Survival of Acid-Adapted Escherichia Coli O157:H7 and Non-O157:H7

The Pennsylvania State University

The Graduate School

College of Agricultural Sciences

SURVIVAL OF ACID-ADAPTED ESCHERICHIA COLI O157:H7 AND NON-O157:H7

SHIGA TOXIN-PRODUCING E. COLI (STEC) DURING PROCESSING OF DRY,

FERMENTED SAUSAGES

A Dissertation in

Food Science

Minerva Rivera

Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

December 2017

The dissertation of Minerva Rivera was reviewed and approved* by the following:

Catherine N. Cutter Professor of Food Science Food Safety Extension Specialist – Muscle Foods Dissertation Advisor Chair of Committee

Jonathan A. Campbell Assistant Professor of Meat Science Extension Meat Specialist

Edward G. Dudley Associate Professor of Food Science

Sara Milillo Food Science Faculty Director of Math and Science at Bay Path University

Robert F. Roberts Professor of Food Science Head of the Department of Food Science

*Signatures are on file in the Graduate School

Abstract

Escherichia coli O157:H7 was first recognized as a human pathogen following outbreaks of hemorrhagic colitis in the early 1980s. Due to the infectious dose of E. coli O157:H7, the severity of the disease, and the possibility of lifelong sequalae, the U. S. Department of

Agriculture-Food Safety and Inspection Service (USDA-FSIS) declared it as an adulterant in raw ground beef in 1994. In 2012, non-O157:H7 Shiga toxin-producing E. coli (STEC) serogroups (O26,

O45, O103, O111, O121, and O145) also were declared adulterants in raw beef products by USDA-

FSIS. Until recently, the majority of the research done on interventions used to control STEC has been done on E. coli O157:H7. However, recent research suggests that some non-O157:H7 STEC may be more resistant to some interventions than E. coli O157:H7. The purpose of this study was to assess the differences in survival between E. coli O157:H7 and non-O157:H7 STEC when exposed to acid adaptation, followed by various stressors typically encountered during processing of dry, fermented sausages (DFS), including fermentation, lowered water activity (aw), and long-term, vacuum packaged storage (VPS).

Experiments were conducted in vitro and in situ by exposing STEC to low pH, low water activity, and/or desiccation. In a series of in vitro experiments, control (non-acid adapted) and acid-adapted STEC cocktails were exposed to modified TSB (lowered pH and lowered aw; 24°C for

4 days), subjected to desiccation on paper disks, and evaluated for survival. In situ experiments were carried out in modified ground beef slurries (with lowered pH and lowered aw; 24°C for 4 days). Finally, acid-adapted E. coli O157:H7 and non-O157:H7 STEC were evaluated for survival throughout processing of DFS, including fermentation, drying, and long-term VPS.

iii

Results from the first in vitro experiments showed that populations of the control non-

O157:H7 STEC serogroups O45, O103, O111 and O121 were significantly different from populations of E. coli O157:H7,and non-O157:H7 STEC serogroups O26 and O145. In contrast, when STEC serogroups were acid-adapted, all populations of non-O157 STEC were significantly different from populations of E. coli O157:H7. There were no significant differences between control (non-adapted) and acid-adapted cells between any individual serogroup. Reduction rates for all serogroups demonstrate that they not only survived well in acidified TSB, but also were able to grow over time, albeit minimally. Results from the second in vitro experiment, TSB with modified water activity (aw=0.88 or 0.78), demonstrate that there was no significant difference between E. coli O157:H7 and any of the non-O157:H7 STEC, regardless of acid-adaptation. In a third in vitro experiment, exposure to desiccation on dry discs, there was no significant difference in survival between E. coli O157:H7 and the non-O157:H7 STEC, regardless of acid-adaptation.

Results from an in situ experiment, acidified ground beef slurries, indicated that all non-O157:H7

STEC behaved similarly to E. coli O157:H7, with the exception of O145 STEC which had a higher reduction than O157:H7. There were no significant differences in ground beef slurries with modified water activity. These results suggest than non-O157:H7 STEC behave similarly to E. coli

O157:H7 when exposed to laboratory stress conditions of low pH (pH 4.5) and low water activity

(0.88 or 0.78). These results are in agreement with other researchers; interventions that work on O157:H7 are likely to work on non-O157:H7 STEC.

Experiments conducted with DFS processing demonstrated that the highest overall reductions in non-O157:H7 STEC was observed for E. coli O26 (2.66 log10 CFU/g), while the lowest were observed for E. coli O111 (2.29 log10 CFU/g) following fermentation, drying, and long-term

VPS. All serogroups demonstrated total reductions >2.0 log10 CFU/g and there was no significant difference between the log reduction rates of non-O157:H7 STEC, when compared to E. coli

O157:H7. These results suggest that non-O157:H7 STEC survival during the production of a DFS was comparable to E. coli O157:H7 and that all tested STEC serogroups are able to survive DFS processing. Overall, results also suggest that acid-adaptation had minimal or no significant effect on the survival of any of the tested serogroups. Moreover, results suggest that interventions that work for E. coli O157:H7 will be equally effective against non-O157 STEC. These results could be useful to regulatory agencies by providing the science needed to make recommendations and/or regulations to mitigate the risk of non-O157:H7 STEC in the food industry. Additionally, these results may be of use to researchers and/or quality control personnel seeking to validate interventions and processes to ensure the safety of dry fermented sausage products for the consuming public.

Table of Contents List of Tables ...... viii Acknowledgements ...... ix Chapter 1 ...... 1 Enterohemorrhagic E. coli (EHEC) ...... 2 History and Description ...... 2 Virulence Factors and STEC Pathogenesis ...... 4 Shiga Toxins ...... 4 Other Virulence Factors ...... 5 History of E. coli O157:H7 as a Pathogen ...... 6 Animal Reservoirs ...... 7 Environmental Reservoirs ...... 9 Epidemiology of E. coli O157:H7 Outbreaks ...... 11 History of non-O157:H7 STEC as Pathogens ...... 13 Non-O157:H7 Shiga toxin-producing E. coli (STEC) ...... 13 STEC Detection Methods ...... 18 Culture Methods ...... 18 Molecular Methods...... 19 Stress Response in E. coli ...... 23 Acid Stress Response ...... 24 Osmotic Stress Response ...... 26 Statement of the Problem ...... 27 References ...... 29 Chapter 2 ...... 42 Abstract ...... 43 Introduction ...... 45 Materials & Methods ...... 47 Bacterial Cultures ...... 47 Acid Adaptation Protocol ...... 48 Experiment 1-Challenge Media Assays ...... 48 Experiment 2-Desiccation Assay ...... 49 Experiment 3-Acidified Ground Beef Slurries ...... 50 Statistical Analysis ...... 50

Results and Discussion ...... 51 Experiment 1-Challenge Media Assays ...... 51 Experiment 2-Desiccation Assay ...... 54 Experiment 3-Acidified Ground Beef Slurries ...... 55 References ...... 57 Chapter 3 ...... 64 Abstract ...... 65 Introduction ...... 67 Materials and Methods ...... 71 Bacterial strains ...... 71 Sausage preparation ...... 73

pH, aw, and microbial analysis...... 74 Statistical analyses ...... 75 Results and Discussion ...... 75 Chapter 4 ...... 92 4.1 Conclusions ...... 93 4.2 Future research ...... 95 References ...... 98 Appendix A Pathogen Reductions Associated with Traditional Processing of Landjäger ...... 99

Appendix B Table B1: Populations (log10 CFU/g) of STEC during processing of dry, fermented sausage...... 124

vii

List of Tables

Chapter 2

Table 1. Serogroups and Strains Used in all Experiments …………………………………………………....61

Table 2. Differences between the means of control (E. coli O157:H7) and acid-adapted non- O157:H7 STEC ………………………………………………………………………………………………………………………. 62

Table 3. Rate of reduction of acid-adapted E. coli O157:H7 and non-O157:H7 STEC ……………………………………………………………………………………………………………………………………..………. 63

Chapter 3

Table 1. Serogroups and Strains Used in all Experiments …………………………………………………….. 86

Table 2. Sausage processing parameters ……………………………………………………………………………. 87

Table 3. Average pH measurements of dry, fermented sausages at various points during processing ………………………………………………………………………………………………………….……………… 88

Table 4. Average aw measurements of dry, fermented sausages at various points during processing ………………………………………………………………………………………………………………………….. 89

Table 5. Log reductions of STEC during production of dry, fermented sausages enumerated on Rainbow Agar O157:H7 ……………………………………………………………………………………………………… 90

Table 6. Means of the log reduction rate of acid-adapted STEC during production of dry, fermented sausages …………………………………………………………………………………………………………. 91

viii

Acknowledgements

I would first like to thank Dr. Catherine Cutter for giving me the opportunity to come to

Penn State to study and conduct valuable research in Food Science under her direction. Dr.

Cutter’s vast knowledge and experience has been an instrumental in guiding my research and I am truly grateful for her mentorship. I would also like to thank the members of my committee,

Dr. Jonathan Campbell, Dr. Edward Dudley, and Dr. Sara Milillo, who have contributed, not only their knowledge, but their valuable time during the course of my stay at Penn State. I would especially like to thank Dr. Campbell for his encouragement and providing hands-on help during research in the food safety/pathogen pilot plant. I especially want to than Dr. Nancy Ostiguy for her valuable help when analyzing statistics and the great conversations. I also would like to thank the Departmental staff who always go above and beyond their duty, in helping graduate students navigate the intricacies of graduate school. I would like to thank Glenn Myers and Jason Monn for their help and support when I needed to work in the Meat Lab. I would like to thank Dr.

Edward Mills for giving me the opportunity to TA for his class; I learned a lot throughout the experience. I would also like to thank Mr. Martin Bucknavage for his mentorship and guidance. I also would like to thank Dr. Emily Furumoto for her friendship and the much-needed lunch meetings! I want to give a heartfelt thank you to all the undergrad research assistants that have in some way or another contributed to my work. Especially I would like to thank Rebecca Hovingh,

Sam Watson, Christine Kosciewicz, Emily Cutter, and Thomas Pastor. Without your hard work cleaning, making media and contributing to work done in the laboratory, it would not have been

ix possible to complete my project. I am so very grateful to the best lab mates a girl could ask for--

Dr. Joshua Sheinberg, Dr. Robson Machado, Mr. Nelson Gaydos, Ms. Samantha McKinney and Dr.

Siroj Pokharel. Particularly, I want to thank Josh and Rob--you guys were unfailingly supportive and helpful and I am truly grateful for your friendship and all the great memories.

I would also like to thank my friends who have been so supportive, helpful and inspiring all throughout my time at Penn State. First I want to thank those of you, who even from afar, kept me in your prayers and thoughts. Thank you Ashlyn Sanchez, Dr. Lurdes G. Siberio-Perez,

Nancy Ortiz, Nydia Muñoz, Aimee Montero-Arce, Jessica Nieves, Henry Velazquez and so many others--it would take another dissertation to name you all. I am particularly grateful to Lurdes-- our long distance decompression chats and conversations kept me sane! I would now like to thank the friends I have made while at Penn State. Thank you Dr. Latha Murugesan, Dr. Lingzi

Xiaoli, Laura Rolon, and Laura Homich. Our days together in school may have come to an end, but the bonds of friendship remain and I take with me all the beautiful memories of our time together. I am also incredibly grateful to my brothers and sisters at Oakwood Presbyterian Church in State College. I especially want to thank the members of the Bible Small Group that I have been part of the past several years. No words can truly express the blessing you have all been to my life. I have learned so much from sharing in scripture and worship with you and I will miss our time together. I particularly want to thank the Eshelman family (Tim, Sue, Kristie, Scott, and

Bethany) who welcomed me and treated me as their own daughter. I am truly humbled by the selfless love you have shown me and I will always be in awe of the joy in which you serve others.

I would also like to thank the Houston family. Pastor Tom Houston was the first person to reach out to me when I visited Oakwood for the first time and in Spanish, no less! I will never forget

x your humble spirit and your complete devotion to the Lord--you are greatly missed dear brother.

I would also like to thank the Bonness and Bennett families--the love and concern you have always shown me has made an enormous impact on my life. I wish to also thank my brethren at

Iglesia de Dios Pentecostal en Arenales, Isabela for their constant prayers and support.

Finally but not least, I would like to thank all of my family for the love, support, and prayers that have kept me going all these years. To my parents (Angel Rivera and Noemi Reyes), thank you for you unconditional love, for believing in me and encouraging me to always go as far as I can go. I want nothing in this life but to be able to make you proud. I want to thank my loving sister Vanesa Rivera Reyes and my brother-in-law Angel O. Ramos. You have always been there when I need you, with love, wisdom, and patience. I want to thank all my uncles/aunts, who from afar, never cease in their prayers, love and support for me. I want to thank my grandmother, Pilar

Rosado. Although you can no longer recall who I am, I will never forget you were the center of our household, a pillar of strength and hope. I want to thank all of my cousins; but being Puerto

Rican, there are way too many to name you all! Thank you Eunice Perez, Abner Perez, Bethzaida

Reyes, Elienisse Reyes, Amisadai Reyes and Abinoam Reyes. I also want to thank my cousins on this side of the pond, Choño y Olga Velzquez (my second set of parents!), Nina and Anthony

Arizmendi and their children, Johnly and Imelda Velazquez and their children, and Janneidy and

Carlos Cruz. You have all been given me so much support and love. I am blessed to be a part of your family. Thank you my Lord and Savior Jesus Christ, the love of my life, the light at my feet and my daily portion.

“The Lord is near to all who call on him, to all who call on him in truth. He fulfills the desire of those who fear him; he also hears their cry and saves them” – Psalm 145:18-19.

In loving memory of Pastor Tom Houston (April 23, 1956 to May 3, 2016)

xii

Chapter 1

Literature Review

Enterohemorrhagic E. coli (EHEC)

History and Description

Escherichia coli is a Gram-negative, facultative anaerobe and rod-shaped bacterium first

described by Theodor Escherich when he isolated it from the feces of newborns in 1885 (26,

90). E. coli is considered a commensal organism of the gastrointestinal tract (GIT), colonizing

within hours after birth. However, some E. coli are pathogenic, capable of causing human

illnesses that range from diarrhea, a urinary tract infection, bloody diarrhea, hemolytic

uremic syndrome (HUS), and in some cases, death (48, 80, 82). Pathogenic E. coli have been

classified according to specific virulence factors into six diarrheagenic pathothypes:

enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli

(ETEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC) and diffusely adherent

E. coli (DAEC). (80).

Enterohemorrhagic E. coli (EHEC) are a group of highly infectious pathogens that are an

increasing cause of concern in the food industry. Cattle are asymptomatic carriers of the

bacterium and the major reservoir of EHEC. There is a group of cattle aptly named “super

shedders” that excrete a higher amount (>104 CFU/g feces) of EHEC than other animals (11).

Although they comprise a small ratio of the cattle population, they may be responsible for

most of the EHEC transmissions (44, 46). EHEC strains comprise a subgroup of Shiga-toxin

(Stx)-producing E. coli (STEC) characterized by particular serotypes which are commonly

associated with outbreaks and severe illness (53).

Humans can become infected with STEC by ingesting contaminated food and water or

undercooked, processed beef products (71). E. coli O157:H7 has a low infectious dose,

2 estimated to be between 10 and 100 cells, but infectious doses for other STEC serogroups are currently unknown (29, 145). Transmission of STEC to the human food chain can occur when intestinal tract contents, cattle feces, or debris present on the animal hide are transferred to the carcass during processing. In addition, contamination of the soil can occur when fields are fertilized with cattle manure, irrigated with water contaminated with cattle feces, or by contact with contaminated water from surface runoff (73, 84). In turn, contaminated soil can become a reservoir for these pathogens and serve as a transmission route into the human food chain by contaminating produce (135).

According to the U. S. Centers for Disease Control and Prevention (CDC), STEC cause an estimated 265,000 illnesses, more than 3,600 hospitalizations and 30 deaths each year in the

United States (126). To date, over 400 STEC serotypes have been isolated from humans and many of these have also been recovered from animals. However, among these, only a few serotypes have been associated with the majority of human disease (48, 82). In 1994, the

United States Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) declared E. coli O157:H7 an adulterant in ground beef and non-intact beef products following a multi-state outbreak linked to hamburgers (10, 149). Non-intact beef products, such as beef that has been injected with solutions, mechanically-tenderized, or beef products in which pathogens may be introduced below the surface by a comminution process, such as chopping, grinding, flaking, or mincing, have a higher risk for contamination (147). In 2012, the USDA-FSIS extended this zero-tolerance policy to include six additional STEC serogroups which are the most common STEC responsible for human illness, after O157:H7. The serogroups O26, O45, O103, O111, O121 and O145 are commonly referred to as the “big six”

and like E. coli O157:H7, can cause severe illnesses in children, the elderly or

immunocompromised people (150).

Virulence Factors and STEC Pathogenesis

Shiga Toxins Shiga toxins are part of the Ab5 family of toxins, so termed because they consist of an A subunit, responsible for disrupting host functions, and the pentameric B subunit, which targets the glycan receptors of the target cell (18, 130) . It is believed that E. coli acquired Shiga toxins from Shigella dysenteriae through bacteriophages, as it evolved from its early ancestor E. coli

O55:H7 (85). Shiga toxins are powerful cytotoxins that block protein synthesis by inactivating ribosomes. The A subunit of the toxin damages the ribosome and stops protein synthesis by removal of an adenine residue from the 28S rRNA of the 60S ribosome (104, 144). Shiga toxins are the major virulence factor of STEC, but they can possess many other virulence factors as well.

Most STEC carry several virulence factors, including multiple Stx subtypes at the same time. In order to cause severe human illness, STEC need to employ other virulence factors aside from Stx.

For instance, STEC that have been associated with HUS colonize the human GIT by inducing, attaching and effacing (A/E) lesions to the enterocyte (intestinal absorptive cells) (83, 143).

The Shiga toxin is structurally and functionally similar to Shiga toxin type 1 from S. dysenteriae. Shiga toxin 1 is virtually identical to the Shiga toxin produced by Shigella, however

Stx2 differs significantly from Stx1 (72). Shiga toxins had been described in the past using different methods and criteria which led to significant confusion regarding Stx nomenclature. In 2012,

Scheutz et al. developed a more consistent nomenclature system based on the phylogenetic sequence relatedness of the proteins. The authors chose the Stx nomenclature (without

4 numbers) for the toxin produced by Shigella spp., and Stx subtypes found in E. coli were designated as Stx1a, Stx1c, Stx1d, Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f and Stx2g (83, 127).

Epidemiological data suggest that isolates producing Stx2 are associated with more serious disease than those that produce Stx1 or both (86). It has been observed that some Stx2 subtypes are highly associated with HUS; in particular, Stx2a is associated with higher number of outbreaks and more severe disease (83, 123, 127).

Other Virulence Factors

The Locus of Enterocyte Effacement (LEE) is a necessary virulence factor in order for

EHEC/STEC to cause A/E lesions. The LEE encodes a Type III Secretion System (T3SS), a key virulence factor of enteric pathogens, which injects bacterial protein into the host cell. T3SS possess a subunit that interacts with the eukaryotic by secretion of proteins that form a translocon, a filamentous extension of the T3SS needle complex. It mediates the delivery of secreted effector proteins which then modulate cellular processes to the pathogen’s benefit

(136). The LEE pathogenicity island (PAI) also encodes numerous adhesins, which help STEC colonize the intestinal epithelium of humans to prevent removal by peristaltic flow. Intimate attachment of EHEC/STEC to host cells is facilitated by interactions between intimin and Tir

(translocated intimin receptor). Intimin (eae) is an outer membrane adhesin encoded by the eae gene and mediates intestinal tissue tropism. The intimin receptor, Tir, has the additional role of activating actin assembly and recruiting cytoskeletal proteins at the site of adhesion. This modulation of the host cell cytoskeleton results in the formation of actin pedestals and enhances bacterial host cell attachment. In addition, an intimin-Tir interaction may help prevent bacteria from detaching under flow, one of the conditions encountered in the GIT (17, 136).

Although intimin is the primary adhesin that promotes attachment of EHEC/STEC, there are other contributing factors. Several studies have also suggested a possible role for curli in facilitating bacterial attachment to vegetable leaves and produce (39, 97). In a recent study, researchers found that curli fimbriae significantly enhanced the ability of E. coli O157:H7 to attach to spinach leaves and stainless steel, by as much as five-fold (39). E. coli O157:H7 also has the ability to attach to meat and meat components using curli (33, 121). In work with extracellular matrix (ECM) proteins, researchers demonstrated that bacterial attachment was influenced by growth conditions, including temperature and pH and by the growth media utilized. For example, it was found that growth in Luria Bertani broth caused the strongest attachment of E. coli

O157:H7 EDL933 stx− to type I or II collagen. It was also demonstrated that maximum specific bacterial adhesion occurred at 25°C. However at lower temperatures (7 or 4°C), specific bacterial adhesion to type I or III collagen could still be observed. Attachment at 25°C was highest at pH 7, and once bacteria had attached at pH 7, exposing them to a lower pH did not affect adherence to collagen I or III, except when exposed to pH 5.5 (42).

