an educational multi-year curriculum focusing on nutrition science and food production for middle and high school students. The project, currently a pilot limited to Kansas and Nebraska, will be expanded once it’s finished and the curriculum validated. The curriculum consists of four courses, each focused on developing skills and knowledge so students are able to ask questions and solve problems. This inquirybased learning method encourages students to view challenges as positive experiences, rather than backing away and becoming disengaged.

Jason D. Ellis

Professor, Agricultural Communications and Journalism Kansas State University

The first course, completed last year, is a foundational course focused on food production, nutrition and health and introduces concepts and terms. Now, the researchers are developing courses 2 and 3, which focus on in-depth science. Course 2 focuses on food sciences. Curriculum writers are developing units on fermentation, food allergies, food safety and product development. It incorporates student projects and relies heavily on instructional aides, such as photos and videos. The aides help teachers both in the classroom and prior to teaching students to understand and gain confidence in the material. In addition, teachers participating in the pilot project attend workshops and spend a week at the University of Nebraska– Lincoln to learn and feel more comfortable teaching these topics in their schools. Course 3 focuses on nutritional science and includes units on losing one’s nutritional balance, science myths and components for a busy family. The course ends with a culminating project called “farm to flush.” Although “farm to fork” is commonly used to refer to the entire food production system, the final “fork to flush” piece, after products reach the consumer, is also important, Ellis said. To teach students about STEC-related health issues, the curriculum includes an outbreak scenario in which students must conduct an inquiry-based study to answer such questions as where the outbreak originated and what caused it. In the process, they learn about biology and pathogens’ effects on the gut. Course 4 will be more advanced and will focus on food nutrition research. To engage students in research, Ellis said they hope to partner students with researchers and other collaborators involved in STEC projects.


Video of Jason Ellis presentation

ugh education & consumers From the audience, Husmann added that encouraging students to ask questions and engage in experiments is important to get them excited in research. “If we can do these kinds of things that you’re doing in the lab,” he told the audience, “and create scenarios or learning events that we can make applicable to the teachers … then you’re going to get students. You’re going to find young men and women that will gravitate toward that.” Although they focused initially on secondary education, other markets and venues exist. Middle schools teach relevant material in family consumer science and agricultural education classes, which are usually shorter and more survey oriented than high school classes. Ellis and Husmann are adapting aspects of their high school curriculum for younger students. The middle school curriculum also introduces students to career possibilities, such as genetics, microbiology, food science, food processing and agriculture. In addition, Kansas State University is developing a summer educational program for 14 to 17 year olds, coordinated through the state 4-H program. The program includes components on food safety and processing. Some of Ellis and Husmann’s curriculum will be integrated into this educational program, which begins in summer 2014, and

Dann E. Husmann

Professor and Associate Dean College of Agricultural Sciences and Natural Resources University of Nebraska-Lincoln

will provide a good learning opportunity to enhance the program’s materials and perhaps expand into other areas, Ellis said. “It’s exciting what I hear and see that people are doing on the research side,” Husmann added. “We just have to be able to do this on the education side, to put that in a high school so that it’s palatable to teachers and, thus, we get that down to the students and get them excited.”

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stEc Consumers and Consumer Advocates Play a Vital Role in Reducing the Public Health Burden Attributed to STEC Illness

preventing outbreaks thro Though sometimes overlooked, consumers and consumer advocates play vital roles in reducing STEC illnesses, said Vanessa Coffman. STOP Foodborne Illness, a consumer policy and advocacy organization, was founded in 1993 by angry parents whose children had died from eating contaminated ground beef during the Jack in the Box outbreak. Those early leaders advocated for new legislation. STOP continues to engage in policy, but has become more involved in consumer advocacy as well. While STOP also provides education and outreach, particularly using social media, changing consumer behavior is difficult, so STOP focuses more on getting safe food into homes and restaurants. “We are the voice of consumers,” Coffman said. STOP also provides support for families affected by outbreaks. It connects people

Vanessa Coffman

Education Manager

STOP Foodborne Illness

to others who’ve gone through similar experiences and provides resources to help families heal. The organization also seeks to instill good, lifelong habits in consumers at an early age and provides educational materials. Social media is now a major vehicle for reaching members. STOP posts E-alerts about new outbreaks on Facebook, twitter and on their website. The website,, also has a policy action center with an interactive database of legislators’ addresses and letters to help people contact them about food safety concerns. STOP listens to and works for members. Consumers care about price, taste and convenience. When it comes to food safety, they want transparent, easy-tounderstand information, so they can make the best choices for their families. But consumers would rather not have to worry about food safety in the first place. “We want to have confidence in our food supply,” Coffman said. Consumers should have a voice, she said. They should feel empowered to speak and be heard. They should also have access to information they want and to information they didn’t know they needed. Ultimately, STOP is working to put itself out of business by ending E. coli outbreaks. The organization is involved in numerous policy initiatives and consumer advocacy work regarding a range of pathogens, but especially STEC. It participates in coalitions, including the Safe Food Coalition, a group of consumer representatives that use their expertise to advocate for important food safety legislation. That’s accomplished through writing congressmen and USDA officials, trips to visit representatives in Washington D.C. and media appearances. People affected by foodborne illness provide powerful stories for STOP’s advocacy work. Its website contains numerous stories, a valuable tool to remind those in the industry, researchers and legislators about the importance of food safety.