History of E. coli O157:H7 as a Pathogen

The emergence of E. coli O157:H7 as a major human pathogen is considered to be relatively new, since examination into culture collections (dating from 1973 in the U.S.) around the world, revealed that only a few isolates had been deposited before 1982 (87, 113). Two major virulence factors are required for EHEC to cause human disease. First, the ability to produce Stx1 and/or Stx2 and second, the presence of LEE and its ability to form A/E lesions (87, 145). Research into the emergence of E. coli O157:H7 has revealed that it evolved from a sorbitol-fermenting precursor related to E. coli O55:H7, a pathogen associated with infant diarrhea; the organism also

6 possessed LEE. The emergence of E. coli O55:H7 occurred in four evolutionary steps. It first acquired the ability to produce Stx2, followed by a change in its somatic antigen. Second, it obtained the virulence plasmid pO157. Subsequently, the organism gained the Stx1-converting bacteriophage. The initial step was the loss of its ability to ferment sorbitol (87, 90, 113). The evolution of E. coli O157:H7 is an ongoing process; not only has it gained foreign DNA through

PAIs, plasmids, or phages, but has also lost genetic material through deletions, commonly referred to as “black holes.” In a study by Maurelli et al. (99) strains of enteroinvasive E. coli and

Shigella had large-scale DNA deletions, which included the gene for lysine decarboxylase, whose end-product, cadaverine, is an enterotoxin inhibitor (99, 100).

Animal Reservoirs

Domesticated cattle and other ruminants are the main reservoirs of E. coli O157:H7, but they are asymptomatic carriers because they lack the Stx vascular receptor, globotriaosylceramide (Gb3) (116). The primary site for STEC colonization in cattle is the mucosa of the recto-anal junction in the cow GIT, allowing the organism to spread to the environment through the shedding of feces. Three different colonization patterns have been described in cattle: transient culture positive or passive shedders; colonization of one to two months; and long duration colonization, in which the animal can shed the bacteria for 3 to 12 months or even longer (90). In addition, researchers have concluded that animal diet is another important factor in STEC colonization. In particular, it has been demonstrated that cattle fed a high grain diet shed more STEC, when compared to cattle fed a diet of hay. E. coli shedding was reduced 1000-fold within five days by switching from grain diet to a forage diet and a decreased tolerance to acid shock was also observed (36, 37). Lowe et al. (92) demonstrated that regardless of lineage type

(I or II), colony forming units (CFU) of E. coli O157:H7 were significantly higher in the feces of cattle fed a grain diet, in contrast to a hay diet (92).

Determining the prevalence of E. coli O157:H7 in various niches has been the focus of many studies, but results can vary depending on sampling technique (anal swabs versus fecal culture), sample type (fecal, animal hide, organs) sampling season (summer versus winter) or methodology used (use of enrichments) (50, 55, 58, 120). In addition, it can be further complicated by use of different detection methods (cultural versus molecular), isolation techniques (isolated colonies or DNA isolation) or target, such as detecting a particular serogroup or determining the presence of Stx1 and/or Stx2 in the sample (61, 89). One study reported a fecal prevalence of 11.5% E. coli O157:H7 in feedlot cattle, whereas another study detected E. coli O157:H7 in 4.7% and 9.5%, by fecal and recto-anal mucosal (RAMS) sampling, respectively

(35, 67). Some researchers using molecular techniques or immunomagnetic separation (IMS) have estimated the prevalence to be closer to 30% in feedlot cattle. In contrast, others have reported a fecal prevalence as low as 0.26% and 0.08%, by culture/latex agglutination and PCR respectively (61, 62, 67). Prevalence of the pathogen in beef cattle feces has been reported to be anywhere between 4% and 7% (119, 128). Conversely, prevalence of E. coli O157:H7 on cattle hides has been reported to be approximately 22% and 3.3%, in the brisket area and rump area, respectively (118). However, it has been reported that survival of E. coli O157:H7 on cattle hide is short-lived, persisting for a period of 9 days or less (12). Prevalence can also be influenced by season, with fecal samples peaking in the summer months, but prevalence on hide being highest from spring through fall. Intervention techniques at the farm or processing facility can greatly influence prevalence. In samples harvested prior to the pre-evisceration wash, prevalence was

6%, 60%, and 27%, in feces, hide and carcasses, respectively. But, prevalence post-intervention was only 1.2% on chilled carcasses (14). In a similar study, prevalence of E. coli O157:H7 in feces and on hides was 28% and 11%, respectively. Prevalence on carcasses prior to processing was found to be approximately 43%, but dramatically reduced to 1.8% in post-processing samples

(56, 62). It is believed that differences associated with hide and carcass prevalence may be attributed to the implementation of antimicrobial interventions employed by beef processors.

Environmental Reservoirs

Several studies have also recognized water and soil to be major secondary reservoirs of

STEC in the environment, where they have been shown to persist for months (27, 60, 73, 96).

Produce can become contaminated by the application of animal manure as a fertilizer, nearby farm runoff, and/or irrigation with a contaminated water source (74). E. coli O157:H7 has been show to survive for a year in manure-treated soil and over 20 months in non-composted raw manure (77). In a recent study, the type of soil management was shown to have an effect on the survival of E. coli O157:H7. Ma et al. (96) noted that E. coli O157:H7 survived longer in organically- managed soils than in conventionally-managed soils (96). Studies have shown that E. coli

O157:H7 also can survive for long periods of time in different water sources; results suggest that survival of the pathogen may be dependent on both temperature and type of water (47, 54). In one study, E. coli O157:H7 was found to survive for a week in dechlorinated tap water incubated at 15°C. Conversely, when inoculated in autoclaved stream water and incubated at 15°C, it was able to survive up to 234 days (160). Similarly, in another study, the pathogen survived weeks in filtered and autoclaved municipal water, reservoir water, and recreational lake water. The

9 greatest survival was found in filtered and autoclaved water, whereas the lowest survival was in lake water. Regardless of water source, survival was greatest in cold water (8°C), when compared to warmer water (15 or 25°C) (155).

Survival of STEC on abiotic surfaces is a major concern in the food industry because processing equipment, food contact, and non-food contact areas can serve as source of cross- contamination of the pathogen into the food (154). The formation of biofilms is particularly worrisome since they are difficult to remove once established (156). The ability to form biofilms is one of the reasons some pathogens are able to persist in the environment (154). It has been demonstrated that E. coli O157:H7 is capable of adhering to several abiotic surfaces, including stainless steel. However, attachment and transfer may be serotype- and strain-dependent, as well as surface type and substrate-dependent. It has also been shown that strains which are curli positive are better able to form biofilms than curli negative strains (39, 131, 156).

It may be that survival of STEC is serotype/strain dependent, but environmental conditions could also influence the persistence of the pathogens in the environment. For example, researchers noted that survival of various STEC serotypes varied depending on the source soil (sandy loam versus clay soil). The sandy loam soil had a neutral pH (7.01); in contrast, the clay sole had a slightly acidic pH (5.02), with data suggesting that some STEC survived longer in the alkaline soil (27, 77). Other factors affecting the survival of STEC in soils include: availability of nutrients (particularly a carbon source), temperature fluctuations, water availability, and the presence of indigenous microflora (153).

Epidemiology of E. coli O157:H7 Outbreaks

E. coli O157:H7 is the best known member of the EHEC group, in part because it was the first to be discovered, but also because is the one that is more often tied to severe illnesses in humans (113). Its discovery as the causative agent of an early 1980s, multi-state outbreak related to undercooked burgers prompted the USDA-FSIS to implement measures to reduce the risk associated with this pathogen. In the mid-1990s, USDA-FSIS ruled that E. coli O157:H7 was an adulterant in raw ground beef. In 1998, USDA-FSIS began a farm-to-fork risk assessment of the public health impact of non-O157:H7 STEC in ground beef. Based on this information, in 1999, the agency also included non-intact beef products as potential vehicles for contamination. In

2000, the USDA-Agricultural Research Service (USDA-ARS) reported that the incidence of E. coli

O157:H7 in live cattle and carcasses was 28% and 43% respectively, and higher than originally reported. In addition to the adulterant rule, USDA-FSIS implemented the Pathogen Reduction

Act-Hazard Analysis and Critical Control Point in 1996, which required federally-inspected plants to address E. coli O157:H7 as a hazard reasonably likely to occur. Establishments and/or their suppliers established critical control points to prevent contamination (147, 149).

Since the 1980s, outbreaks with E. coli O157:H7 have been linked to other foods, such as apple juice, cheeses, cookie dough and, in recent years, fresh produce (7, 60, 113). From 1998 to 2015, the Centers for Disease Control and Prevention (CDC) reported that the pathogen was responsible for 554 outbreaks, resulting in 12,250 illnesses of which 2,005 (16.4%) resulted in hospitalizations and 35 deaths (0.3%) (41). Rangel et al. (117) conducted an epidemiological investigation into outbreaks caused by E. coli O157:H7 from 1982 to 2002. They found that out of 8,598 cases, 1,493 (17%) required hospitalizations, of which 354 (4%) cases developed HUS.

Transmission routes were also investigated and 182 (52%) were foodborne related, 74 (21%) were of unknown route, 50 (14%) were from person-to-person contact, 31 (9%) were waterborne, 11 (3%) were due to animal contact, and 1 (0.3%) was laboratory-related.

Furthermore, of the foodborne outbreaks, ground beef accounted for 75 cases (41%), while 38

(21%) were linked to produce (117).

In a study of 90 outbreaks occurring between 1982 and 2006 and encompassing the U. S.

, Canada, Japan and several European countries, 42.2% were related to food products of which

12% were linked to dairy products, 8% to animal contact, 7% to water sources and 30% were of unknown source (133). Sodha et al. conducted a study to determine national patterns of E. coli

O157:H7 infections from 1996 to 2011. They determined that the annual isolation rate was 0.84, with northern states (1.52) having a higher isolation rate than southern states (0.43). Isolation rate from patients was highest in children aged 1-4 (3.19); seasonality played a major role, with

49% of isolates collected from July to September (134). Nevertheless, both O157:H7 and non-

O157:H7 STEC infection incidence decreased since 2000, due in great part to the implementation of the E. coli O157:H7 zero tolerance rule, as well as concerted efforts of the food industry and regulatory and public health officials (48, 68). The Healthy People 2020 initiative has a projected target of reducing E. coli O157:H7 infections to 0.6 infection cases (per 100, 000 people) by the year 2020. To date, E. coli O157:H7 infection cases have dropped from 1.2 cases (baseline reported from 2006-08) to 0.9 cases (146).

History of non-O157:H7 STEC as Pathogens

Non-O157:H7 Shiga toxin-producing E. coli (STEC)

Non-O157:H7 Shiga toxin-producing E. coli have become an increasing problem in recent years and in 2011, the USDA-FSIS expanded the 1994 E. coli O157:H7 “zero tolerance” policy to include the “big six” non-O157:H7 STEC as adulterants when present in non-intact raw beef products (150). Evidence suggests that the incidence of non-O157:H7 STEC in some locations in the U. S. is on par to that of Shigella, which is further indication of the significant disease burden associated with these pathogens (70). According to the latest CDC published data, E. coli O157:H7 cause approximately 63,153 illnesses a year, whereas the non-O157:H7 STEC are responsible for over a hundred thousand cases annually in the U.S. (126). From 1990 to 2007, there were 23 outbreaks caused by non-O157:H7 STEC in the U.S., and the “big six” serotypes accounted for

75% of the reported cases. It is likely that illnesses caused by non-O157:H7 STEC are underreported primarily because they are harder to differentiate from E. coli O157:H7 (66). Of the cases reported to FoodNet in 2011, 463 where caused by E. coli O157:H7, whereas 521 (0.97 per 100,000 people) where caused by non-O157:H7 STEC (incidence of 1.10); though hospitalizations and case-fatality rates were approximately 2-fold higher for E. coli O157:H7 than for the non-O157:H7 STEC (48). Gould et al. (65) has noted that the incidence of the “big six” in illnesses were approximately 26 (O26), 22 (O103), 19 (O111), 6 (O121), 5 (O45) and 4% (O145)

(65). Interestingly, the majority of the outbreaks associated with non-O157:H7 STEC have been linked to serogroups O26, O103 and O111 (21). Some evidence also suggests that infection with serogroups O26, O103 and O145 are more likely to result in HUS (70).

Serogroup O26

Enterohemorrhagic E. coli, belonging to serogroup O26, has been used to study the effects of Shiga toxins for over 25 years (76). In some countries, like Australia, O26 along with

O111, are the most commonly identified non-O157:H7 serogroups (48). In sporadic illnesses reported to CDC from 1983 through 2002, serogroup O26 is among the “big six” non-O157:H7

STEC isolated from infected individuals (28). In Scotland, the prevalence of O26 in cattle farms is similar to that of E. coli O157:H7, but infections of the former in humans are fewer (45). STEC

O26 has been increasingly associated with diarrheal disease, outbreaks and the development of

HUS (57, 125, 159). In 2002, a multi-state outbreak in Germany was linked to beef products from the same retailer, and through molecular subtyping, was attributed to E. coli O26:H11 (159).

A study to determine the fecal prevalence of E. coli O26 in commercial feedlot cattle found that O26 was higher by culture-based methods immunomagnetic separation (IMS) with

Dynabeads VTEC/ STEC O26 and plating on Sorbitol MacConkey agar (SMAC) as compared to the

PCR assays (22.7 versus 10.5%). However, only 7 of the 260 isolates were positive for the Stx gene and 90.1% of the isolates possessed an eaeβ gene that codes for intimin subtype β (110).

The CDC has reported on various outbreaks caused by E. coli O26 in recent years. In 2012, there was a multi-state outbreak linked to raw clover sprouts used in a fast-food chain restaurant.

This outbreak resulted in 29 cases, 24% of which needed to be hospitalized, but no HUS cases were reported. In late 2015, two multi-state outbreaks, one large outbreak followed by a smaller one, were linked to two fast-food restaurants of the same chain. However, each outbreak was caused by distinct and rare strains of E. coli O26 that were not related to each other, based on

Pulse Field Gel Electrophoresis (PFGE) patterns and Whole Genome Sequence (WGS) data (2). In

2016, a multi-state outbreak linked to flour was associated with both E. coli O26 and O121. Sixty- three people became ill, 17 were hospitalized and one case developed HUS, but no deaths were reported (5).

Serogroup O45

E. coli serogroup O45, both enterotoxigenic (ETEC) and STEC, have been isolated from animals and humans (51). There have been a few reported outbreaks with this organism in recent years. While food has been involved in some, a common trend seems to be transmission by person-to-person or by direct animal contact. In 2010, a multi-state outbreak linked to smoked game meat was attributed to E. coli O45:H2 and seven people became ill. In a 2005 outbreak, ill correctional food workers were the source and 52 people became ill. One outbreak was linked to visiting a farm and possibly petting goats, which then resulted in person-to-person transmission in a childcare facility. In another outbreak, animal-to-human transmission occurred after visiting a fair in New Hampshire (93, 98).

Serogroup O103

In recent years, due to increasing awareness of the importance of non-O157:H7 STEC, more public health laboratories are making the effort to identify these serogroups (24). E. coli

O103 has been isolated from a variety of healthy and sick animals, including, cattle, sheep and rabbits (20, 75, 129). Strains of E. coli O103, particularly Shiga toxin-producing strains, such as

O103:H2, O103:H-, O103:H18, O103:H21, and O103:H25, have been associated with bloody diarrhea, hemorrhagic colitis, and HUS cases around the world (22, 94, 140, 142). In 2000, an outbreak linked to a water-based punch served at an event was attributed to O103:H2, in which

18 people became ill and two developed HUS. In 2010, an outbreak linked to venison was also associated with E. coli O103:H2 and resulted in 29 illnesses (93).

Serogroup O111

Some non-O157:H7 serotypes are more commonly-associated with sporadic cases of outbreak-associated HUS. In a study conducted by the CDC on 940 non-O157:H7 isolates of human origin obtained from public health labs across the nation from 1983 through 2002, 21 isolates were linked to HUS. Of these isolates, serogroup O111 accounted for 48% of those cases.

Similarly, O111 is one of the serogroups that is most often associated with HUS cases in Germany and Australia (28, 70). Two O111 serotypes seem to predominate among the cases reported; the non-motile O111:H- and the motile O111:H8, yet this serogroup is isolated infrequently. For example, the first reported O111 isolate in Australia yielded only four colonies obtained from a cow with profuse watery diarrhea. Yet serogroup O111 is the predominant non-O157:H7 STEC associated with major outbreaks in Australia (21). Though this disparity could be the result of culture and isolation methods as previously noted, non-O157:H7 STEC might be underreported, due to the lack of proper isolation methods. In 2011, Japan reported a large outbreak linked to beef products consumed at barbecue restaurants and isolated O111:H8 and O157:H7 with variable Stx patterns. The isolates resulted in 181 illnesses, 34 (19%) of which developed HUS.

Among HUS patients, 21 developed acute encephalopathy and five died (158).

Serogroup O121

Enterohemorrhagic E. coli O121 has been linked to hemorrhagic colitis and HUS worldwide. In the Akita prefecture of Japan, two distinct O121:H19 strains were isolated from a

15-year-old female and a 20-year-old male patients suffering from bloody diarrhea. These closely related strains were responsible for sporadic infectious cases in 1999 (163). In 1999, follow-up routine of surveillance reports of HUS cases identified three children who developed onset of disease following time spent at a lake in Connecticut. Illnesses were positively- associated with swimming and swallowing water while at the lake (102). In 2001, a case of hemorrhagic colitis in a transplant patient was the result of an infection with EHEC O121:H19 (138). More recently, the

CDC has reported several outbreaks related to serogroup O121. In 2013, an outbreak of O121 was linked to frozen food products, resulting in 35 people becoming ill, 31 were hospitalized and two developed HUS (4). In a multi-state outbreak in 2014, 19 people became ill after consuming raw clover sprouts, 44% were hospitalized, but no HUS cases were reported (1). In the 2015-2016 outbreak linked to flour, serogroup O121 was one of the etiological agents associated with disease (5).

Serogroup O145

Shiga toxin-producing E. coli O145 is one of the “big six” serogroups most commonly associated with disease in the U.S. (28, 65). Several O145 strains including, O145:NM, O145:H–,

O145:H8, O145:H16, O145:H25, and O145:H28 have been associated with numerous illnesses, ranging from bloody diarrhea to the development of HUS (22, 25, 142). In a 1999 outbreak in a childcare facility, two people became ill; the mode of transmission was person-to-person, but the vehicle for infection could not be determined. A multi-state outbreak in 2010 was linked to shredded romaine lettuce and among 30 cases, 40% were hospitalized and three patients developed HUS (3, 93). In 2012, a multi-state outbreak of unknown source was reported in which

19 people became ill, 4 were hospitalized and one died (6).

STEC Detection Methods

To date, there are well over 400 STEC serotypes which have been isolated from human patients and over a 100 E. coli O groups have been associated with Stx production. Yet, many

STEC isolated from patients, animals, and foodstuffs remain untypeable for the O antigen, highlighting the fact that current serotyping schemes might fail to detect these strains (21, 23).

Despite the vast amount of STEC serotypes, epidemiological data suggest that only a few serotypes predominate in severe human illnesses (24, 28, 81). These predominant serotypes are mainly from the EHEC group and include the serogroups O26, O45, O103, O111, O121, O145 and

O157:H7 (28). Given that there are so many STEC strains that are yet untypeable, but which may have the potential to cause severe human illness or evolve to acquire virulence traits, it might be wise to test for important virulence traits such as stx and eae genes and not only limit identification to serotyping.

Culture Methods

Culture-dependent methods are still routinely used in many laboratories since they are usually inexpensive and do not require highly skilled personnel. These methods remain an important step in isolating and detecting bacteria and are typically used first to obtain presumptive isolates that can be further studied. Cultural methods can be used to quantitatively or qualitatively detect bacteria from many different sources (food, feces and soil samples). Some drawbacks of culture-dependent methods include lack of sensitivity, time, difficulty in growing fastidious organisms, or the inability to culture some organisms (32). Depending on the sample,

18 e.g. a cooked product where cell injury might be high, culture methods may require incubation in enrichment broths prior to plating on solid media to enhance isolation. There is a wide range of media, both liquid and solid, used in the isolation, detection, differentiation and enumeration of E. coli O157:H7, some of which can also be used to detect other non-O157:H7 STEC.

E. coli O157:H7 is unable to ferment sorbitol and this trait is used extensively to differentiate it from other E. coli that may be present in food samples. Sorbitol MacConkey agar

(SMAC) was developed for isolating E. coli O157:H7 and is considered a differential and selective media. Unfortunately, SMAC can be easily overcrowded by background flora naturally present in a food sample. The use of cefixime, which suppresses the growth of Proteus spp., and tellurite, which suppresses growth of most Gram-negative organisms (CT-SMAC), can overcome this problem. However, non-O157:H7 STEC present in the sample also may be inhibited (76). Another popular selective and differential media is Rainbow Agar O157:H7 (Biolog, Hayward, CA), which is a chromogenic agar that uses β-galactosidase and β-glucuronidase to differentiate metabolic characteristics. According to the manufacturer, it is able to detect and differentiate between

O157:H7, O26:H11, O48:H21, O111:H- and O111:H8 serotypes based on color, due to their lack of β-glucuronidase activity, when compared to non-toxigenic strains. USDA-FSIS has used this agar to differentiate other non-O157:H7 STEC as well (148). Similarly, CHROMagar™ STEC

(CHROMagar Microbiology, Paris, France) is meant to detect all STEC serotypes based on proprietary chromogenic substrates. Enrichment broths or modified media commonly employ the addition of novobiocin to inhibit background flora or other antibiotics, such as vancomycin, cefixime, and cefsulodin to inhibit Gram-positive bacteria (105, 111).

Molecular Methods

Phenotypic Assays

Cytotoxicity tests are the gold standard when testing for Stx, since they are able to determine the cytotoxic effect on mammalian cells. They are commonly performed on Vero

(African green monkey kidney) cells, although HeLa cells have been used as well, but have been shown to be less sensitive (65, 117). These cell lines have high concentrations of globotriaosylcermaides Gb3 and Gb4, known receptors of Stx in eukaryotic cells. These types of tests are an effective method for detecting Stx in cell extracts, fecal filtrates, food, and environmental samples. However, the presence of cytotoxicity in a sample could be due to other bacterial toxins. Therefore, positive samples should be confirmed for cytotoxic specificity by neutralizing with Stx1- and Stx2-specific antisera. However, this technique is not routinely used since it is labor-intensive and requires specific equipment and trained personnel familiarized with tissue culture (64, 112).

Immunological assays are another way to detect Shiga toxins and many commercial kits are available. These assays range from enzyme-linked immunosorbent assays (ELISAs), immunochromatographic lateral flow tests and reverse passive latex agglutination assays. ELISA is a technique that can quantify the concentration of an analyte, usually antibodies or antigens, present in a solution. ELISA relies on the use of antigens attached to a solid surface, typically a

96-well polystyrene plate, which are then complexed with an enzyme-linked antibody. Then, detection can be achieved by direct, indirect, or sandwich formats. The direct method uses a labeled antibody, which reacts directly with the antigen, whereas the last two use a secondary- labeled antibody which is specific to the primary antibody. Commercial products that rely on

ELISA are the Premier EHEC (Meridian Diagnostics, Cincinnati, Ohio) and the ProSpecT Shiga Toxin

E. coli Microplate Assay (Remel, Lenexa, Kansas). The Immunocard STAT! EHEC (Meridian

Diagnostics, Cincinnati, Ohio) and the Duopath Verotoxins Gold Labeled Immunosorbent Assay

(Merck, Germany) are lateral flow immunoassays which are able to detect qualitatively and differentiate between Stx1 and Stx2. These and other similar products have been used successfully to detect Stx in samples (38, 43). The RIDA® QUICK Verotoxin/O157 Combi R- biopharm AG (Darmstadt, Germany) is an immunochromatographic assay that is simple and fast

(ex. dipstick). In principle, specific antibodies are attached to red (Stx specific) and green

(O157:H7 specific) latex particles. The sample is enriched overnight in modified Tryptic Soy Broth supplemented with mitomycin at 37°C. The sample is then centrifuged and aliquots are diluted

(1:2) with the provided buffer. The test strip is then introduced and the antibody-coated, colored particles attach to the antigens present in the sample and flow via the stick membrane to specific collection bands. The development of a red and/or green band along with a blue control band denotes a positive sample (31).

DNA-Based Methods of Detection

DNA-based methods for detecting STEC virulence markers are rapid to perform and do not rely on the use of specific Stx antisera. General polymerase chain reactions (PCR) for the detection of Stx from a variety of samples have been developed and are widely-used in many laboratories. PCR relies on the amplification of specific DNA templates, which can be observed by subsequent gel electrophoresis. Over the years, several modifications to this simple technique have only increased its usefulness as a rapid way to discriminate Stx positive samples. The development of a multiplex PCR (mPCR) method targeting the wzx gene of the O-antigen cluster

21 was able to detect not only O157:H7, but all “big six” non-O157:H7 serogroups as well (52, 152).

It has been successfully used to detect the presence of STEC in beef-processing plants and ground beef products (139).

Real-time PCR (RT-PCR) is a modification of the common PCR method which eliminates the need to run a gel in order to detect the amplicon. Many variations of the method exist, but most researchers use the SYBR green and TaqMan® systems. Several commercial RT-PCR systems are available, such as the GeneDisc® (GeneDisc® Technolgies Pall Corporation, NY, USA) and BAX®

System (DuPont Nutrition and Health, Wilmington, DE, USA). They include a panel for rapid screening of Stx1, Stx2, and eae or other genes and panels that target serotype-specific genes of

O157:H7 and non-O157:H7 STEC (111, 157).

Pulse Field Gel Electrophoresis (PFGE) is considered the gold standard for genotyping, and it is used extensively in epidemiological investigations and in public health surveillance. This method involves creating DNA plugs by extracting DNA from the isolate coated with agarose. The plugs are then treated with restriction enzymes and loaded onto an agarose gel; the DNA pieces are separated by size using an electric field PFGE can separate very large fragments to generate a specific DNA fingerprint pattern by constantly changing the direction of the electric field.

Finally, the gel is stained and band patterns are observed under UV light. PFGE is primarily used to determine relatedness between isolates of the same serotype. Public health laboratories from around the world that form part of the PulseNet Organization are able to upload the PFGE profiles of their isolates, facilitating the identification of outbreaks that otherwise may go unnoticed.

PFGE can be combined with multiple locus variable number of tandem repeats analysis (MLVA), which can help differentiate rapidly evolving bacterial strains in an ongoing outbreak (8). In

22 recent years, whole genome sequencing (WGS) has surpassed PFGE as the preferred method for

DNA fingerprinting of outbreak isolates. The CDC is in the process of switching from PFGE to this more powerful tool using the commercial MiSeq™ (Illumina Inc., San Diego, USA) platform(9).

The advantage of this tool is the ability to compare millions of bases at a time, instead of being limited to 15-30 bands available from a PFGE pattern (9, 111).

Stress Response in E. coli

A crucial component for stress adaptation is RpoS or stationary phase sigma factor that is induced automatically upon entry to the stationary phase of growth. In addition, it has been suggested that SOS, which is induced by DNA damage and RpoS can be complementary systems in response to some stressors (13, 15). Moreover, it has been suggested that the RpoS regulon can be transiently induced upon DNA damage, independent of the SOS system (106). During exponential growth and favorable conditions, the concentration of RpoS is very low but increases as cells enter into stationary phase. Unfavorable conditions that come about as a result of growth during the exponential phase, such as nutrient depletion and acidification of the growth media can induce the activation of RpoS. The activation of the RpoS system can result in protection against a varied range of stressors. Consequently, RpoS-dependent genes are induced in response to these stressors. However, it has been suggested that although a core set of RpoS genes could be responsible for cross-protection, this core set might not be activated in all cases.

It appears that some genes may be activated only after facing a particular type of stress (16).

Acid Stress Response

In order to cause disease, STEC must first survive their passage through the acidic environment of the rumen or human stomach. While many organisms require large infectious doses (109 cells) to survive the acidic environment in the stomach (pH 1.5-3.5), Shiga toxin- producing E. coli have lower infectious dose (102 cells), due in part to their ability to survive and resist acid environments, since they must pass through the acidic environment of the stomach in order to cause disease (86, 108). It has been suggested that this ability to adapt to and survive acidic conditions increases the virulence of E. coli O157:H7. In a study by House et al., exposure to extreme acidic conditions (pH 3), preceded by adaptation at pH 5, enhanced the adhesion of the pathogen to epithelial cells and induction of host-cell apoptosis (69). The ability of E. coli

O157:H7 to adapt to acidic conditions relies on various mechanisms that have been well documented. E. coli can use physiological, metabolic and proton-consuming mechanisms to survive acidic conditions (79, 91). In order to survive acidic environments, bacterial cells use a combination of passive (cell components buffering capacity) and active acid resistance (AR) systems. The AR can be further divided into physiological, metabolic and proton-consuming mechanisms. Physiological responses to acid stress include changes to the outer membrane of the cell, such as decreasing membrane fluidity and permeability. Metabolic changes in response to acid stress include the activation of stress response systems, such as osmotic and heat shock systems. In addition, exposure to low pH can increase the regulation of genes involved in transport and metabolism of secondary carbon sources, whereas exposure to higher pH can accelerate proton import (79, 101).

The first acid resistance (AR1) system to be discovered in E. coli is still not well understood, but it has been shown to require the stationary phase sigma factor (σs) or RpoS, as well as the global regulatory protein CRP (59). The sigma factor-dependent oxidative system (AR1) is glucose-repressed, which led to the discovery of the amino acid dependent systems. However, entry into the stationary-phase induces σs system, regardless of media pH (40). The proton- consuming response systems are amino acid decarboxylase/antiporter dependent systems, of which there are four known to date. Briefly, these systems are the glutamic-acid dependent acid resistance (GDAR) system, the arginine-dependent AR (ADAR) system, the lysine-dependent AR

(LDAR) system and the ornithine-dependent AR (ODAR) system. Of these, the GDAR and ADAR systems provide stronger protection in extreme acidic conditions, whereas the LDAR and ODAR provide protection under mild acid conditions. The regulation of these AR systems are complex and are dependent on the specifics of the growth media, as well and the challenge media (40,

59). The existence of all these overlapping systems ensures that upon exposure to acidic stress, at least one of these systems will activate in order to protect the cell.

The real-world implications of acid resistance by STEC are evident in the several outbreaks involving acidic foods, such as apple cider (pH 3.5). A study was conducted to determine which

AR systems are important for survival in acidic foods and the bovine gastrointestinal tract.

Interestingly, mutants defective in AR2- or AR3-dependent systems survived as well as the wild- type in apple cider. However, the RpoS mutants that lacked AR1 failed to survive in the apple cider and became undetectable after 24 h. Furthermore, when the pH of the apple cider was neutralized, the RpoS mutant was able to grow (115). This study highlighted the fact that the use of the AR system will depend on the conditions the pathogen faces under acid stress. Finally,

25 once any of the AR systems are induced, they will remain active for long periods of time during cold storage (4°C) conditions (91).

Osmotic Stress Response

Exposure to low moisture environments is a challenge that pathogens must overcome in order to survive host-to-host transmission and to persist in dry environments such as soil. In such instances, bacteria activate systems, that aid in the retention or acquisition of water from their surroundings (161). This approach is achieved by obtaining or synthesizing compatible solutes such as glycine betaine, proline and trehalose (114). In addition, the proteins involved in the synthesis or transport of these solutes play an important role. In fact, ProP a broad-spectrum transporter and ProU, an ABC transporter of glycine betaine and proline, are produced in response to osmotic stress (95, 162). It has also been suggested that these solutes are responsible for conferring cross-protection to other stressors (19, 114).

The general stress response, regulated by RpoS, is also found to be activated in low moisture environments or high osmolarity (16). A study conducted with RpoS deletion mutants

(ΔrpoS), aimed to determine the importance of the RpoS regulon in surviving conditions similar to those encountered during host-to-host transmission. The parent and mutant were inoculated in sterile bovine feces adjusted to a water activity (aw) between 0.87 and 0.89. The water activity of the inoculated feces continued to decrease during incubation at 30°C from aw 0.81 (day 1) to aw <0.50 (≥3 days). The parent strain decreased 1 log10 CFU/g after 3 days of incubation and nearly

2 logs after 21 days. In contrast, the ΔrpoS strain was reduced by 4 log10 CFU/g after 3 days of incubation and was undetectable after 21 days. Results clearly demonstrated that RpoS is integral

26 to the survival of E. coli O157:H7 in desiccated feces and may be necessary for prolonged survival in desiccated environments (114). It has been shown that RpoS expression during osmotic stress is increased and that it positively regulates the expression of OtsBA (trehalose synthesis) and

ProP (proline uptake) (49). Similarly, others have observed that ΔrpoS mutants are significantly less able to tolerate osmotic and desiccation stress. When exposed to desiccating and osmotically-challenging environments, E. coli O157:H7 was found to survive for prolonged periods of time (30, 137).

Statement of the Problem

There is ample research exploring the ability of pathogenic E. coli to adapt to stressful conditions. It has also been suggested that adaptation to one type of stress may lead to cross- adaptation to other stressors (103, 132, 141). In a recent study, researchers explored the synergistic effects of combined treatments of sodium chloride (NaCl) and acetic acid (AA) on the survival of E. coli O157:H7. Results demonstrated that high concentrations of NaCl significantly increased the survival of the bacterium in solutions containing 1.5% AA (88). Recently, researchers have started to explore the survival of the other “big six” non-O157:H7 STEC, when compared to E. coli O157:H7, in artificial or meat systems (63, 78). The majority of these studies have employed lethal treatments (i.e. cooking) in combination with process interventions which can effectively control the pathogen. However, the effect of the effect of acid-adaptation and the use of sub-lethal interventions in minimally processed foods (i.e. no heat step) has not been fully explored (34, 109). The effect of acid resistance and/or acid-adaptation has been studied mainly in E. coli O157:H7 in both artificial and simulated food systems (107, 124, 151). The impact of

27 acid-resistance on the survival of non-O157:H7 STEC under simulated conditions (low pH and low water activity) associated with dry, fermented sausages is not known. Similarly, the effect of fermentation, drying and long-term vacuum packaged storage of non-O157:H7 STEC in dry, fermented sausages (DFS) has not been documented. Previous research conducted in our lab utilized E. coli O157:H7 to validate a traditional process for a fermented semi-dry sausage, but did not evaluate the survival of the “big six” non-O157:H7 under similar conditions (122).

The overarching aim of this research is to determine if differences in survival exist between control or acid-adapted E. coli O157:H7 and non-O157:H7 STEC. The information obtained can be useful for determining if any particular serogroup is a better candidate for a

“worst case scenario” used in the validation of interventions employed in sausage production.

The information from this type of research may be of interest to regulatory officials, researchers, and food industry personnel who are interested in the validation of process interventions used to control non-O157:H7 STEC in food systems.

References

1. Anonymous. 2014. Escherichia coli O121 infections linked to raw clover sprouts. https://www.cdc.gov/ecoli/2014/O121-05-14/index.html 2. Anonymous. 2016. Escherichia coli O26 infections linked to Chipotle Mexican Grill restaurants (Final Update). https://www.cdc.gov/ecoli/2015/o26-11-15/index.html 3. Anonymous. 2010. Multistate outbreak of human E. coli O145 infections linked to shredded romaine lettuce from a single processing facility (Final Update). https://www.cdc.gov/ecoli/2010/shredded-romaine-5-21-10.html 4. Anonymous. 2013. Multistate outbreak of Shiga toxin-producing Escherichia coli O121 infections linked to Farm Rich Brand frozen food products. https://www.cdc.gov/ecoli/2013/O121-03-13/index.html 5. Anonymous. 2016. Multistate outbreak of Shiga toxin-producing Escherichia coli O121 infections linked to flour (Final Update). https://www.cdc.gov/ecoli/2016/o121-06- 16/index.html 6. Anonymous. 2012. Multistate outbreak of Shiga toxin-producing Escherichia coli O145. https://www.cdc.gov/ecoli/2012/O145-06-12/index.html 7. Anonymous. 2014. Pathogenic E. coli binds to fresh vegetables. Soc. Gen. Microbiol. www.sciencedaily.com/releases/2014/04/140415203813.htm 8. Anonymous. 2016. PulseNet Methods. https://www.cdc.gov/pulsenet/pathogens/index.html 9. Anonymous. PulseNet Methods- Whole Genome Sequencing (WGS). https://www.cdc.gov/pulsenet/pathogens/wgs.html 10. Anonymous. 1993. Update: multistate utbreak of Escherichia coli O157:H7 infections from hamburgers -- Western United States, 1992-1993. Morb. Mortal. Wkly. Rep. 42:258–263. 11. Arthur, T. M., D. M. Brichta-Harhay, J. M. Bosilevac, N. Kalchayanand, S. D. Shackelford, T. L. Wheeler, and M. Koohmaraie. 2010. Super shedding of Escherichia coli O157:H7 by cattle and the impact on beef carcass contamination. Meat Sci. 86:32–37. 12. Arthur, T. M., X. Nou, N. Kalchayanand, J. M. Bosilevac, T. Wheeler, and M. Koohmaraie. 2011. Survival of Escherichia coli O157:H7 on cattle hides. Appl. Environ. Microbiol. 77:3002–3008. 13. Baharoglu, Z., and D. Mazel. 2014. SOS, the formidable strategy of bacteria against aggressions. FEMS Microbiol. Rev. 38:1126–1145. 14. Barkocy-Gallagher, G. A., T. M. Arthur, M. Rivera-Betancourt, X. Nou, S. D. Shackelford, T. L. Wheeler, and M. Koohmaraie. 2003. Seasonal prevalence of Shiga toxin-producing

Escherichia coli, including O157:H7 and non-O157 serotypes, and Salmonella in commercial beef processing plants. J. Food Prot. 66:1978–86. 15. Battesti, A., N. Majdalani, and S. Gottesman. 2011. The RpoS-Mediated General Stress Response in Escherichia coli. Annu. Rev. Microbiol. 65:189–213. 16. Battesti, A., N. Majdalani, and S. Gottesman. 2011. The RpoS-mediated general stress sesponse in Escherichia coli. Annu. Rev. Microbiol. 65:189–213. 17. Battle, S. E., M. J. Brady, S. K. Vanaja, J. M. Leong, and G. A. Hecht. 2014. Actin pedestal formation by Enterohemorrhagic Escherichia coli enhances bacterial host cell attachment and concomitant Type III translocation. Infect. Immun. 82:3713–3722. 18. Beddoe, T., A. W. Paton, J. Le Nours, J. Rossjohn, and J. C. Paton. 2010. Structure, biological functions and applications of the AB5 toxins. Trends Biochem. Sci. 35:411–418. 19. Begley, M., and C. Hill. 2015. Stress adaptation in foodborne pathogens. Annu. Rev. Food Sci. Technol. 6:191–210. 20. Bettelheim, K., M. Hornitzky, and S. Djordjevic. 2001. First bovine and ovine isolations of Shiga toxin-producing Escherichia coli O103:H2 in Australia. Aust. Vet. J. 79:289–290. 21. Bettelheim, K. A. 2007. The non-O157 Shiga-toxigenic (Verocytotoxigenic) Escherichia coli ; under-rated pathogens. Crit. Rev. Microbiol. 33:67–87. 22. Beutin, L., S. Zimmermann, and K. Gleier. 1998. Human infections with Shiga toxin- producing Escherichia coli other than serogroup O157 in Germany. Emerg. Infect. Dis. 4:635–639. 23. Beutin, L., and P. Fach. 2014. Detection of Shiga toxin-producing Escherichia coli from nonhuman sources and strain typing. Microbiol. Spectr. 2:299–319. 24. Beutin, L., G. Krause, S. Zimmermann, S. Kaulfuss, and K. Gleier. 2004. Characterization of Shiga toxin-producing Escherichia coli strains isolated from human patients in Germany over a 3-year period. J. Clin. Microbiol. 42:1099–1108. 25. Blanco, M., J. E. Blanco, A. Mora, G. Dahbi, M. P. Alonso, E. A. González, M. I. Bernárdez, and J. Blanco. 2004. Serotypes, virulence genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from cattle in Spain and identification of a new intimin variant gene (eae-xi). J. Clin. Microbiol. 42:645–651. 26. Blount, Z. D. 2015. The unexhausted potential of E. coli. Elife 4:1–12. 27. Bolton, D. J., A. Monaghan, B. Byrne, S. Fanning, T. Sweeney, and D. A. McDowell. 2011. Incidence and survival of non-O157 verocytotoxigenic Escherichia coli in soil. J. Appl. Microbiol. 111:484–490. 28. Brooks, J. T., E. G. Sowers, J. G. Wells, K. D. Greene, P. M. Griffin, R. M. Hoekstra, and N. A. Strockbine. 2005. Non‐O157 Shiga toxin–producing Escherichia coli infections in the United States, 1983–2002. J. Infect. Dis. 192:1422–1429.