ugh education & consumers Recently, STOP advocated for labeling mechanically tenderized beef. In 2009, nearly 250,000 pounds of mechanically tenderized steaks were recalled. The outbreak sickened 21 people in 16 states and made people aware of the dangers of tenderized beef. The Safe Food Coalition came together and wrote letters to the USDA requesting labeling rules. The Conference for Food Protection also requested a labeling rule, and in 2013 the government issued a proposed rule. STOP comments on proposed rules and encourages members to comment. For example, when the 2011 Food Safety Modernization Act was proposed, hundreds of people signed STOP’s letters or sent their own in support. STOP also organized visits to Congress and attended Food and Drug Administration public meetings with members, who shared their stories. “It’s a nice, good, huge voice and an important one,” she said. “Consumer participation and perspectives are keys to successful initiatives.” Coffman encouraged audience members to engage with consumers and consumer advocates and to contact STOP to learn about consumers’ perspectives on issues. STOP members are passionate about food safety and would appreciate the

opportunity to provide feedback on ideas or share information as well. STOP’s philosophy is not to antagonize industry, but to work as partners fighting for the same goal. For example, organization representatives and members affected by the 2006 spinach outbreak visited farmers and others in California. The farmers were nervous, Coffman said, but it was a positive and productive experience of sharing ideas and learning about each other’s perspectives.

Consumer participation and perspectives are keys to successful

Coffman encouraged participants to visit the website and use the stories to provide


context for their work, such as educators for their classroom. She also encouraged participants to get in touch: “If there’s some way that we can work together, I would love to do that for you.”

Video of Vanessa Coffman presentation

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stEc Progress on Development and Improvement of Detection and Quantification Methods for STEC

i m p rov i n g s t ec d e t ec t io n The ability to detect E. coli O157:H7 (O157) and other STEC is a critical component to catching pubic health threats before they become outbreaks, to develop economically feasible interventions at meat processing facilities and to aid research. But determining whether STEC are present, as well as identifying specific strains, is challenging. Several speakers described their studies to improve detection methods and other resources to aid STEC research. Rodney Moxley’s research focuses on identifying improved methods to grow and detect STEC, conducting STEC detection and quantification tests on samples as a part of epidemiological and intervention studies, and identifying mechanisms of STEC colonization of the bovine intestine in order to develop improved pre-harvest interventions. His lab has collected 80 strains across eight STEC O groups and 38 non-STEC strains for use in test validation. He described several studies comparing

Rodney Moxley

STEC CAP Project Director, School of Veterinary Medicine and Biomedicine, University of Nebraska-Lincoln

the effectiveness of different growth and culture plating media as well as commonly used methods to differentiate between different STEC strains. Moxley reported on the prevalence of Top 7 EHEC in hide and carcass samples at harvest in a 2013 beef feedlot study using different detection methods. Culture methods were very insensitive for detection of EHEC. In contrast, results with a highly sensitive commercially-available test method that utilizes PCR and mass spectroscopy on DNA extracted from enrichment broth cultures (NeoSEEKTM STEC Detection and Identification Test) indicated that the cumulative prevalence of Top 7 EHEC on beef cattle hides at harvest was high, approximately 78 percent. On hides in this study, EHEC O145 and O45 were the most prevalent, followed in order by O103, O157, O111, O121, and O26. On carcass surfaces of the same cattle, the cumulative prevalence of the Top 7 EHEC was approximately 11 percent, with O145 and O157 being most prevalent, followed in order by O103, O45, O26, O111 and O121. These results indicate that non-O157 and O157 EHEC are highly prevalent on the hides of beef feedlot cattle and that continued improvement and validation of non-O157 culture methods are needed. Confirming the presence of Top 7 EHEC in broth enrichment samples without isolating the organism is difficult due to the contribution of gene targets from nonpathogenic bacteria, which may lead to false positive results, Moxley said. Further developing tests that recognize unique markers in EHEC, especially those that are non-O157 adulterants, is needed. Moxley is collaborating with STEC CAP researcher, Harshini Mukundan, at Los Alamos National Laboratory, and researchers at the monoclonal antibody laboratory at the Dana-Farber Cancer Institute at Harvard University, to develop more discriminatory biomarkers for non-O157 EHEC to improve detection. Preliminary results are encouraging, and studies are ongoing.