29. Buchanan, R. L., and M. P. Doyle. 1997. Foodborne disease significance of Escherichia coli O157:H7 and other enterohemorrhagic E. coli. Food Technol. Mag. 51:69–76. 30. Burgess, C. M., A. Gianotti, N. Gruzdev, J. Holah, S. Knøchel, A. Lehner, E. Margas, S. S. Esser, S. Sela (Saldinger), and O. Tresse. 2016. The response of foodborne pathogens to osmotic and desiccation stresses in the food chain. Int. J. Food Microbiol. 221:37–53. 31. Burgos, Y., and L. Beuti. 2012. Evaluation of an Immuno-Chromatographic Detection System for Shiga Toxins and the E. coli O157 Antigen, p. 29–40. In E. Abuelzein (ed.), Trends in Immunolabelled and Related Techniques. InTech, Berlin. 32. Bursle, E., and J. Robson. 2016. Non-culture methods for detecting infection. Aust. Prescr. 39:171–175. 33. Cabedo, L., J. N. Sofos, G. R. Schmidt, and G. C. Smith. 1997. Attachment of Escherichia coli O157:H7 and other bacterial cells grown in two media to beef adipose and muscle tissues. J. Food Prot. 60:102–106. 34. Calicioglu, M., N. G. Faith, D. R. Buege, and J. B. Luchansky. 2002. Viability of Escherichia coli O157:H7 during manufacturing and storage of a fermented, semidry soudjouk-style sausage. J. Food Prot. 65:1541–1544. 35. Callaway, T. R., T. S. Edrington, A. D. Brabban, J. E. Keen, R. C. Anderson, M. L. Rossman, M. J. Engler, K. J. Genovese, B. L. Gwartney, J. O. Reagan, T. L. Poole, R. B. Harvey, E. M. Kutter, and D. J. Nisbet. 2006. Fecal prevalence of Escherichia coli O157, Salmonella, Listeria, and bacteriophage infecting E. coli O157:H7 in feedlot cattle in the southern plains region of the United States. Foodborne Pathog. Dis. 3:234–244. 36. Callaway, T. R., R. O. Elder, J. E. Keen, R. C. Anderson, and D. J. Nisbet. 2003. Forage feeding to reduce preharvest Escherichia coli populations in cattle, a review. J. Dairy Sci. 86:852–860. 37. Callaway, T. R., M. A. Carr, T. S. Edrington, R. C. Anderson, and D. J. Nisbet. 2009. Diet, Escherichia coli O157:H7, and cattle: a review after 10 years. Curr. Issues Mol. Biol. 11:67–79. 38. Carroll, K. C., K. Adamson, K. Korgenski, A. Croft, R. Hankemeier, J. Daly, and C. H. Park. 2003. Comparison of a commercial reversed passive latex agglutination assay to an enzyme immunoassay for the detection of Shiga toxin-producing Escherichia coli. Eur. J. Clin. Microbiol. Infect. Dis. 22:689–692. 39. Carter, M. Q., J. W. Louie, D. Feng, W. Zhong, and M. T. Brandl. 2016. Curli fimbriae are conditionally required in Escherichia coli O157:H7 for initial attachment and biofilm formation. Food Microbiol. 57:81–89. 40. Castanie-Cornet, M.-P., T. A. Penfound, D. Smith, J. F. Elliott, and J. W. Foster. 1999. Control of acid resistance in Escherichia coli. J. Bacteriol. 181:3525–3535. 41. Centers for Disease Control and Prevention (CDC). 2017. Foodborne Outbreak Online

Database (FOOD Tool). https://wwwn.cdc.gov/foodborneoutbreaks/ 42. Chagnot, C., A. Agus, S. Renier, F. Peyrin, R. Talon, T. Astruc, and M. Desvaux. 2013. In vitro colonization of the muscle extracellular matrix components by Escherichia coli O157:H7: the influence of growth medium, temperature and pH on initial adhesion and induction of biofilm formation by collagens I and III. PLoS One 8:e59386. 43. Chart, H., G. A. Willshaw, and T. Cheasty. 2001. Evaluation of a reversed passive latex agglutination test for the detection of Verocytotoxin (VT) expressed by strains of VT- producing Escherichia coli. Lett. Appl. Microbiol. 32:370–374. 44. Chase-Topping, M. E., I. J. McKendrick, M. C. Pearce, P. MacDonald, L. Matthews, J. Halliday, L. Allison, D. Fenlon, J. C. Low, G. Gunn, and M. E. J. Woolhouse. 2007. Risk factors for the presence of high-level shedders of Escherichia coli O157 on Scottish farms. J. Clin. Microbiol. 45:1594–1603. 45. Chase-Topping, M. E., T. Rosser, L. J. Allison, E. Courcier, J. Evans, I. J. McKendrick, M. C. Pearce, I. Handel, A. Caprioli, H. Karch, M. F. Hanson, K. G. J. Pollock, M. E. Locking, M. E. J. Woolhouse, L. Matthews, J. C. Low, and D. L. Gally. 2012. Pathogenic potential to humans of bovine Escherichia coli O26, Scotland. Emerg. Infect. Dis. 18:439–448. 46. Chase-Topping, M., D. Gally, C. Low, L. Matthews, and M. Woolhouse. 2008. Super- shedding and the link between human infection and livestock carriage of Escherichia coli O157. Nat. Rev. Microbiol. 6:904–912. 47. Chauret, C. 2011. Survival and control of Escherichia coli O157:H7 in foods, beverages, soil and water. Virulence 2:593–601. 48. Croxen, M. A., R. J. Law, R. Scholz, K. M. Keeney, M. Wlodarska, and B. B. Finlay. 2013. Recent Advances in Understanding Enteric Pathogenic Escherichia coli. Clin. Microbiol. Rev. 26:822–880. 49. Culham, D. E., A. Lu, M. Jishage, K. A. Krogfelt, A. Ishihama, and J. M. Wood. 2001. The osmotic stress response and virulence in pyelonephritis isolates of Escherichia coli: contributions of RpoS, ProP, ProU and other systems. Microbiology 147:1657–1670. 50. Davis, M. A., D. H. Rice, H. Sheng, D. D. Hancock, T. E. Besser, R. Cobbold, and C. J. Hovde. 2006. Comparison of cultures from rectoanal-junction mucosal swabs and feces for detection of Escherichia coli O157 in dairy heifers. Appl. Environ. Microbiol. 72:3766– 3770. 51. DebRoy, C., P. M. Fratamico, E. Roberts, M. A. Davis, and Y. Liu. 2005. Development of PCR assays targeting genes in O-antigen gene clusters for detection and identification of Escherichia coli O45 and O55 serogroups. Appl. Environ. Microbiol. 71:4919–4924. 52. DebRoy, C., E. Roberts, A. M. Valadez, E. G. Dudley, and C. N. Cutter. 2011. Detection of Shiga toxin–producing Escherichia coli O26, O45, O103, O111, O113, O121, O145, and O157 serogroups by Multiplex Polymerase Chain reaction of the wzx gene of the O- antigen gene cluster. Foodborne Pathog. Dis. 8:651–652.

53. Delannoy, S., L. Beutin, and P. Fach. 2013. Discrimination of enterohemorrhagic Escherichia coli (EHEC) from non-EHEC strains based on detection of various combinations of type III effector genes. J. Clin. Microbiol. 51:3257–3262. 54. Duffitt, A. D., R. T. Reber, A. Whipple, and C. Chauret. 2011. Gene expression during survival of Escherichia coli O157:H7 in soil and water. Int. J. Microbiol. 2011:1–12. 55. Edrington, T. S., T. R. Callaway, S. E. Ives, M. J. Engler, M. L. Looper, R. C. Anderson, and D. J. Nisbet. 2006. Seasonal shedding of Escherichia coli O157:H7 in ruminants: a new hypothesis. Foodborne Pathog. Dis. 3:413–421. 56. Elder, R. O., J. E. Keen, G. R. Siragusa, G. A. Barkocy-Gallagher, M. Koohmaraie, and W. W. Laegreid. 2000. Correlation of enterohemorrhagic Escherichia coli O157 prevalence in feces, hides, and carcasses of beef cattle during processing. Proc. Natl. Acad. Sci. U. S. A. 97:2999–3003. 57. Ethelberg, S., B. Smith, M. Torpdahl, M. Lisby, J. Boel, T. Jensen, E. M. Nielsen, and K. Mølbak. 2009. Outbreak of non-O157 Shiga toxin-producing Escherichia coli infection from consumption of beef sausage. Clin. Infect. Dis. 48:e78–e81. 58. Ferens, W. A., and C. J. Hovde. 2011. Escherichia coli O157:H7: animal reservoir and sources of human infection. Foodborne Pathog. Dis. 8:465–487. 59. Foster, J. W. 2004. Escherichia coli acid resistance: tales of an amateur acidophile. Nat. Rev. Microbiol. 2:898–907. 60. Fremaux, B., C. Prigent-Combaret, and C. Vernozy-Rozand. 2008. Long-term survival of Shiga toxin-producing Escherichia coli in cattle effluents and environment: an updated review. Vet. Microbiol. 132:1–18. 61. Galland, J. C., D. R. Hyatt, S. S. Crupper, and D. W. Acheson. 2001. Prevalence, antibiotic susceptibility, and diversity of Escherichia coli O157:H7 isolates from a longitudinal study of beef cattle feedlots. Appl. Environ. Microbiol. 67:1619–1627. 62. Gansheroff, L. J., and A. D. O’Brien. 2000. Escherichia coli O157:H7 in beef cattle presented for slaughter in the U.S.: higher prevalence rates than previously estimated. Proc. Natl. Acad. Sci. U. S. A. 97:2959–2961. 63. Glass, K. a, C. W. Kaspar, J. J. Sindelar, A. L. Milkowski, B. M. Lotz, J. Kang, N. G. Faith, E. Enache, a I. Kataoka, and C. Henry. 2012. Validation of Pepperoni process for control of Shiga toxin-producing Escherichia coli. J. Food Prot. 75:838–846. 64. Gould, L. H., C. Bopp, N. Strockbine1, R. Atkinson, V. Baselski, B. Body, R. Carey, C. Crandall, S. Hurd, R. Kaplan, M. Neill, S. Shea, P. Somsel, M. Tobin-D’Angelo, P. M. Griffin, and P. Gerner-Smidt. 2009. Recommendations for diagnosis of Shiga toxin-producing Escherichia coli infections by clinical laboratoriesMMWR - Recomendations and Reports. 65. Gould, L. H., R. K. Mody, K. L. Ong, P. Clogher, A. B. Cronquist, K. N. Garman, S. Lathrop, C. Medus, N. L. Spina, T. H. Webb, P. L. White, K. Wymore, R. E. Gierke, B. E. Mahon, and

P. M. Griffin. 2013. Increased recognition of non-O157 Shiga toxin-producing Escherichia coli infections in the United States during 2000-2010: epidemiologic features and comparison with E. coli O157 infections. Foodborne Pathog. Dis. 10:453–460. 66. Grant, M. A., C. Hedberg, R. Johnson, J. Harris, M. Jianghong, C. M. Logue, J. N. Sofos, and J. S. Dickson. 2011. The significance of non-O157 Shiga toxin-producing Escherichia coli in food. Food Prot. Trends 33–45. 67. Greenquist, M. A., J. S. Drouillard, J. M. Sargeant, B. E. Depenbusch, X. Shi, K. F. Lechtenberg, and T. G. Nagaraja. 2005. Comparison of rectoanal mucosal swab cultures and fecal cultures for determining prevalence of Escherichia coli O157:H7 in feedlot cattle. Appl. Environ. Microbiol. 71:6431–6433. 68. Hadler, J. L., P. Clogher, S. Hurd, Q. Phan, M. Mandour, K. Bemis, and R. Marcus. 2011. Ten-year trends and risk factors for non-O157 Shiga toxin-producing Escherichia coli found through Shiga toxin testing, Connecticut, 2000-2009. Clin. Infect. Dis. 53:269–276. 69. House, B., J. V. Kus, N. Prayitno, R. Mair, L. Que, F. Chingcuanco, V. Gannon, D. G. Cvitkovitch, and D. Barnett Foster. 2009. Acid-stress-induced changes in enterohaemorrhagic Escherichia coli O157 : H7 virulence. Microbiology 155:2907–2918. 70. Hughes, J. M., M. E. Wilson, K. E. Johnson, C. M. Thorpe, and C. L. Sears. 2006. The emerging clinical importance of non-O157 Shiga toxin-producing Escherichia coli. Clin. Infect. Dis. 43:1587–1595. 71. Hunt, J. M. 2010. Shiga toxin–producing Escherichia coli (STEC). Clin. Lab. Med. 30:21–45. 72. Hurley, B. P., M. Jacewicz, C. M. Thorpe, L. L. Lincicome, A. J. King, G. T. Keusch, and D. W. Acheson. 1999. Shiga toxins 1 and 2 translocate differently across polarized intestinal epithelial cells. Infect. Immun. 67:6670–6677. 73. Ibekwe, A. M., J. Ma, D. E. Crowley, C.-H. Yang, A. M. Johnson, T. C. Petrossian, and P. Y. Lum. 2014. Topological data analysis of Escherichia coli O157:H7 and non-O157 survival in soils. Front. Cell. Infect. Microbiol. 4:122. 74. Islam, M., M. P. Doyle, S. C. Phatak, P. Millner, and X. Jiang. 2004. Persistence of enterohemorrhagic Escherichia coli O157:H7 in soil and on leaf lettuce and parsley grown in fields treated with contaminated manure composts or irrigation water. J. Food Prot. 67:1365–1370. 75. Jenkins, C., M. C. Pearce, A. W. Smith, H. I. Knight, D. J. Shaw, T. Cheasty, G. Foster, G. J. Gunn, G. Dougan, H. R. Smith, and G. Frankel. 2003. Detection of Escherichia coli serogroups O26, O103, O111 and O145 from bovine faeces using immunomagnetic separation and PCR/DNA probe techniques. Lett. Appl. Microbiol. 37:207–212. 76. Jenkins, C., J. Evans, H. Chart, G. A. Willshaw, and G. Frankel. 2007. Escherichia coli serogroup O26 - a new look at an old adversary. J. Appl. Microbiol. 104:14–25. 77. Jiang, X., J. Morgan, and M. P. Doyle. 2002. Fate of Escherichia coli O157:H7 in manure-

amended soil. Appl. Environ. Microbiol. 68:2605–2609. 78. Jiang, Y., J. A. Scheinberg, R. Senevirathne, and C. N. Cutter. 2015. The efficacy of short and repeated high-pressure processing treatments on the reduction of non-O157:H7 shiga-toxin producing Escherichia coli in ground beef patties. Meat Sci. 102:22–26. 79. Kanjee, U., and W. A. Houry. 2013. Mechanisms of acid resistance in Escherichia coli. Annu. Rev. Microbiol. 67:65–81. 80. Kaper, J. B., J. P. Nataro, and H. L. T. Mobley. 2004. Pathogenic Escherichia coli. Nat. Rev. Microbiol. 2:123–140. 81. Käppeli, U., H. Hächler, N. Giezendanner, L. Beutin, and R. Stephan. 2011. Human infections with non-O157 Shiga toxin–producing Escherichia coli , Switzerland, 2000– 2009. Emerg. Infect. Dis. 17:180–185. 82. Karmali, M. A., V. Gannon, and J. M. Sargeant. 2010. Verocytotoxin-producing Escherichia coli (VTEC). Vet. Microbiol. 140:360–370. 83. Krüger, A., and P. M. A. Lucchesi. 2015. Shiga toxins and stx phages: highly diverse entities. Microbiology 161:451–462. 84. Kudva, I. T., K. Blanch, and C. J. Hovde. 1998. Analysis of Escherichia coli O157:H7 survival in ovine or bovine manure and manure slurry. Appl. Environ. Microbiol. 64:3166–3174. 85. Kyle, J. L., C. A. Cummings, C. T. Parker, B. Quiñones, P. Vatta, E. Newton, S. Huynh, M. Swimley, L. Degoricija, M. Barker, S. Fontanoz, K. Nguyen, R. Patel, R. Fang, R. Tebbs, O. Petrauskene, M. Furtado, and R. E. Mandrell. 2012. Escherichia coli serotype O55:H7 diversity supports parallel acquisition of bacteriophage at Shiga toxin phage insertion sites during evolution of the O157:H7 lineage. J. Bacteriol. 194:1885–1896. 86. Law, D. 2000. Virulence factors of Escherichia coli O157 and other Shiga toxin-producing E. coli. J. Appl. Microbiol. 88:729–745. 87. Law, D. 2000. The history and evolution of Escherichia coli O157 and other Shiga toxin- producing E. coli. World J. Microbiol. Biotechnol. 16:701–709. 88. Lee, S.-Y., and D.-H. Kang. 2016. Survival mechanism of Escherichia coli O157:H7 against combined treatment with acetic acid and sodium chloride. Food Microbiol. 55:95–104. 89. Lim, J. Y., J. Li, H. Sheng, T. E. Besser, K. Potter, and C. J. Hovde. 2007. Escherichia coli O157:H7 colonization at the rectoanal junction of long-duration culture-positive cattle. Appl. Environ. Microbiol. 73:1380–1382. 90. Lim, J. Y., J. Yoon, and C. J. Hovde. 2010. A brief overview of Escherichia coli O157:H7 and its plasmid O157. J. Microbiol. Biotechnol. 20:5–14. 91. Lin, J., M. Smith, K. Chapin, H. Baik, G. Bennett, and J. Foster. 1996. Mechanisms of acid resistance in enterohemorrhagic Escherichia coli. Appl. Envir. Microbiol. 62:3094–3100. 92. Lowe, R. M. S., K. Munns, L. B. Selinger, L. Kremenik, D. Baines, T. A. McAllister, and R. 35

Sharma. 2010. Factors influencing the persistence of Escherichia coli O157:H7 lineages in feces from cattle fed grain versus grass hay diets. Can. J. Microbiol. 56:667–675. 93. Luna-Gierke, R. E., P. M. Griffin, L. H. Gould, K. Herman, C. A. Bopp, N. Strockbine, and R. K. Mody. 2014. Outbreaks of non-O157 Shiga toxin-producing Escherichia coli infection: USA. Epidemiol. Infect. 142:2270–2280. 94. Luzzi, I., A. E. Tozzi, G. Rizzoni, A. Niccolini, I. Benedetti, G. Minelli, and A. Caprioli. 1995. Detection of serum antibodies to the lipopolysaccharide of Escherichia coli 0103 in patients with Hemolytic- Uremic Syndrome. J. lnfectious Dis. 171:514–515. 95. Ly, A., J. Henderson, A. Lu, D. E. Culham, and J. M. Wood. 2004. Osmoregulatory systems of Escherichia coli: identification of betaine-carnitine-choline transporter family member BetU and distributions of betU and trkG among pathogenic and nonpathogenic isolates. J. Bacteriol. 186:296–306. 96. Ma, J., A. Mark Ibekwe, D. E. Crowley, and C. H. Yang. 2014. Persistence of Escherichia coli O157 and non-O157 strains in agricultural soils. Sci. Total Environ. 490. 97. Macarisin, D., J. Patel, G. Bauchan, J. A. Giron, and V. K. Sharma. 2012. Role of curli and cellulose expression in adherence of Escherichia coli O157:H7 to spinach leaves. Foodborne Pathog. Dis. 9:160–167. 98. Mathusa, E. C., Y. Chen, E. Enache, and L. Hontz. 2010. Non-O157 shiga toxin-producing Escherichia coli in foods. J. Food Prot. 73:1721–1736. 99. Maurelli, A. T., R. E. Fernández, C. A. Bloch, C. K. Rode, and A. Fasano. 1998. Black holes and bacterial pathogenicity: a large genomic deletion that enhances the virulence of Shigella spp. and enteroinvasive Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 95:3943– 3948. 100. Maurelli, A. T. 2007. Black holes, antivirulence genes, and gene inactivation in the evolution of bacterial pathogens. FEMS Microbiol. Lett. 267:1–8. 101. Maurer, L. M., E. Yohannes, S. S. Bondurant, M. Radmacher, and J. L. Slonczewski. 2005. pH regulates genes for flagellar motility, catabolism, and oxidative stress in Escherichia coli K-12. J. Bacteriol. 187:304–319. 102. McCarthy, T. A., N. L. Barrett, J. L. Hadler, B. Salsbury, R. T. Howard, D. W. Dingman, C. D. Brinkman, W. F. Bibb, and M. L. Cartter. 2001. Hemolytic-Uremic Syndrome and Escherichia coli O121 at a lake in Connecticut, 1999. Pediatrics 108:1–7. 103. Mei, G.-Y., J. Tang, C. Carey, S. Bach, and M. Kostrzynska. 2015. The effect of oxidative stress on gene expression of Shiga toxin-producing Escherichia coli (STEC) O157:H7 and non-O157 serotypes. Int. J. Food Microbiol. 215:7–15. 104. Melton-Celsa, A. R. 2014. Shiga Toxin (Stx) Classification, Structure, and Function. Microbiol. Spectr. 2. 105. Meng, J., P. M. Fratamico, and P. Feng. 2015. 34. Pathogenic Escherichia coli, p. 411–424.

In Y. Salfinger, and M. Lou Tortorello (eds.), Compendium of Methods for the Microbiological Examination of Foods, 5th ed. American Public Health Association. 106. Merrikh, H., A. E. Ferrazzoli, A. Bougdour, A. Olivier-Mason, and S. T. Lovett. 2009. A DNA damage response in Escherichia coli involving the alternative sigma factor, RpoS. Proc. Natl. Acad. Sci. 106:611–616. 107. Montet, M. P., S. Christieans, D. Thevenot, V. Coppet, S. Ganet, M. L. D. Muller, L. Duniere, S. Miszczycha, and C. Vernozy-Rozand. 2009. Fate of acid-resistant and non-acid resistant shiga toxin-producing Escherichia coli strains in experimentally contaminated French fermented raw meat sausages. Int. J. Food Microbiol. 129:264–270. 108. Nguyen, Y., and V. Sperandio. 2012. Enterohemorrhagic E. coli (EHEC) pathogenesis. Front. Cell. Infect. Microbiol. 2:90. 109. Omer, M. K., O. Alvseike, A. Holck, L. Axelsson, M. Prieto, E. Skjerve, and E. Heir. 2010. Application of high pressure processing to reduce verotoxigenic E. coli in two types of dry-fermented sausage. Meat Sci. 86:1005–1009. 110. Paddock, Z. D., D. G. Renter, C. A. Cull, X. Shi, J. Bai, and T. G. Nagaraja. 2014. Escherichia coli O26 in feedlot cattle: fecal prevalence, isolation, characterization, and effects of an E. coli O157 vaccine and a direct-fed microbial. Foodborne Pathog. Dis. 11:186–193. 111. Parsons, B. D., N. Zelyas, B. M. Berenger, and L. Chui. 2016. Detection, characterization, and typing of Shiga toxin-producing Escherichia coli. Front. Microbiol. 7:478. 112. Paton, J. C., and A. W. Paton. 1998. Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli infections. Clin. Microbiol. Rev. 11:450–479. 113. Pennington, H. 2010. Escherichia coli O157. Lancet 376:1428–1435. 114. Pratt, Z., M. Shiroda, A. J. Stasic, J. Lensmire, and C. W. Kaspar. 2016. Osmotic and desiccation tolerance in Escherichia coli O157:H7 and Salmonella Enterica requires RpoS (σ 38 ), p. 716–724. In Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria. Hoboken, NJ, USA. 115. Price, S. B., J. C. Wright, F. J. DeGraves, M.-P. Castanie-Cornet, and J. W. Foster. 2004. Acid resistance systems required for survival of Escherichia coli O157:H7 in the bovine gastrointestinal tract and in apple cider are different. Appl. Environ. Microbiol. 70:4792– 4799. 116. Pruimboom-Brees, I. M., T. W. Morgan, M. R. Ackermann, E. D. Nystrom, J. E. Samuel, N. A. Cornick, and H. W. Moon. 2000. Cattle lack vascular receptors for Escherichia coli O157:H7 Shiga toxins. Proc. Natl. Acad. Sci. U. S. A. 97:10325–10329. 117. Rangel, J. M., P. H. Sparling, C. Crowe, P. M. Griffin, and D. L. Swerdlow. 2005. Epidemiology of Escherichia coli O157:H7 outbreaks, United States, 1982–2002. Emerg. Infect. Dis. 11:603–609. 118. Reid, C.-A., A. Small, S. . Avery, and S. Buncic. 2002. Presence of food-borne pathogens

on cattle hides. Food Control 13:411–415. 119. Reinstein, S., J. T. Fox, X. Shi, and T. G. Nagaraja. 2007. Prevalence of Escherichia coli O157:H7 in gallbladders of beef cattle. Appl. Environ. Microbiol. 73:1002–1004. 120. Rice, D. H., H. Q. Sheng, S. A. Wynia, and C. J. Hovde. 2003. Rectoanal mucosal swab culture is more sensitive than fecal culture and distinguishes Escherichia coli O157:H7- colonized cattle and those transiently shedding the same organism. J. Clin. Microbiol. 41:4924–9. 121. Rivas, L., G. A. Dykes, and N. Fegan. 2006. Attachment of Shiga toxigenic Escherichia coli to beef muscle and adipose tissue. J. Food Prot. 69:999–1006. 122. Rivera-Reyes, M., J. A. Campbell, and C. N. Cutter. 2017. Pathogen reductions associated with traditional processing of landjäger. Food Control 73:768–774. 123. Russo, L. M., A. R. Melton-Celsa, M. J. Smith, and A. D. O’Brien. 2014. Comparisons of native Shiga toxins (Stxs) type 1 and 2 with chimeric toxins indicate that the source of the binding subunit dictates degree of toxicity. PLoS One 9:e93463. 124. Samelis, J., P. Kendall, G. C. Smith, and J. N. Sofos. 2004. Acid tolerance of acid-adapted and nonadapted Escherichia coli O157:H7 following habituation (10 degrees C) in fresh beef decontamination runoff fluids of different pH values. J. Food Prot. 67:638–645. 125. Sayers, G., T. Mccarthy, M. O’connell, M. O’leary, D. O’brien, M. Cafferkey, and E. Mcnamara. 2006. Haemolytic uraemic syndrome associated with interfamilial spread of E. coli O26:H11. Epidemiol. Infect. 134:724. 126. Scallan, E., R. M. Hoekstra, F. J. Angulo, R. V. Tauxe, M. A. Widdowson, S. L. Roy, J. L. Jones, and P. M. Griffin. 2011. Foodborne illness acquired in the United States-Major pathogens. Emerg. Infect. Dis. 17. 127. Scheutz, F., L. D. Teel, L. Beutin, D. Piérard, G. Buvens, H. Karch, A. Mellmann, A. Caprioli, R. Tozzoli, S. Morabito, N. A. Strockbine, A. R. Melton-Celsa, M. Sanchez, S. Persson, and A. D. O’Brien. 2012. Multicenter evaluation of a sequence-based protocol for subtyping Shiga toxins and standardizing Stx nomenclature. J. Clin. Microbiol. American Society for Microbiology (ASM) 50:2951–2963. 128. Schurman, R. D., H. Hariharan, S. B. Heaney, and K. Rahn. 2000. Prevalence and characteristics of shiga toxin-producing Escherichia coli in beef cattle slaughtered on Prince Edward Island. J. Food Prot. 63:1583–6. 129. Sekse, C., K. O’Sullivan, P. E. Granum, L. M. Rørvik, Y. Wasteson, and H. J. Jørgensen. 2009. An outbreak of Escherichia coli O103:H25 - bacteriological investigations and genotyping of isolates from food. Int. J. Food Microbiol. 133:259–264. 130. Shen, J., L. Rump, W. Ju, J. Shao, S. Zhao, E. Brown, and J. Meng. 2015. Virulence characterization of non-O157 Shiga toxin-producing Escherichia coli isolates from food, humans and animals. Food Microbiol. 50:20–27.

131. Simpson Beauchamp, C., D. Dourou, I. Geornaras, Y. Yoon, J. A. Scanga, K. E. Belk, G. C. Smith, G.-J. E. Nychas, and J. N. Sofos. 2012. Transfer, attachment, and formation of biofilms by Escherichia coli O157:H7 on meat-contact surface materials. J. Food Sci. 77:M343–M347. 132. Smith, J. L., P. M. Fratamico, and N. W. Gunther. 2014. Shiga Toxin-Producing Escherichia coli, p. 145–197. In Advances in Applied MicrobiologyFirst. San Diego, CA. 133. Snedeker, K. G., D. J. Shaw, M. E. Locking, and R. J. Prescott. 2009. Primary and secondary cases in Escherichia coli O157 outbreaks: a statistical analysis. BMC Infect. Dis. 9:144– 154. 134. Sodha, S. V., K. Heiman, L. H. Gould, R. Bishop, M. Iwamoto, D. L. Swerdlow, and P. M. Griffin. 2015. National patterns of Escherichia coli O157 infections, USA, 1996–2011. Epidemiol. Infect. 143:267–273. 135. Solomon, E. B., S. Yaron, and K. R. Matthews. 2002. Transmission of Escherichia coli O157:H7 from contaminated manure and irrigation water to lettuce plant tissue and its subsequent internalization. Appl. Environ. Microbiol. 68:397–400. 136. Sperandio, V., and C. J. Hovde. 2015. Enterohemorrhagic Escherichia coli and other shiga toxin-producing E. coli. American Society of Microbiology, Washington, DC. 137. Stasic, A. J., A. C. L. Wong, and C. W. Kaspar. 2012. Osmotic and desiccation tolerance in Escherichia coli O157:H7 requires RpoS (σ38). Curr. Microbiol. 65:660–665. 138. Stock, K. J. K. J., J. S. S. Dummer, M. A. M. A. Scott, S. F. S. F. Davis, R. N. R. N. Pierson, and J. S. S. Dummer. 2001. Hemorrhagic colitis due to a novel Escherichia coli serotype (O121:H19) in a transplant patient. Transpl. Int. 14:44–47. 139. Svoboda, A. L., E. G. Dudley, C. DebRoy, E. W. Mills, and C. N. Cutter. 2013. Presence of Shiga toxin–producing Escherichia coli O-groups in small and very-small beef-processing plants and resulting ground beef detected by a Multiplex Polymerase Chain Reaction assay. Foodborne Pathog. Dis. 10:789–795. 140. Tarr, P. I., L. S. Fouser, A. E. Stapleton, R. A. Wilson, H. H. Kim, J. C. Vary, and C. R. Clausen. 1996. Hemolytic–Uremic Syndrome in a six-year-old girl after a Urinary Tract Infection with Shiga-toxin–producing Escherichia coli O103:H2. N. Engl. J. Med. 335:635– 638. 141. Topalcengiz, Z., and M. D. Danyluk. 2017. Thermal inactivation responses of acid adapted and non-adapted stationary phase Shiga toxin-producing Escherichia coli (STEC), Salmonella spp. and Listeria monocytogenes in orange juice. Food Control 72:73–82. 142. Tozzi, A. E., A. Caprioli, F. Minelli, A. Gianviti, L. De Petris, A. Edefonti, G. Montini, A. Ferretti, T. De Palo, M. Gaido, and G. Rizzoni. 2003. Shiga toxin–producing Escherichia coli infections associated with Hemolytic Uremic Syndrome, Italy, 1988–2000. Emerg. Infect. Dis. 9:106–108.

143. Tozzoli, R., L. Grande, V. Michelacci, P. Ranieri, A. Maugliani, A. Caprioli, and S. Morabito. 2014. Shiga toxin-converting phages and the emergence of new pathogenic Escherichia coli: a world in motion. Front. Cell. Infect. Microbiol. 4:80. 144. Tumer, N. E., and X.-P. Li. 2012. Interaction of ricin and Shiga toxins with ribosomes. Curr. Top. Microbiol. Immunol. 357:1–18. 145. U.S. Food and Drug Administration. 2012. Bad bug book: handbook of foodborne pathogenic microorganisms and natural toxins, p. 69–81. In Keith A. Lampel, S. Al-Khaldi, and S.M. Cahill (eds.), Bad bug book: Handbook of Foodborne Pathogenic Microorganisms and Natural Toxins, 2nd ed. 146. U.S. Food and Drug Administration. 2010. Healthy People Initiative. 147. United States Department of Agriculture - Food Safety and Inspection Service. 1999. FSIS policy: non-intact raw beef products and E. coli O157:H7. Backgrounder. 148. United States Department of Agriculture - Food Safety and Inspection Service. 2012. Morphologies of representative strains from six non-O157 Shiga toxin- producing Escherichia coli (STEC) grown on modified Rainbow Agar, p. 1–7. In Microbiological Laboratory Guidebook. 149. United States Department of Agriculture - Food Safety and Inspection Service. 2002. New measures to address E. coli O157:H7 contamination. Backgrounder - Congr. Public Aff. 150. United States Department of Agriculture - Food Safety and Inspection Service. 2012. USDA Targeting six additional strains of E. coli in raw beef trim starting Monday. News Release - Congr. Public Aff. 151. Usaga, J., R. W. Worobo, and O. I. Padilla-Zakour. 2014. Effect of acid adaptation and acid shock on thermal tolerance and survival of Escherichia coli O157:H7 and O111 in apple juice. J. Food Prot. 77:1656–1663. 152. Valadez, A. M., C. Debroy, E. Dudley, and C. N. Cutter. 2011. Multiplex PCR detection of Shiga toxin–producing Escherichia coli strains belonging to serogroups O157, O103, O91, O113, O145, O111, and O26 on experimentally inoculated in beef carcass swabs, beef trim, and ground beef. J. Food Prot. 74:228–239. 153. van Elsas, J. D., A. V Semenov, R. Costa, and J. T. Trevors. 2011. Survival of Escherichia coli in the environment: fundamental and public health aspects. ISME J. 5:173–183. 154. Vogeleer, P., Y. D. N. Tremblay, A. A. Mafu, M. Jacques, and J. Harel. 2014. Life on the outside: role of biofilms in environmental persistence of Shiga-toxin producing Escherichia coli. Front. Microbiol. 5:1–12. 155. Wang, G., and M. P. Doyle. 1998. Survival of enterohemorrhagic Escherichia coli O157:H7 in water. J. Food Prot. 61:662–667. 156. Wang, R., J. L. Bono, N. Kalchayanand, S. Shackelford, and D. M. Harhay. 2012. Biofilm formation by Shiga toxin–producing Escherichia coli O157:H7 and non-O157 strains and

their tolerance to sanitizers commonly used in the food processing environment. J. Food Prot. 75:1418–1428. 157. Wasilenko, J. L., P. M. Fratamico, C. Sommers, D. R. DeMarco, S. Varkey, K. Rhoden, and G. Tice. 2014. Detection of Shiga toxin-producing Escherichia coli (STEC) O157:H7, O26, O45, O103, O111, O121, and O145, and Salmonella in retail raw ground beef using the DuPont BAX® system. Front. Cell. Infect. Microbiol. 4:1–7. 158. Watahiki, M., J. Isobe, K. Kimata, T. Shima, J. Kanatani, M. Shimizu, A. Nagata, K. Kawakami, M. Yamada, H. Izumiya, S. Iyoda, T. Morita-Ishihara, J. Mitobe, J. Terajima, M. Ohnishi, and T. Sata. 2014. Characterization of enterohemorrhagic Escherichia coli O111 and O157 strains isolated from outbreak patients in Japan. J. Clin. Microbiol. 52:2757– 2763. 159. Werber, D., A. Fruth, A. Liesegang, M. Littmann, U. Buchholz, R. Prager, H. Karch, T. Breuer, H. Tschäpe, and A. Ammon. 2002. A multistate outbreak of Shiga toxin– producing Escherichia coli O26:H11 infections in Germany, detected by molecular subtyping surveillance. J. Infect. Dis. 186:419–422. 160. Wojcicka, L., R. Hofmann, C. Baxter, R. C. Andrews, I. Auvray, J. Lière, T. Miller, C. Chauret, and H. Baribeau. 2007. Inactivation of environmental and reference strains of heterotrophic bacteria and Escherichia coli O157:H7 by free chlorine and monochloramine. J. Water Supply Res. Technol. - Aqua 56. 161. Wood, J. M. 2007. Bacterial Osmosensing Transporters, p. 77–107. In J.N. Abelson, M.I. Simon, S.P. Colowick, and N.O. Kaplan (eds.), Methods in Enzymology. Elsevier Masson SAS, Ontario, CA. 162. Wood, J. M., E. Bremer, L. N. Csonka, R. Kraemer, B. Poolman, T. van der Heide, and L. T. Smith. 2001. Osmosensing and osmoregulatory compatible solute accumulation by bacteria. Comp. Biochem. Physiol. Part A Mol. Integr. Physiol. 130:437–460. 163. Yatsuyanagi, J., Y. Kinouchi, S. Saito, Y. Suzuki, H. Sato, and Y. Miyajima. 1999. Epidemiological characteristics and virulence factor of Verotoxin-producing Eschericia coli O121:H19 isolated in Akita Prefecture in July 1997. Kansenshogaku Zasshi. 73:218– 224.

Chapter 2

Survival of Acid-Adapted and Non-Adapted Shiga toxin-producing E. coli Using an in vitro Model

Abstract

One important characteristic of Shiga toxin-producing Escherichia coli (STEC) is the ability to adapt and survive acidic conditions, such as the gastrointestinal tract of mammals or acidified food products. It has been reported that acid-adaptation may also result in cross-protection against other stressors present in foods, such as lowered water activity (aw), refrigerated conditions, or antimicrobials. Limited research has been conducted on acid-adaptation of non-

O157:H7 STEC in meat systems, because it is believed that interventions needed to control E. coli

O157:H7 should also control non-O157:H7 STEC. In the present study, three separate experiments were conducted with non-adapted (control) and acid-adapted E. coli O157:H7 and non-O157:H7 serogroups (E. coli O26, O45, O103, O111, O121 and O145) in vitro and in situ. For the first experiment, STEC cocktails were left untreated (non-adapted; control) or adapted (pH

5.0), and then exposed to mild acidic conditions (pH 4.5) and a reduced water activity (aw 0.88 and aw 0.75) environment. Pathogen cocktails were then stored for 4 days at 24°C in tryptic soy broth (TSB) and evaluated for survival. In a second experiment, non-adapted and acid-adapted

STEC cocktails were subjected to desiccation (28 days at 24°C) on sterile paper disks, and remaining populations of the pathogens were determined. Finally, control and acid-adapted STEC were subjected to mild acidic conditions (pH 4.5) and low water activity (0.88 or 0.78) in ground beef slurries (GBS) (4 days at 24°C), and surviving populations were enumerated. Results from the first experiment showed that populations of the control non-O157:H7 STEC serogroups O45,

O103, O111 and O121 were significantly different from populations of E. coli O157:H7,and non-

O157:H7 STEC serogroups O26 and O145. In contrast, when STEC serogroups were acid-adapted, all populations of non-O157 STEC were significantly different from populations of E. coli O157:H7.

There were no significant differences between control (non-adapted) and acid-adapted cells between any individual serogroup. Rates of reduction for all serogroups demonstrate that they not only survived well in acidified TSB, but also were able to grow minimally over time. Results from TSB with modified water activity (aw=0.88 or 0.78) demonstrate that there was no significant difference between E. coli O157:H7 and any of the non-O157:H7 STEC, regardless of acid- adaptation. In a second experiment, exposure to desiccation on dry discs, there was no significant difference in survival between E. coli O157:H7 and the non-O157:H7 STEC, regardless of acid- adaptation. Results also suggest that acid-adaptation had no significant effect on the survival of any of the serogroups. Results from acidified GBS indicated that all non-O157:H7 STEC behaved similarly to E. coli O157:H7, with the exception of O145 STEC which had a higher reduction than

O157:H7. There were no significant differences in ground beef slurries with modified water activity.

These results suggest than non-O157:H7 STEC behave similarly to E. coli O157:H7 when exposed to laboratory stress conditions of low pH (pH 4.5) and low water activity (0.88 or 0.78).

These results are in agreement with other researchers; interventions that work on O157:H7 are likely to work on non-O157:H7 STEC. The information obtained in this study may be of interest to regulatory officials, researchers, or the food industry that conduct process control validations or challenge studies with E. coli O157:H7 and non-O157:H7 STEC.

Introduction

Shiga toxin-producing Escherichia coli (STEC) have been recognized as important human foodborne pathogens since 1982 (14). STEC have been associated with a number of gastrointestinal disease symptoms, including abdominal pain, vomiting, diarrhea, hemorrhagic colitis, as well as more severe sequelae, such as hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) (9, 28). Shiga toxins (Stx1 and/or Stx2) are the major virulence factors of STEC and the more virulent strains harbor Stx2 and intimin genes (32).

According to the U. S. Department of Agriculture, Economic Research Service, the economic burden of STEC-associated illnesses is estimated to be approximately $15.5 billion dollars (2013 dollars), whereas others have estimated the cost to be around $280 million (13, 16, 17, 46).

STEC have also been isolated from a variety of food and livestock. However, cattle and other ruminants remain the major reservoir for these pathogens (10, 20). Cattle are normally asymptomatic carriers of STEC and shed the organism in their feces. Raw food products can become contaminated by direct contact with fecal material (e.g. during carcass evisceration) or indirectly by contact with contaminated water from processing plants or manure application (e.g. soil and produce) (10). Infections typically occur after consumption of a contaminated food product. The most common foods associated with STEC are of bovine origin. However, in more recent years, many infections have been linked to produce, dairy products, and other foodstuffs

(48).

The safety of many food products relies heavily on the use of hurdle technology; that is, using more than one method or intervention to control pathogen growth by exposure to treatments in a sequential manner (e.g. fermentation and ripening of sausages, drying, etc.) or

45 simultaneously (24). While some techniques rely on sub-lethal treatments to kill or inhibit the pathogens, no single technique can be used to ensure product safety, apart from heating. The sequence of these hurdles has also been shown to play a role in pathogen survival. In a study by

Shadbolt et al. (13), researchers investigated the effects of reduced pH and low water activity

(aw), followed by exposure to a second lethal stress (SLS). The researchers found that E. coli M23 cells exposed first to medium-low water activity (aw 0.90), resulted in a more resistant subpopulation that was able to persist for 50 h after exposure to the SLS (pH 3.5). In contrast, cells that were first exposed to pH 3.50 were inactivated rapidly to undetectable levels upon exposure to the SLS (aw 0.90). The researchers concluded that a lethal pH imposed a higher energetic drain to the bacteria, which sensitized cells to future stressful conditions (41). The aforementioned study highlighted the effect of subsequent lethal stress on pathogen survival.