Video of Rod Moxley presentation

Shiga toxin and intimin virulence genes, as well as the gene identifying O-group type, are key targets for detection of EHEC and are easy to find by the genetic-based PCR test. But the method can’t distinguish if the genes belong to the same bacterium. Therefore, finding all three virulence factors within a sample is considered only a “potential positive,” Bosilevac said. He described several approaches to improve on these “classic” targets to detect non-O157 STEC and to help reduce the number of potentially positive samples during

The U.S. MARC Experience of Applying Non-O157 STEC Detection and Enumeration

STEC monitoring.

Methods to the Beef

One approach targets intimin subgroups. EHEC O-groups generally carry specific


intimin, or eae, subtypes. For example, O26 nearly always carries eae beta, and so on. Therefore, if a sample contains an O26 and an eae beta, there is a greater probability that those genes are carried within the same bacteria. A second method looks at single-nucleotide polymorphisms, or SNPs, a variation in the DNA sequence of one base pair within members of the same strain. Previous work sequencing E. coli genomes has identified the SNPs that correlate with EHEC types. Testing these known genetic variations allows EHEC O-groups to be distinguished

Mick Bosilevac

Research Microbiologist, Meat Safety and Quality Research Unit USDA’s U.S. Meat Animal Research Center

from non-EHEC of the same O-group type. The commercially available NeoSEEKTM STEC Detection and Identification Test incorporates both methods. A study comparing the methods found that using eae subtypes alone detected 31 “potential positives” compared to NeoSEEK’s six potential positives. While both methods captured the actual positive sample, it’s less burdensome for a beef processor to deal with six potential positives than 31 potentials. Researchers have identified other genetic differences associated with virulence that, when combined, can be used as tools to more reliably predict EHEC presence, Bosilevac said. He and his team are evaluating these genetic targets. “A combination of these targets may be what’s useful to come up with a really good prediction of presence for detection,” Bosilevac said.

Video of Mick Bosilevac presentation

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stEc STEC Detection and Characterization using Multiplex Oligonucleotide Ligation-PCR (MOL-PCR)

i m p rov i n g s t ec d e t ec t io n Alina Deshpande and her team have developed the first of two genetic-based MOL-PCR tests to detect the Top 7 EHEC, STEC O104:H4, plus three virulence genes. MOL-PCR, or multiplexed oligonucleotide ligation polymerase chain reaction assay, simultaneously screens samples for multiple pathogens or toxin profiles. It enables large-scale sample screening, which would help during outbreaks or other times when large numbers of samples must be screened quickly, Deshpande said. The assay is also flexible and can easily be adjusted to screen for new virulence markers or pathogens. Preliminary results evaluating the assay are promising. The researchers continue to test the assay and are developing an automated data processing algorithm that will process the samples and “call� a positive rather than require manual identification. Deshpande and her team are refining assay conditions to make them more robust and

Alina Deshpande

DSA-3, Systems Analysis and Surveillance Defense Systems and Analysis Division Los Alamos National Laboratory


are currently developing a SNP-based characterization assay.

Video of Alina Deshpande presentation

T. G. Nagaraja described research comparing three methods for screening to detect the Top 7 EHEC strains in cattle feces: traditional PCR, real-time PCR and the culture method. To detect the STEC and virulence genes (Shiga toxins, intimin, and enterohemolysin), the team designed several Real-Time PCR (RT-PCR) assays, a modification of traditional PCR that are more sensitive. The studies demonstrate that RT-PCR is indeed the most sensitive screening method,

PCR- and Culture-based Methods of Detection of STEC-7 in Cattle Feces

detecting more potential positives than the other two. However, the culture method, although less sensitive, sometimes may detect STEC or EHEC in samples that test negative by RT-PCR. The greatest advantage of culture is that when EHEC is isolated from a sample, the result definitively confirms a positive. “It may be a good idea to subject a fecal sample to both PCR and culture method. That way we get the best estimate,” Nagaraja said, adding that “the culture method, in my opinion, requires further refinement.” The researchers also looked at the performance of immunomagnetic separation (IMS) beads to identify specific STEC serogroups during PCR. Results suggest that using multiple IMS bead types improved detection. However, using individual IMS beads on a fecal sample requires much time and effort. Preliminary results from studies looking at pooling beads suggest that two sets of beads may be used for the Top 6 non-O157

T. G. Nagaraja

Distinguished Professor of Microbiology, Department of Diagnostic Medicine/Pathobiology College of Veterinary Medicine Kansas State University

STEC and a separate IMS bead for O157. That work is ongoing.

Video of T.G. Nagaraja presentation

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20 1 4 G ov e r n o r ’ s co n f e r e n ce/ S T EC C A P A n n ua l co n f e r e n ce

This project was supported by Agriculture and Food Research Initiative Grant No. 2012-68003-30155 from the USDA National Institute of Food and Agriculture, Prevention, Detection and Control of Shiga Toxin-Producing Escherichia coli (STEC) from Pre-Harvest Through Consumption of Beef Products Program –A4101.


Managing the Risk of Shiga ToxinProducing Escherichia coli in Beef