E. coli prefer neutral pH (6.5 – 7.5) environments for optimum growth. However, this organism has been shown to adapt to acidic conditions upon entry to stationary phase or prior to exposure to a sub-lethal acidic environment (12, 21). In turn, it has been suggested that stress- adapted bacteria could become more resistant to subsequent stressors (24, 39). This cross- protection is particularly worrisome in the case of foods that rely on sub-lethal sequential hurdles to ensure product safety. Moreover, pathogens present in food processing environments already face many stressors, ranging from starvation, drying, and competition with commensal microflora or starter cultures, all of which could induce stress-adaptation by activation of the general stress response system RpoS (47).

Changes to aw is a hurdle commonly used to control food pathogens. The aw in foods is intrinsically related to the amount of humectants (i.e. sodium chloride or sucrose) present, which

46 bind available water, making it unavailable for use by microorganisms in their metabolic activities

(37). Recently, there have been several outbreaks of STEC linked to low moisture foods (2, 3). In a recent study, E. coli O157:H7 was inoculated onto walnut kernels and pathogen survival was monitored during storage. After inoculation, the kernels were dried and stored at 23°C for up to one year. Although, a rapid decrease (up to 4 log CFU/g) was observed during drying, E. coli

O157:H7 was able to survive long term in this product (5). In another study carried out using potato starch, survival of E. coli O157:H7 was evaluated as affected by aw, pH, and temperature.

Survival was found to be adversely affected by higher temperatures, enhanced as aw decreased, and unaffected by pH (31).

The majority of the research on STEC stress-adaptation and behavior has been done with

E. coli O157:H7 (9, 18). Some authors have suggested that E. coli O157:H7 and non-O157:H7 STEC could behave differently (27, 29, 34). Therefore, if some serogroups are able to survive better, food processes may not be effective to control these pathogens, resulting in unsafe food products. As such, there is a need to understand stress adaptation and subsequent behavior of non-O157:H7 STEC in vitro and in situ and compare them to E. coli O157:H7. The main objective of this study was to evaluate the effects of stress-adaptation of E. coli O157:H7 and non-O157:H7

STEC when exposed to subsequent stressors (acidic and low water activity; aw) in broth, on paper disks, and in a meat system.

Materials & Methods Bacterial Cultures Five strains each of Shiga toxin-producing E. coli O26, O45, O103, O111, O121, O145 and O157:H7 were obtained from the E. coli Reference Center (ECRC; Department of Veterinary and Biomedical

Sciences), the Dudley Lab, and the Cutter Lab (both in the Department of Food Science,

Pennsylvania State University) (Table 1). These isolates were selected based on serotype, the presence of toxin-producing genes (stx1 and stx2), and the presence of other virulence factors

(eae) via PCR (8). All cultures were stored at -80°C in tryptic soy broth (TSB) (Difco, Detroit, MI) supplemented with 20% glycerol (Sigma Aldrich; St. Louis, MO) to ensure viability. Frozen stock cultures were revived by transferring 0.1 ml of each thawed culture to 10 ml of fresh TSB and incubated at 37 °C for 24 h. A loopful of the overnight culture was streaked onto Tryptic Soy agar

(TSA) plates and incubated at 37°C for 24 h to obtain isolated colonies. Working stock cultures were maintained on refrigerated (4°C) TSA plates and sub-cultured in TSB at 37°C for 24 h twice before subsequent use. Each serogroup cocktail was made by combining equal amounts (5 ml) of five isolates in a sterile tube prior to use in every experiment.

Acid Adaptation Protocol Bacterial isolates were grown individually in TSB, incubated overnight at 37°C, and transferred twice prior to adapting. Acid resistance was induced by culturing bacteria in mildly acidic media (TSB, pH 5.0) and culturing cells to stationary phase at 37°C (18h). A solution of 50%

(v/v) lactic acid (pH 1.67) was prepared by diluting 88% DL-lactic acid (Birko Corp., Denver, CO) with sterile distilled water. TSB (pH 7.13, 100 ml) was acidified with the 50% lactic acid solution to the target pH (~0.5 ml = pH 5.07 ± 0.02) and sterilized (121°C, 15 psi, 15 min). Non-adapted

(control) cells were grown individually in TSB (pH 7.3) and incubated at 37°C for 18h. Non- adapted and acid-adapted isolates were combined in equal volumes respectively to prepare serogroup cocktails for use in each experiment.

Experiment 1-Challenge Media Assays Bacterial acid adaptation was performed as described by Cheng et al. (2003) with some modifications; TSB was adjusted to pH 5.0 with a sterile lactic acid solution (50%; pH 1.67) (6).

Acidic challenge media (TSB pH 4.5) was prepared following the same protocol for TSB acidification to pH 5 (above) and sterilized (121°C, 15 psi, 15 min). Low water activity challenge media (TSB+NaCl) was prepared by adjusting TSB with sodium chloride to obtain TSB with aw of

0.78 ± 0.02 and 0. 88 ± 0.02. Sterile fresh TSB was modified by adding 175.32g/L (TSB88) or

350.64g/L (TSB78). Nine ml aliquots of each media type were dispensed aseptically into sterile test tubes. Non-acid adapted and acid-adapted serogroup cocktails were inoculated into tubes containing challenge media and a 1 ml aliquot sample was taken at time intervals of 0, 24, 48, and 72h. The samples were serially diluted in 9 ml of 1X strength (0.1M) phosphate buffered saline (PBS, pH 7.5), spread plated in duplicate onto TSA, and incubated at 37°C for 24h.

Experiment 2-Desiccation Assay The method by Stasic et al. (42) was used with some modifications. Briefly, autoclaved

(121°C, 15 min) paper disks (2.1cm) (VWR® Glass Fiber Filters, US) were placed in sterile petri dishes (5 pieces/dish). Serogroup cocktails were prepared as described above. Equal amounts of each serogroup isolate were combined in a sterile tube. Cells were pelleted, washed once with a volume of sterile 1X PBS and resuspended in 0.5ml of 1X PBS (0.01M; pH=7.4). The dry paper disks were inoculated with 100 µl (ca. 7 log CFU/ml) of acid-adapted and non-adapted E. coli

O157:H7 and non-O157:H7 STEC cocktails. Petri dishes with inoculated paper disks were incubated at 24°C for a period of 28 days. The plates were not sealed to allow for the natural loss of moisture and to reach increasingly low aw as time progressed. One paper disk was removed at each sampling time and rehydrated in a sterile tube containing 5 ml of 1X PBS + 0.02 % Tween

80. Tubes were incubated at room temperature for 10 min, followed by 30 s of vigorous shaking by vortex to facilitate the release of cells into solution. Serial dilutions (1:5) were subsequently performed and 0.1 ml aliquots were spread plated on TSA and sorbitol MacConkey (SMAC; Difco) 49 plates. The paper disks were removed for enumeration on days 0, 4, 8, 14, 21, and 28. The aw was measured at each time point during the experiment (AquaLab 4TE, WA).

Experiment 3-Acidified Ground Beef Slurries The method by Abdul-Raouf et al. (1) was used with some modifications. Irradiated ground beef (80% lean/20% fat) was purchased from a local retail supermarket, transported to the lab and kept under refrigeration until use (≤ 24 h). The ground beef package was sanitized with alcohol wipes prior to opening with a sterile scalpel. Ground beef slurries were prepared by aseptically transferring 25 g of irradiated ground beef into a filtered, sterile stomacher bag

(Interscience Laboratories, Woburn, MA) and re-suspending in 100 ml of sterile 1X PBS. Acidified ground beef slurries (pH 4.5) were prepared by adding a volume of autoclave sterilized 10% (v/v) lactic acid solution. Slurries were inoculated with 10 ml of non-adapted or acid-adapted serogroup cocktails and homogenized for 30s in a stomacher (Stomacher 400 Circulator, Seward,

UK) to ensure proper distribution. Slurries were then incubated at 24°C and samples were taken at time intervals of 0, 24, 48, and 72 h. A of 10 ml sample was removed at each time interval, from which a 1 ml aliquot was serially diluted in 9 ml of 1X PBS. Samples were plated in duplicate on TSA and incubated for 24 h at 37°C.

Statistical Analysis Bacterial populations (averaged duplicates) were converted to log10 CFU/g or log10

CFU/ml to allow for analysis and transformation of data in order to meet the assumptions of the statistical test. Resulting data were analyzed using the SAS statistical program version 9.4 (SAS

Institute, Cary, NC). The general lineal model procedure with Dunnett’s test was used to analyze all non-O157:H7 STEC, using E. coli O157:H7 as the control. The data set (all serogroups) were analyzed for significant difference from O157 by dividing the data sets by type (non-acid adapted

50 or adapted). The data set was also analyzed by distinct serogroup against (non-acid adapted vs adapted) to determine if acid adaptation had an effect on survival. The analysis was done separately for control (non-adapted) and acid-adapted cells. The general lineal model with

Tukey’s test was used to analyze differences between non-adapted and acid-adapted cells for each serogroup. Data was also analyzed to determine the rate of reduction by generating the regression line for each serogroup, type and repetition. Log reduction rates (log CFU g- time) were obtained by generating a linear regression best-fit line for each serogroup and type (control or acid-adapted). Significance was declared at p=0.01.

Results and Discussion Experiment 1-Challenge Media Assays

Although, E. coli are relatively resistant to mildly acidic environments, and a lower pH could have been used to test extreme acid survival in acid-adapted cells, the pH (4.5) used in this study was chosen to represent the final pH of typical dry, fermented sausage (45). Results indicate that only non-adapted non-O157:H7 STEC serogroups O45, O103, O111 and O121 were significantly different from E. coli O157:H7. Differences between the means (Table 2) demonstrate that serogroup O157:H7 was better able to survive all treatments than the non-

O157:H7 STEC. In contrast, when STEC serogroups were acid-adapted, all non-O157 STEC were significantly different from O157:H7. Again, E. coli O157:H7 survived better than all the non-

O157:H7 STEC. This finding could be an indication that acid-adaptation likely injured these cells or that E. coli O157:H7 is better able to tolerate acid exposure. The majority of the published research has shown that acid adaptation of E. coli O157:H7 allows the pathogen to survive

51 subsequent stressors (39, 43). However, the rate of reduction (slope) for all serogroups exposed to this treatment (pH 4.5) was positive, indicating that all organisms were not only able to survive the exposure to pH 4.5, but also started to grow over time (Table 3). However, there was no significant difference in the rate of reduction between any of the non-O157:H7 serogroups. It is evident that the low pH used in this study failed to control pathogen growth for all serogroups and strains tested. It is possible that the pH level used in the present study was not low enough to observe any differences between control and acid-adapted cells. Exposure to lower pH or different acids could result in different survival patterns. For example, it has been reported that following adaptation in meat washings, acidified with either lactic acid or acetic acid, E. coli

O157:H7 from acetate washings (pH 3.7 to 4.7) survived a subsequent exposure to pH of 3.5 better than E. coli O157:H7 from lactate washings (pH 3.1 to 4.6) (39). Lactic acid was chosen for the current study since it is the main organic acid produced by starter cultures during fermentation. Pathogen survival can also be improved by the activation of different acid resistance (AR) system dependent on the type of acidic environment the cells are faced with (30).

Results from a study comparing different AR systems suggest that the type of acid employed influences cell survival exposed to different environments (cattle gastrointestinal tract versus apple cider). Furthermore, the AR1 (oxidative system; glucose repressed), which is managed by

RpoS, provides more protection in apple cider (pH 3.5) than activation of AR2 (glutamic-acid dependent) or AR3 (arginine-dependent ) (35). However, it is not likely that the AR2 or AR3 system were induced in this experiment. Even though TSB contains sufficient amounts of each amino acid, it is unlikely the internal cell pH was low enough (pH 4.0) to elicit this responses (36).

Results from TSB with a modified water activity (TSB88 or TSB78) demonstrate that there was no significant difference between E. coli O157:H7 and any of the non-O157:H7 STEC, regardless of acid-adaptation (Table 2). Moreover, there were no significant differences between control or acid-adapted serogroups. Results from the rate of reduction calculations demonstrate that all serogroups were reduced (negative slope). However, only the rate of reduction of control serogroup O121 was significantly different from its acid-adapted counterpart (Table 3). Although acid-adapted E. coli O121 had higher survival than its control counterpart, in the present study it could not be ascertained if this finding is a serogroup trait or if it its dependent on the strains used in this study.

In a recent study, high concentrations (9–15%) of sodium chloride significantly increased

E. coli O157:H7 survival in Luria-Bertani (LB) broth solutions containing 1.5% acetic acid (2.74 -

3.32), when compared to solutions without sodium chloride (23). While the synergetic effect of acid and salt were investigated previously, in the current study, cells were exposed to acid stress first and then to osmotic stress. It has been suggested that the order of hurdle implementation could affect the survival of pathogens. Shadbolt et al. (41) concluded that exposure to lethal acidic conditions (pH 3.5), followed by exposure to lethal sodium chloride (15%) was more harmful than the reverse order. However, the acidic condition used to adapt cells in the present study is considered mild (pH 5.0). Conversely, the concentrations of NaCl (15% & 20%) used in the present study were lethal, as evidenced by the rate of reduction of all serogroups (Table 3).

Experiment 2-Desiccation Assay

It has been reported that aw < 0.95 inhibits the growth of E. coli and that exposure to aw of ≤ 0.53, results in DNA damage (11, 42, 44). Limited research suggests that E. coli O157:H7 may be able to survive exposure to dry conditions during extended periods of time (4, 40). However, limited information exists with regard to the survival of the non-O157:H7 STEC under similar conditions. Research conducted on both artificial and food matrices have focused for the most part on E. coli O157:H7. In a study using a desiccation model, the survival of STEC was assessed on dry paper disks. Results demonstrated that STEC strains (O26, O111 and O157) survived with a population of 103 to 104 CFU/disk after 24 months of storage at 4°C (15). In another study, the survival of E. coli O157:H7 in potato starch powder, as affected by aw, pH, and temperature, was evaluated. Results demonstrated that survival was enhanced at lower aw (0.24), was not affected by pH, and was reduced at higher temperatures (37°C) (31). Results from the present study suggest that non-O157:H7 STEC are as equally susceptible to desiccation as E. coli O157:H7. There was no significant difference in survival between E. coli O157:H7 and the non-O157:H7 STEC, regardless of acid-adaptation (Table 2). There also was no significant difference in the rate of reduction of the non-O157:H7 STEC serogroups tested, regardless of acid adaptation (Table 3).

These results suggest that acid-adaptation had no significant effect on the survival of any of the serogroups following desiccation. In another relevant study, the effect of acid-adaptation on the viability of E. coli O157:H7 in beef powder, as affected by aw, sodium chloride content, and temperature, was evaluated (38). Interestingly, there were no significant differences in survival among the type of cells (acid-adapted or acid shocked) when subjected to all tested parameters.

The authors observed that results may suggest the mechanisms associated with acid-adaptation

54 do not cross-protect against dehydration or osmotic stress (38). Results obtained in the current study are in agreement with this observation: that prior acid-adaptation does not seem to provide an advantage over non -adapted cells when exposed to extreme, low moisture environments. In this study, there were no solutes or food components present that could either enhance or diminish the survival of the pathogens. However, more research is needed to address this issue under real-world conditions. In particular, cross adaptation experiments involving desiccation and/or acidification should be conducted in complex food systems to better understand the implications of aw, pH, and the presence of food components on the survival of these foodborne pathogens.

Experiment 3-Acidified Ground Beef Slurries

Results from experiments performed on acidified ground beef slurries (GBS) indicate that all non- adapted non-O157:H7 STEC behaved similarly to non-acid adapted E. coli O157:H7, with the exception of O145, which demonstrated the highest difference between the means (Table

2). However, this difference was not evident in acid-adapted O145, suggesting that all serogroups were equally injured, and therefore, differences were not detected. There was no significant difference between control and acid-adapted cells of any STEC serogroup. Similarly, GBS with modified water activity showed no significant differences between serogroups, regardless of prior acid adaptation. As with experiments in modified TSB (pH 4.5), there was growth over time, as evidenced by the positive rate of reduction (slope) values of acidified GBS (Table 3). Results obtained in the present study differ slightly from others. In a study by Abdul-Raouf (1), a decrease in cell population over time was observed in GBS acidified to 4.7 with lactic acid and stored at 55

21°C. In contrast, GBS acidified to 4.7 with lactic acid and stored at 30°C did not prevent pathogen growth. In the present study, GBS were acidified to 4.5 with lactic acid and stored at 24°C to mimic the temperature used to ferment sausages. Results demonstrated that this pH and temperature combination also failed to control STEC growth, regardless of prior adaptation.

In recent years, there has been increased attention to non-O157:H7 STEC. They appear to cause more illnesses than E. coli O157:H7, although the illnesses tend to be less severe (25).

Several studies have suggested that interventions that work on E. coli O157:H7 are likely to work on the “big six” non-O157:H7 STEC as well (7, 19). However, other studies have found some non-

O157:H7 STEC serogroups behave differently, and in some cases, survive better than E. coli

O157:H7 (28, 29, 33). Results from the present set of experiments suggest that the non-O157:H7

STEC behave similarly to E. coli O157:H7 when exposed to the stress conditions used in these studies, regardless of acid-adaptation. In the future, conditions that could enhance the survival of STEC in real foods should be explored. The role of different types of stressors and subsequent adaptation may need to be explored in combination to multiple hurdles. The use of salt, which has been observed to increase tolerance to acidity, as well as increased survival to hurdles, are dependent on the order in which they are applied (23, 41). In addition, the role of stx genotypes and genetic lineages and how these traits might provide an advantage to stressors encountered in real world processing conditions (dry, fermented meat systems) should be explored, since some evidence suggests that resistance might be dependent of these factors (9, 22, 26).

References 1. Abdul-Raouf, U. M., L. R. Beuchat, and M. S. Ammar. 1993. Survival and growth of Escherichia coli O157:H7 in ground, roasted beef as affected by pH, acidulants, and temperature. Appl. Environ. Microbiol. 59:2364–2368. 2. Anonymous. 2016. Multistate outbreak of Shiga toxin-producing Escherichia coli O121 infections linked to flour (Final Update). http://meathaccp.wisc.edu/validation/fermentation.html 3. Anonymous. 2017. Multistate Outbreak of Shiga toxin-producing Escherichia coli O157:H7 Infections Linked to I.M. Healthy Brand SoyNut Butter. https://www.cdc.gov/ecoli/2017/o157h7-03-17/index.html 4. Beuchat, L. R., E. Komitopoulou, H. Beckers, R. P. Betts, F. Bourdichon, S. Fanning, H. M. Joosten, and B. H. Ter Kuile. 2013. Low–Water Activity Foods: Increased Concern as Vehicles of Foodborne Pathogens. J. Food Prot. 76:150–172. 5. Blessington, T., E. J. Mitcham, and L. J. Harris. 2012. Survival of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes on inoculated walnut kernels during storage. J. Food Prot. 75:245–254. 6. Cheng, H.-Y., R.-C. Yu, and C.-C. Chou. 2003. Increased acid tolerance of Escherichia coli O157:H7 as affected by acid adaptation time and conditions of acid challenge. Food Res. Int. 36:49–56. 7. Cutter, C. N., and B. M. Rivera. 2000. Interventions for the reduction of Salmonella Typhimurium DT 104 and non-O157:H7 enterohemorrhagic Escherichia coli on beef surfaces. J. Food Prot. 63:1326–1332. 8. DebRoy, C., E. Roberts, A. M. Valadez, E. G. Dudley, and C. N. Cutter. 2011. Detection of Shiga toxin–producing Escherichia coli O26, O45, O103, O111, O113, O121, O145, and O157 serogroups by Multiplex Polymerase Chain reaction of the wzx gene of the O- antigen gene cluster. Foodborne Pathog. Dis. 8:651–652. 9. Elhadidy, M., and A. Álvarez-Ordóñez. 2016. Diversity of survival patterns among Escherichia coli O157:H7 genotypes subjected to food-related stress conditions. Front. Microbiol. 7:322. 10. Ferens, W. A., and C. J. Hovde. 2011. Escherichia coli O157:H7: animal reservoir and sources of human infection. Foodborne Pathog. Dis. 8:465–487. 11. Fontana, A. J. 2008. Appendix D: minimum water activity limits for growth of microorganisms, p. 405–405. In Gustavo V. Barbosa-Cánovas, A.J. Fontana, S.J. Schmidt, and T.P. Labuza (eds.), Water Activity in Foods. Blackwell Publishing Ltd, Oxford, UK. 12. Foster, J. W. 2004. Escherichia coli acid resistance: tales of an amateur acidophile. Nat. Rev. Microbiol. 2:898–907. 13. Frenzen, P. D., A. Drake, F. J. Angulo, and Emerging Infections Program FoodNet Working

Group. 2005. Economic cost of illness due to Escherichia coli O157 infections in the United States. J. Food Prot. 68:2623–2630. 14. Guraya, R., J. F. Frank, and A. N. Hassan. 1998. Effectiveness of salt, pH, and diacetyl as inhibitors for Escherichia coli O157:H7 in dairy foods stored at refrigeration temperatures. J. Food Prot. 61:1098–1102. 15. Hiramatsu, R., M. Matsumoto, K. Sakae, and Y. Miyazaki. 2005. Ability of shiga toxin- producing Escherichia coli and Salmonella spp. to survive in a desiccation model system and in dry foods. Appl. Environ. Microbiol. 71:6657–6663. 16. Hoffmann, S., M. B. Batz, and J. G. Morris. 2012. Annual cost of illness and quality- adjusted life year losses in the United States due to 14 foodborne pathogens. J. Food Prot. 75:1292–1302. 17. Hoffmann, S., B. Maculloch, and M. Batz. 2015. USDA ERS - Economic Burden of Major Foodborne Illnesses Acquired in the United States. https://www.ers.usda.gov/publications/pub-details/?pubid=43987 18. House, B., J. V. Kus, N. Prayitno, R. Mair, L. Que, F. Chingcuanco, V. Gannon, D. G. Cvitkovitch, and D. Barnett Foster. 2009. Acid-stress-induced changes in enterohaemorrhagic Escherichia coli O157 : H7 virulence. Microbiology 155:2907–2918. 19. Kalchayanand, N., T. M. Arthur, J. M. Bosilevac, J. W. Schmidt, R. Wang, S. D. Shackelford, and T. L. Wheeler. 2012. Evaluation of commonly used antimicrobial interventions for fresh beef inoculated with Shiga toxin–producing Escherichia coli serotypes O26, O45, O103, O111, O121, O145, and O157:H7. J. Food Prot. 75:1207–1212. 20. Karmali, M. A., V. Gannon, and J. M. Sargeant. 2010. Verocytotoxin-producing Escherichia coli (VTEC). Vet. Microbiol. 140:360–370. 21. Lacour, S., and P. Landini. 2004. SigmaS-dependent gene expression at the onset of stationary phase in Escherichia coli: function of SigmaS-dependent genes and identification of their promoter sequences. J. Bacteriol. 186:7186–7195. 22. Lee, K. I., N. P. French, G. Jones, Y. Hara-Kudo, S. Iyoda, H. Kobayashi, Y. Sugita-Konishi, H. Tsubone, and S. Kumagai. 2012. Variation in stress resistance patterns among stx genotypes and genetic lineages of shiga toxin-producing Escherichia coli O157. Appl. Environ. Microbiol. 78:3361–3368. 23. Lee, S.-Y., and D.-H. Kang. 2016. Survival mechanism of Escherichia coli O157:H7 against combined treatment with acetic acid and sodium chloride. Food Microbiol. 55:95–104. 24. Leistner, L. 2000. Basic aspects of food preservation by hurdle technology. Int. J. Food Microbiol. 55:181–186. 25. Mathusa, E. C., Y. Chen, E. Enache, and L. Hontz. 2010. Non-O157 shiga toxin-producing Escherichia coli in foods. J. Food Prot. 73:1721–1736. 26. McLeod, A., I. Måge, E. Heir, L. Axelsson, and A. L. Holck. 2016. Effect of relevant

environmental stresses on survival of enterohemorrhagic Escherichia coli in dry- fermented sausage. Int. J. Food Microbiol. 229:15–23. 27. Miszczycha, S. D., N. Bel, P. Gay-Perret, V. Michel, M. C. Montel, and D. Sergentet- Thevenot. 2016. Short communication: Behavior of different shiga toxin-producing Escherichia coli serotypes (O26:H11, O103:H2, O145:H28, O157:H7) during the manufacture, ripening, and storage of a white mold cheese. J. Dairy Sci. 99:5224–5229. 28. Miszczycha, S. D., F. Perrin, S. Ganet, E. Jamet, F. Tenenhaus-Aziza, M.-C. Montel, and D. Thevenot-Sergentet. 2013. Behavior of different shiga toxin-producing Escherichia coli serotypes in various experimentally contaminated raw-milk cheeses. Appl. Environ. Microbiol. 79:150–158. 29. Miszczycha, S. D., J. Thévenot, S. Denis, C. Callon, V. Livrelli, M. Alric, M.-C. Montel, S. Blanquet-Diot, and D. Thevenot-Sergentet. 2014. Survival of Escherichia coli O26:H11 exceeds that of Escherichia coli O157:H7 as assessed by simulated human digestion of contaminated raw milk cheeses. Int. J. Food Microbiol. 172:40–48. 30. Nguyen, Y., and V. Sperandio. 2012. Enterohemorrhagic E. coli (EHEC) pathogenesis. Front. Cell. Infect. Microbiol. Frontiers Media SA 2:90. 31. Park, C.-M., and L. R. Beuchat. 2000. Survival of Escherichia coli O157:H7 in potato starch as affected by water activity, pH and temperature. Lett. Appl. Microbiol. 31:364–367. 32. Peng, S., T. Tasara, J. Hummerjohann, and R. Stephan. 2011. An overview of molecular stress response mechanisms in Escherichia coli contributing to survival of shiga toxin– producing Escherichia coli during raw milk cheese production. J. Food Prot. 74:849–864. 33. Pokharel, S., J. C. Brooks, J. N. Martin, A. Echeverry, A. Parks, B. Corliss, and M. M. Brashears. 2015. Shiga-toxin producing Escherichia coli (STEC) serogroups exhibit varied thermal susceptibility in marinated beef products. Meat Sci. 101:154. 34. Pokharel, S., J. C. Brooks, J. N. Martin, A. Echeverry, A. R. Parks, B. Corliss, and M. M. Brashears. 2016. Internalization and thermal susceptibility of shiga toxin-producing Escherichia coli (STEC) in marinated beef products. Meat Sci. 116:213–220. 35. Price, S. B., J. C. Wright, F. J. DeGraves, M.-P. Castanie-Cornet, and J. W. Foster. 2004. Acid resistance systems required for survival of Escherichia coli O157:H7 in the bovine gastrointestinal tract and in apple cider are different. Appl. Environ. Microbiol. 70:4792– 4799. 36. Richard, H., and J. W. Foster. 2004. Escherichia coli glutamate- and arginine-dependent acid resistance systems increase internal pH and reverse transmembrane potential. J. Bacteriol. American Society for Microbiology (ASM) 186:6032–6041. 37. Russell, N. J., L. Leistner, and G. W. Gould. 2003. Solutes and low water activity, p. 119– 145. In N.J. Russell, and G.W. Gould (eds.), Food Preservatives. Springer US, Boston, MA. 38. Ryu, J.-H., Y. Deng, and L. . Beuchat. 1999. Survival of Escherichia coli O157:H7 in dried

beef powder as affected by water activity, sodium chloride content and temperature. Food Microbiol. 16:309–316. 39. Samelis, J., P. Kendall, G. C. Smith, and J. N. Sofos. 2004. Acid tolerance of acid-adapted and nonadapted Escherichia coli O157:H7 following habituation (10 degrees C) in fresh beef decontamination runoff fluids of different pH values. J. Food Prot. 67:638–645. 40. Santillana Farakos, S. M., and J. F. Frank. 2014. Challenges in the Control of Foodborne Pathogens in Low-Water Activity Foods and Spices, p. 15–34. In The Microbiological Safety of Low Water Activity Foods and Spices. New York, NY. 41. Shadbolt, C., T. Ross, and T. A. McMeekin. 2001. Differentiation of the effects of lethal pH and water activity: food safety implications. Lett. Appl. Microbiol. 32:99–102. 42. Stasic, A. J., A. C. L. Wong, and C. W. Kaspar. 2012. Osmotic and desiccation tolerance in Escherichia coli O157:H7 requires RpoS (σ38). Curr. Microbiol. 65:660–665. 43. Stopforth, J. D., J. Samelis, J. N. Sofos, P. A. Kendall, and G. C. Smith. 2003. Influence of extended acid stressing in fresh beef decontamination runoff fluids on sanitizer resistance of acid-adapted Escherichia coli O157:H7 in biofilms. J. Food Prot. 66:2258– 2266. 44. Tapia, M. S., S. M. Alzamora, and J. Chirife. 2008. Effects of water activity (aw) on microbial stability: as a hurdle in food preservation, p. 239–271. In Gustavo V. Barbosa- Cánovas, A.J. Fontana, S.J. Schmidt, and T.P. Labuza (eds.), Water Activity in Foods. Oxford, UK. 45. Toldrá, F., Y. H. Hui, I. Astiasarán, J. G. Sebranek, and R. Talon. 2014. Handbook of Fermented Meat and Poultry2nd ed. 46. United States Department of Agriculture - Economic Research Service. ERS Reports. https://www.ers.usda.gov/topics/food-safety/foodborne-illness/readings/ 47. van Elsas, J. D., A. V Semenov, R. Costa, and J. T. Trevors. 2011. Survival of Escherichia coli in the environment: fundamental and public health aspects. ISME J. 5:173–183. 48. Wang, R., J. L. Bono, N. Kalchayanand, S. Shackelford, and D. M. Harhay. 2012. Biofilm formation by Shiga toxin–producing Escherichia coli O157:H7 and non-O157 strains and their tolerance to sanitizers commonly used in the food processing environment. J. Food Prot. 75:1418–1428.

Table 1. Serogroup and strains used in all performed experiments Serogroup Isolate Designation Source Virulence indicators O26 6.1592 test strain Stx1, eae O26 7.3964 unknown unknown O26 8.0176 unknown unknown O26 10.0701 cow Stx1, eae O26 10.1400 cow Stx1, eae O45 Lab strain** unknown unknown O45 050E01736* human unknown O45 M11027674001A* human unknown O45 11.0709 cow Stx1, Stx2 eae O45 14.0151 cow Stx1, eae O103 5.1658 cow Stx1, Stx2 O103 6.1623 test strain Stx1, Stx2 O103 9.0036 test strain Stx1, eae O103 9.0108 cow Stx1, eae O103 90.0393 unknown Stx1, Stx2 eae O111 4.0522 cow unknown O111 5.1636 cow Stx1, eae O111 7.1639 cow unknown O111 10.0705 cow Stx1, Stx2 eae O111 10.0731 test strain unknown O121 Lab strain** unknown unknown O121 7.1732 cow unknown O121 7.1678 cow unknown O121 10.0709 cow unknown O121 05E02072* human unknown O145 4.0907 unknown Stx2, eae O145 6.1598 test strain unknown O145 10.0707 cow Stx1, Stx2 O145 10.0708 cow Stx1 O145 10.2421 test strain Stx2, eae O157 EDL 933** food Stx1, Stx2 O157 PA 2* human Stx2 O157 Sakai* human Stx1, Stx2 O157 6.1593 test strain unknown O157 7.1495 ground beef unknown Note: * denotes strains obtained from Dudley lab and ** denotes strains obtained from Cutter lab (Food Science Department, Penn State).

Table 2. Differences between the means of control (E. coli O157:H7) and acid-adapted non-O157:H7 STEC Dunnett TSB (pH =4.5) TSB (aw=0.88) GBS (pH 4.5) Paper Disk Non-Acid Non-Acid Non-Acid Non-Acid Pairs Adapted Adapted Adapted Adapted Adapted Adapted Adapted Adapted O26-O157 -0.33 -0.48* 0.20 0.04 0.14 0.10 A -0.56 -0.33 O45-O157 -0.43* -0.45* 0.58 0.23 -0.26 -0.30 -0.28 0.18 O103-O157 -0.49* -0.45* -0.06 -0.44 -0.50 0.53 -0.60 -0.29 O111-O157 -0.46* -0.47* -0.34 -0.21 -0.78 -0.77 -0.47 -0.20 O121-O157 -0.41* -0.35* 0.40 0.39 -0.53 -0.54 -0.21 0.50 O145-O157 -0.32 -0.40* 0.37 0.29 -1.67 -1.22 -0.67 -0.21 Note: * signifies significant difference between the non-O157 STEC and O157 in the pair comparison specified (p=0.01). The GBS experiment was the only to have a significant different pair (O145-O157) from all the others in both non-acid adapted and acid- adapted cells. (p=0.01).

Table 3. Rate of reduction of acid-adapted E. coli O157:H7 and non-O157:H7 STEC TSB (pH 4.5) TSB (0.88) GBS (pH 4.5) Paper Disk Non-Acid Non-Acid Non-Acid Non-Acid Serogroup Adapted Adapted Adapted Adapted Adapted Adapted Adapted Adapted O26 + 0.48 ± 0.39 + 0.30 ± 0.35 2.83 ± 0.47 1.91 ± 0.09 1 + 0.56 ± 0.27 + 0.57 ± 0.29 5.92 ± 0.36 5.81 ± 0.40 O45 + 0.19 ± 0.04 + 0.05 ± 0.07 2.06 ± 0.19 1.57 ± 0.12 1 + 0.58 ± 0.17 + 0.96 ± 0.10 5.76 ± 0.41 5.09 ± 0.39 O103 + 0.08 ± 0.09 + 0.18 ± 0.10 2.93 ± 0.31 2.90 ± 0.43 1 + 0.32 ± 0.36 + 0.99 ± 0.28 6.24 ± 0.44 5.77 ± 0.19 O111 + 0.34 ± 0.07 + 0.14 ± 0.06 3.53 ± 0.26 2.21 ± 0.53 1 + 0.14 ± 0.33 + 0.31 ± 0.42 6.04 ± 0.12 5.78 ± 0.13 O121 + 0.06 ± 0.17 + 0.37 ± 0.10 2.33 ± 0.08 1.55 ± 0.07 2 + 0.89 ± 0.24 0.10 ± 0.38 5.02 ± 0.63 4.79 ± 0.75 O145 + 0.35 ± 0.10 + 0.11 ± 0.03 2.26 ± 0.25 1.50 ± 0.12 1 0.71 ± 0.13 0.34 ± 0.03 6.79 ± 0.10 5.99 ± 0.31 O157 + 0.63 ± 0.08 + 0.69 ± 0.11 3.44 ± 0.42 2.34 ± 0.25 1 + 0.81 ± 0.15 + 0.75 ± 0.31 4.96 ± 0.44 5.23 ± 0.84 Note: Different numbers in a row indicate significant difference between control and acid-adapted STEC (p=0.01). + signifies a positive slope, indicating growth instead of reduction.

Chapter 3

Survival of Acid-Adapted Shiga toxin-producing Escherichia coli during Processing of Dry, Fermented Sausage

Abstract

It is believed that interventions used to control Escherichia coli O157:H7 are likely to control non-O157:H7 Shiga toxin-producing E. coli (STEC). However, some research indicates that non-O157:H7 STEC can survive better than E. coli O157:H7 under similar conditions. In this study,

E. coli O157:H7 and non-O157:H7 STEC (O26, O145, O45, O103, O111, O121) were acid-adapted under laboratory conditions, subjected to processes associated with the manufacture of dry, fermented sausages (DFS) and evaluated for pathogen survival. Briefly, bacterial cultures were exposed to mildly acidic conditions (pH 5.0) in broth (20 h at 37°C), five-strain serogroup cocktails were made by combining equal amounts of each, added to raw sausage meat batters with a known starter culture to obtain ca. 7 log10 CFU/g, stuffed into natural casings, fermented (60 h at 70°C to a final pH ≤ 5.33), dried (9 days at average 73°C; water activity of ≤ 0.90), vacuum packaged, stored (VPS) up to 4 weeks at 23°C, and evaluated for surviving microbial populations, pH, and water activity over time. Using these procedures, the highest reduction in non-O157:H7

STEC was observed for O121 (0.66 log10 CFU/g) while the lowest reductions were observed for

O26 and O45, at 0.10 and 0.07 log10 CFU/g,, respectively. Meanwhile, E. coli O157:H7 was reduced

0.51 log10 CFU/g during fermentation. However, these reductions were not considered significant for any of the pathogens. During the drying process, non-O157:H7 STEC and E. coli O157:H7 were reduced an additional 0.40 and 0.84 log10 CFU/g, respectively. Following VPS, populations of non-

O157:H7 STEC were reduced an additional 0.80 log10 CFU/g, while E. coli O157:H7 was reduced

2.07 log10 CFU/g. All non-O157:H7 STEC demonstrated total reductions of >2.0 log10 CFU/g, while

E. coli O157:H7 was reduced >3.0 log10 CFU/g. There was no significant difference between the log reduction rates of non-O157:H7 STEC, when compared to E. coli O157:H7. These results

65 suggest that non-O157:H7 STEC survival during the production of a DFS was comparable to E. coli

O157:H7 and that all tested STEC serogroups are able to survive DFS processing. The results from this study may be of interest to researchers, lawmakers and regulatory agencies, or food industry personnel who wish to ascertain the safety of these products, especially with regard to validation of processes (ex. fermentation, drying, vacuum-packaged storage) used by the meat industry.

Introduction Shiga toxin-producing Escherichia coli (STEC) are a highly virulent group of pathogens that can cause severe disease in humans including abdominal pain, vomiting, diarrhea, hemorrhagic colitis, as well as more severe sequelae, such as hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) (14, 36). The serious consequences that can follow an infection with a low infectious dose (10-100 cells) of STEC include: hemolytic colitis, hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP), and even death, making

STEC a significant public health issue (34, 37, 41). Although, the majority of outbreaks and severe cases of STEC infections have been attributed to E. coli O157:H7, in recent years other STEC serogroups have also emerged as important foodborne pathogens (30, 33). In the United States

(U.S.), apart from E. coli O157:H7, six additional STEC serogroups (O26, O45, O103, O111, O121 and O145) have been recognized and classified as adulterants in raw beef products by the U. S.

Department of Agriculture-Food Safety Inspection Service (USDA-FSIS) (48, 51).

STEC are known to adapt to acidic environments, allowing them to persist and survive in otherwise unfavorable conditions (11, 16, 27). Adaptation to a particular stressor has also been linked to cross-adaptation to other stressful conditions, such as enhanced thermal tolerance (6,

37, 52). It has also been suggested that acid stress adaptation may influence the virulence of STEC

(20). Adaptation of STEC to stressors is of concern, because the safety of many food products relies on the implementation of the “hurdle effect,” or subsequent exposure to several sub-lethal stressors in order to control foodborne pathogens (24, 25). One such product, dry, fermented sausages (DFS), relies on a number of hurdles for stability and safety, including the following: acid production and subsequent reduction in pH that results from fermentation by lactic acid bacteria;

67 reduced water activity (aw) due to the presence of solutes (ex. salts, sugars) and/or the drying process; presence of other antimicrobial components (ex. nitrites, nitrates, bacteriocins); and/or processes (ex. cold smoking, vacuum packaging) in order to control pathogens (45, 46).

There exists a wide variety of fermented sausages with distinct formulations, and to some extent, varied processing parameters, which are reflective of the country or region of the world from where these products may have originated. Of these, the majority of DFS are being produced in the Mediterranean region. Typically, these products are dried, but rarely smoked.

While pork is the main meat protein source of DFS, beef has been used to enhance color retention in some products (32). Some processors also may inoculate fungi onto the external surface of the sausages to comply with standards of identification, impart particular flavor profiles, control the rate of drying and prevent toxigenic mold growth (8, 31). No single “hurdle” is able to control all pathogens; rather, it is the combination of factors that help preserve and keep the products safe.

In recent years the inherent safety of these products has been dispelled, given several notable

STEC outbreaks linked to DFS (15, 42, 43). In 2011, an outbreak of E. coli O157:H7 was linked to

Lebanon bologna. After an investigation had been conducted, several factors that could have contributed to the outbreak were identified; most notably, the plant’s failure to validate their process properly. In addition, the establishment’s supporting documents for their HACCP plan did not match their processing protocol. For example, the casing diameter used in the establishment was larger than the diameter used in the research paper. Therefore, having a larger diameter casing may have impacted the processes necessary to reach the required temperature needed for fermentation to the desired pH, thus leading to process failure (47).

Similarly, an outbreak of E. coli O26:H11 associated with organic beef sausages was reported in

Denmark in 2007. The likely cause was improper pathogen control interventions, since these products often use ingredients, such as celery juice/powder, to provide “natural” nitrite in the product, making the real amount of nitrite formed in these products by these ingredients unknown. Lower amounts of nitrite could allow pathogens to survive, resulting in adverse human health reactions (44).

Recently, the importance of non-O157:H7 STEC as foodborne pathogens has been recognized such that USDA-FSIS considers them adulterants in raw ground beef (51). However, there is limited knowledge on the behavior of these pathogens in other food systems, including experimental or real-world food processing conditions. Prior research has suggested that interventions used to control E. coli O157:H7 on beef carcass surfaces are likely to control the non-O157:H7 STEC as well (10, 23). Other research suggests that when heat treatments or high- pressure processing (HPP) are applied to meat systems experimentally inoculated with non-

O157:H7 STEC, reductions are comparable, if not higher, than those observed for E. coli O157:H7

(22). More recently, new research has focused on the behavior of non-O157:H7 STEC and survival under different experimental conditions to assess if there are significant differences between them. In a study by Pokharel et al. (39), the internalization and thermal susceptibility of STEC on marinated beef products was evaluated. Results suggested that there were differences in the internalization and survival among various non-O157:H7 STEC (39). The researchers observed that serogroup O26, O1O3, and O111 survived in meat cuts after cooking to 55°C and 60°C, while

E. coli O157:H7 survived in cuts cooked to 60°C and 65°C. However, only E. coli O145 was able to survive all cooking temperatures, whereas E. coli O121 was not detected in any cooked products.

Given these discrepancies, more research is needed to ascertain if the non-O157:H7 STEC behave

69 differently than E. coli O157:H7 since, results have indicated there may be inherent differences between STEC serogroups (9, 17, 26).

The USDA-FSIS requires that manufacturers of dry and semi-dry sausage products

demonstrate the ability to reduce E. coli O157:H7 by ≥ 5 log10 CFU (1, 50). To date, most of the published data have demonstrated that unless a heat step is employed, multi-hurdle approaches are unable to achieve the desired 5 log10 reduction of E. coli O157:H7 (19, 41) in these products.

Other researchers have observed that controlling fermentation (using a starter culture), modifying levels of ingredients (i.e. NaCl, NaNO2 or glucose), higher storage temperature, or adding additional interventions, prior to or after processing, can improve pathogen reductions

(7, 18, 19, 40). In a recent publication, pre-treating meat trim with a solution of lactic acid (4.5%), fermentation by a starter culture, lowered water activity by drying, and vacuum-packaged storage, affected a ca. 5 log10 CFU/g overall reduction of E. coli O157:H7 (40). In another study, researchers demonstrated higher reductions when the sausage batter was heated lightly (13°C), frozen, and thawed prior to stuffing (19).

Stress adaptation mechanisms used by pathogenic bacteria are critical for survival upon exposure to detrimental environmental conditions. It has been suggested that more than one mechanism can be employed at the same time in order to maximize survival. For example, both the SOS response and RpoS can act in a complimentary way under certain conditions (3, 35). RpoS concentration increases as cells enter into stationary phase; it then promotes gene expression that provides general stress resistance to cells (5). It can be argued then, that E. coli present throughout the food processing chain will encounter detrimental conditions and will employ stress adaptation mechanisms. In addition, some researchers have argued that pretreating cells

70 with a sub-lethal level stress can improve resistance to a subsequent, and otherwise, lethal stress or provide cross-protection to other stressors (5, 6). In a recent study, researchers compared the survival of non-acid resistant (NAR) and acid resistant (AR) non-pathogenic STEC during the production of a fermented sausage. Results showed that NAR cells were completely destroyed by day 60; in contrast, AR cells were still detected (37). In another study, salt and acid adaptation had limited effects on survival of EHEC during production of DFS and during post-processing treatments. Researchers concluded that prior stress adaptation had limited and a strain- dependent effect on cell reductions (34). In addition to recent outbreaks, some studies have reported that E. coli O157:H7 may be able to grow during the initial stages of sausage fermentation (28, 38). If acid-adapted E. coli O157:H7 are present during fermentation, surviving cells may overcome subsequent hurdles during processing. However, very little information exists as to the survivability or adaptation of non-O157:H7 STEC in DFS under similar processing conditions. Therefore, the following research was conducted to determine if there are differences in the survival of acid-adapted, non-O157:H7 STEC and acid-adapted, E. coli O157:H7 during the production of DFS under simulated laboratory conditions.

Materials and Methods Bacterial strains

Five strains each of Shiga toxin-producing E. coli O157:H7, O26, O45, O103, O111, O145 and O121 (Table 1) were obtained from the E. coli Reference Center (Department of Veterinary and Biomedical Sciences) or the Food Microbiology Culture Collection (Department of Food

Science) of the Pennsylvania State University (University Park, PA). All cultures were stx1 and/or stx2 and eae positive. All cultures were stored at - 80°C in fresh sterile tryptic soy broth (TSB)

(Difco, Detroit, MI) supplemented with 20% glycerol (Sigma Aldrich, St. Louis, MO) to ensure viability. Prior to any experiments, frozen stock cultures were revived by transferring 0.1 ml of each thawed frozen culture to 10 ml of fresh TSB and incubated at 37°C for 24 h. A loopful of the overnight culture was streaked onto Tryptic Soy agar (TSA) plates and incubated at 37°C for 24 h to obtain isolated colonies. An overnight culture was prepared from isolated colonies for each organism, transferred 0.1ml to 10 ml of fresh TSB, and incubated at 37°C for 24 h. The inoculum for the experiments used in this study were prepared by transferring 0.5 ml of the overnight cultures into 50 ml of fresh, TSB acidified with sterile 80% lactic-acid solution (pH 5.0) and

incubating overnight at 37°C for 18 h to obtain ca. 9 log10 CFU/ml. Individual cocktails were made with two of the organisms and used for each experiment so that bacterial populations could be enumerated on Rainbow Agar O157 (RBA; Biolog, CA), a chromogenic differential media, to distinguish between STEC (22). Using this procedure, four separate STEC cocktails were prepared as follows: O26 and O145; O103 and O111; O121; O45 and O157. Serogroup cocktails were prepared by combining equal amounts of all five strains in a previously sterilized bottle. Using this procedure, the optical density of all the cocktails was adjusted with fresh acidified TSB to obtain an OD600 of ca. 0.25, which equated to ca. 8 log10 CFU/ml of culture. Cocktails were stored at 4°C for up to 60 min until added to the sausage batter (see below). Overnight single pathogen cocktails were found to be ca. 8 to 9 log10 CFU/ml for E. coli O103, O111 and O157, E. coli O26, E. coli O45 and O145 and E. coli O121.

Sausage preparation

Trim containing pork shoulder, boneless (406A;2.3 kg [5 lbs]; 80% lean) and beef chuck roll (116K) was obtained from Indiana Packers (Delphi, IN) and Cargill Meat Solutions (Wichita,

KS), respectively. The trim (22.7 kg [50 lbs]; 4°C) was ground once through a 4.7 mm plate and the resulting batter was seasoned with salt (NaCl; 2.93% wt/wt), dextrose (0.60% wt/wt), curing salt (6.25% NaNO2;156 ppm), and sodium erythorbate (546 ppm). The mixture was hand massaged using sterile gloves until all ingredients were mixed uniformly into the batter. A commercial, freeze-dried starter culture (Safepro® B-LC-007; CHR Hansen, Denmark) containing

Pediococcus acidilactici, Staphylococcus carnosus, Staphylococcus xylosus, Lactobacillus sakei, and Pediococcus pentosaceus was revived following the manufacturer's recommendations. The revived starter culture was added to the meat batter, ca. 1.1 g per 5 kg (11lbs) of meat to obtain

7.2 ± 0.17 log10 CFU/g inoculation level.

After all of the ingredients were mixed together, serogroup cocktails were added (250 ml) to individual sausage batters and, using sterile gloves, mixed by hand under a biological safety cabinet (BSL02) until the liquid was absorbed into the batter. Control and inoculated batters were hand-stuffed (The Sausage Maker, Buffalo, NY) into 32-35 mm pre-tubed, natural hog casings

(Sausage Maker, Buffalo, NY). Linked sausages, ca. 20 cm (8 in) long and weighing ca. 120 g each, were looped around stainless-steel rods and placed in the drying cabinet (ICS Drying Cabinet

Model AS50/TC; ICS, Italy). Sausages were processed (Table 2) by fermenting at an average temperature of 23.3°C (74°F) and a relative humidity (RH) average of 65% to an internal target pH of ≤5.0 (Testo pH2 model 206, Sparta, NJ). After fermentation, sausages were dried at an average temperature of 16.1°C (average 61°F, RH 76%) to a target aw of <0.90 (AquaLab 4 TE;

Decagon, Pullman, WA), up to 9 days. After drying, sausages were removed aseptically from the drying cabinet, allowed to acclimate to ambient temperature (ca. 23°C), transferred to the laboratory, and vacuum packaged in 3-mil standard barrier vacuum bags (15.2 cm x 20.3 cm; OTR approx. 0.5-0.83 cc/100in2/24hr; Prime Source, Kansas City, MO) using an Ultravac UV-250 (Koch

Equipment; Kansas City, MO, USA). After packaging, samples were stored at 24°C for up to 30 days and evaluated for microbial populations, aw, and pH, at 0, 96,168, 264, 312, 480, 648, 816 and 984 h.

pH, aw, and microbial analysis

At each processing step (Table 2), three samples (individual sausages) each from the control (uninoculated) and inoculated sausages were aseptically removed from the cabinet or packaging for internal pH, aw measurements, and microbial analyses. Internal pH measurements of the sausages were assessed by cutting sausages in half with a sterile scalpel and inserting the pH probe (Testo 206 ph2, Sparta, NJ) in the approximate geometric center until a stable reading was obtained. aw measurements (Aqualab 4TE, Pullman, WA) were assessed by cutting sausages in half with a sterile scalpel and obtaining an interior round cut (5g) to place on the aw cup.

For microbial analysis, individual sausages were placed into sterile, filtered stomacher bags (BagFilter, InterScience; St. Norm, France), weighed, and diluted 1:10 with sterile buffered peptone water (BPW, Difco). Samples were stomached for 1 min at 230 rpm. The resulting stomachate of each sausage was serially diluted in 9 ml of BPW and aliquots of 0.1 ml were spread plated in duplicate onto selective agars to achieve a detection limit of ca. 0.5 log10 CFU/g. Plates were incubated aerobically at 37°C for 24 h, after which typical looking colonies (color) were enumerated manually (22, 49). Five colonies were picked of each color to perform serogroup 74 confirmation by multiplex-PCR targeting the wzx gene of the O-antigen (12). When no bacteria were detected during standard plating, a value of 1 CFU/g was used to allow for conversion to log10 CFU/g. Average counts were determined from duplicate plates and three individual replications of the experiment were conducted.

Statistical analyses

Bacterial populations (averaged duplicates) were converted to log10 CFU/g to allow for analysis and transformation of data was done in order to meet the assumptions of the statistical test. Log reductions were calculated by subtracting final log counts (B) from initial log counts (A):

Log reduction (LR) = log10 (A) - log10 (B). Resulting data were analyzed using the SAS statistical program version 9.4 (SAS Institute, Cary, NC). The general lineal model procedure with Dunnett’s test was used to analyze all non-O157:H7 STEC using E. coli O157:H7 as the control. Log reduction rates (log CFU g- time) were obtained by generating a linear regression best-fit line for each serogroup and stage of DFS processing. Significance was declared at p=0.01.

Results and Discussion To ascertain the effect of fermentation, drying, and vacuum-packaged storage on populations of acid-adapted non-O157:H7 STEC and E. coli O157:H7 in DFS, a number of parameters were evaluated, including pH and aw. Internal pH and aw of the DFS was measured at various stages during processing and averages determined. Initially, the internal pH of the DFS was an average of 5.73, with an average aw of 0.98. By day 4 (T96), the pH dropped below 5.0 and aw reached <0.90 by the end of the drying period (T312). The internal pH of the dry, fermented sausage increased after drying and during vacuum-packaged storage, but remained below 5.5

(Table 3). This increase in pH occurs as protein breaks down during ripening stage, yielding

75 polypeptides, peptides, and free amino acids which can increase the pH, but also impacts flavor and texture development (13, 21). While aw did not exceed 0.90 (Table 4). Viable cell counts of E. coli O157:H7 and the other STEC were monitored at different stages during the production of the

DFS. Reductions of STEC during the initial stages of production (fermentation steps) were not significant; for serogroup O145, minimal growth was observed. Pathogen growth during fermentation has been observed by other researchers under similar experimental conditions (7,

28, 38). Thus, STEC are likely to survive in DFS during the early stages of processing, and could remain in the final product.

The highest reduction in non-O157:H7 STEC during fermentation was observed for E. coli

O121 (0.66 log10 CFU/g), while the lowest were observed for E. coli O26 and O45 at, 0.10 and 0.07 log10 CFU/g, respectively. Meanwhile, E. coli O157:H7 was reduced 0.51 log10 CFU/g during fermentation. However, these reductions were not considered significant for any of the pathogens.

During the drying process, non-O157:H7 STEC and E. coli O157:H7 were reduced an additional 0.40 and 0.84 log10 CFU/g, respectively. Following vacuum packaged storage, populations of non-O157:H7 STEC were reduced an additional 0.80 log10 CFU/g, while E. coli

O157:H7 was reduced 2.07 log10 CFU/g. All serogroups demonstrated total reductions of >2.0 log10 CFU/g, except E. coli O157:H7 which was reduced >3.0 log10 CFU/g (Table 5). There were no significant differences for reductions of pathogen populations between any of the serogroups at any given stage.

Throughout processing, all experimentally-inoculated STEC behaved similarly and all were able to survive processing of a DFS. Results obtained indicate that there is no significant difference between the means of any of the non-O157:H7 STEC and E. coli O157:H7. Therefore, all of the tested non-O157:H7 STEC survived similarly to O157:H7 during the production of a DFS.

When analyzing the rates of reduction for the overall process and by each processing stage

(fermentation, drying and storage), there were no significant differences between the non-

O157:H7 STEC and E. coli O157:H7 (Table 6). These results are in agreement with those of others which have found that non-O157:H7 STEC survival is comparable to that of E. coli O157:H7 (17,

22). Recently, researchers demonstrated that some non-O157:H7 STEC had significantly higher resistance, when compared to E. coli O157:H7 (p < 0.05), during sausage fermentation. However, they reported that non-O157:H7 STEC reductions during the drying stage were comparable or higher than E. coli O157:H7 (4). Nonetheless, the authors concluded that considering the whole process of DFS production, results suggest that the fate of non-O157:H7 STEC is comparable to that of E. coli O157:H7.

In another study validating a pepperoni process, it was reported that E. coli O103 and

O157:H7 had the greatest survival during production, although all serogroups were recovered during drying. In this study, sausages were fermented to a lower pH, were heated to 55°C (1 h) and dried longer (20 days) (17). The authors concluded that non-O157:H7 STEC has less or comparable survival to E. coli O157:H7. Therefore, our results are in agreement with others, and indicate that STEC are able to survive dry, fermented sausage processing conditions. In addition, evidence suggests that non-O157:H7 STEC behave similarly to E. coli O157:H7; thus conditions that control the latter are likely to control the former as well. It must be noted, that although

STEC were able to survive during dry, fermented sausage processing in the present study, the inoculum used in this and many other validation studies are artificially high. In a real world processing scenario, contamination levels of this magnitude are unlikely. The reason such high experimental inoculums are used in these type of studies is to evaluate if a 5 log10 CFU/g of E. coli

O157:H7 can be obtained during processing.

In the current research project, the highest log reductions were observed for E. coli O26 and O157:H7, but all serogroups remained viable and were recovered at the end of the experiment (Table 5). Prior research has determined that a traditional process for landjäger, a semi-dry, cold-smoked, fermented sausage, coupled with lactic acid (4.5%) spray treatment of the beef and pork trim prior to grinding, resulted in combined reduction of ca. 5 log10 CFU/g for

E. coli O157:H7 (40). In the present study, the same level of reduction was not observed and could be attributed to several reasons. First, the meat batter formulation used for the DFS in the present study was limited only to salt, dextrose, sodium nitrite, sodium erythorbate and starter culture; whereas, the formulation for landjäger utilized red wine and other spices, which may impart antimicrobial properties. Secondly, a pre-programmed drying cabinet was used in the current research, resulting in a fermentation time of <60 h (on average), whereas 72h of fermentation was used previously (40) in a manually-controlled smokehouse. It is also important to emphasize, that when validating new food processing parameters they should be validated in equipment with conditions that closely resemble the real life scenario in which the specific food product is to be produced.

In the present study, all serogroups were acid-adapted prior to raw sausage inoculation to represent a worst case scenario, since pathogens in a food processing environment may be

78 subjected to stressful conditions. Although previous in vitro and in situ results demonstrated that acid-adaptation did not appear to provide an advantage over non-acid-adapted cells (Chapter 2), cultures used in this study were acid-adapted. This approach was done to represent a worst case scenario and to mimic industry practices, such as lactic acid washes that are applied to beef carcasses during slaughter (10). Competition between paired serogroups in dry, fermented sausages was not evaluated in the present study. Therefore, it is unknown if co-culturing of STEC leads to enhanced or decreased survival. Despite this limitation, results from the current study indicate that all serogroups can survive in dry, fermented sausages and be found after a month of vacuum-packaged, ambient storage.

In some recent outbreaks involving STEC, more than one serogroup was linked to the same outbreak, suggesting that more than one serogroup can be present and survive in a food product (2). These results demonstrate that all serogroups survived the DFS process, although their ability to survive throughout the process was variable. Recent research suggests that not accounting for serogroup variability, as affected by specific treatment or intervention, may lead to the conclusion that all serogroups are equally or more susceptible than E. coli O157:H7 (29).

The majority of research regarding STEC behavior and survival during exposure to stress or hurdle interventions has been done on E. coli O157:H7. However, some researchers have suggested that non-O157:H7 STEC may behave differently than E. coli O157:H7. Therefore using

E. coli O157:H7 as the only STEC organism for validation studies may lead to biased results. It is recommended that representative strains of the non-O157 STEC be included in future research to account for serogroup variability and improve the power of these types of validation studies.

The results obtained in the present research may be useful for regulatory officials who are tasked

79 with determining if current validation protocols are enough to account for food safety risks. In addition, quality control professionals seeking to validate particular processes may wish to consider using more than one serogroup to strengthen their confidence in their interventions and food processes.

References

1. American Meat Institute Foundation. 1997. Good Manufacturing Practices for Fermented Dry and Semi-Dry Sausage Products. http://meathaccp.wisc.edu/validation/fermentation.html 2. Anonymous. 2016. Multistate outbreak of Shiga toxin-producing Escherichia coli O121 infections linked to flour (Final Update). https://www.cdc.gov/ecoli/2016/o121-06- 16/index.html 3. Baharoglu, Z., and D. Mazel. 2014. SOS, the formidable strategy of bacteria against aggressions. FEMS Microbiol. Rev. 38:1126–1145. 4. Balamurugan, S., R. Ahmed, A. Gao, and P. Strange. 2017. Comparison of the fate of the top six non-O157 shiga-toxin producing Escherichia coli (STEC) and E. coli O157:H7 during the manufacture of dry fermented sausages. Int. J. Food Microbiol. 259:14–21. 5. Battesti, A., N. Majdalani, and S. Gottesman. 2011. The RpoS-mediated general stress sesponse in Escherichia coli. Annu. Rev. Microbiol. 65:189–213. 6. Begley, M., and C. Hill. 2015. Stress adaptation in foodborne pathogens. Annu. Rev. Food Sci. Technol. 6:191–210. 7. Calicioglu, M., N. G. Faith, D. R. Buege, and J. B. Luchansky. 2002. Viability of Escherichia coli O157:H7 during manufacturing and storage of a fermented, semidry soudjouk-style sausage. J. Food Prot. 65:1541–1544. 8. Canel, R. S., J. R. Wagner, S. A. Stenglein, and V. Ludemann. 2013. Indigenous filamentous fungi on the surface of Argentinean dry fermented sausages produced in Colonia Caroya (Córdoba). Int. J. Food Microbiol. 164:81–86. 9. Corliss, B., J. C. Brooks, J. N. Martin, A. Echeverry, A. R. Parks, S. Pokharel, and M. M. Brashears. 2015. The influence of beef quality characteristics on the internalization and thermal susceptibility of Shiga toxin-producing Escherichia coli (STEC) in blade-tenderized beef steaks. Meat Sci. 110:85–92. 10. Cutter, C. N., and B. M. Rivera. 2000. Interventions for the reduction of Salmonella Typhimurium DT 104 and non-O157:H7 enterohemorrhagic Escherichia coli on beef surfaces. J. Food Prot. 63:1326–1332. 11. De Biase, D., and P. A. Lund. 2015. The Escherichia coli acid stress response and its significance for pathogenesis, p. 49–88. In Sima Sariaslani, and G.M. Gadd (eds.), Advances in applied microbiology. Waltham, MA. 12. DebRoy, C., E. Roberts, A. M. Valadez, E. G. Dudley, and C. N. Cutter. 2011. Detection of Shiga toxin–producing Escherichia coli O26, O45, O103, O111, O113, O121, O145, and O157 serogroups by Multiplex Polymerase Chain reaction of the wzx gene of the O- antigen gene cluster. Foodborne Pathog. Dis. 8:651–652.

13. Díaz, O., M. Fernandez, G. D. G. De Fernando, L. de la Hoz, and J. A. Ordoñez. 1997. Proteolysis in dry fermented sausages: The effect of selected exogenous proteases. Meat Sci. 46:115–128. 14. Elhadidy, M., and A. Álvarez-Ordóñez. 2016. Diversity of survival patterns among Escherichia coli O157:H7 genotypes subjected to food-related stress conditions. Front. Microbiol. 7:322. 15. Ethelberg, S., B. Smith, M. Torpdahl, M. Lisby, J. Boel, T. Jensen, E. M. Nielsen, and K. Mølbak. 2009. Outbreak of non‐O157 shiga toxin–producing Escherichia coli infection from consumption of beef sausage. Clin. Infect. Dis. 48:e78–e81. 16. Foster, J. W. 2004. Escherichia coli acid resistance: tales of an amateur acidophile. Nat. Rev. Microbiol. 2:898–907. 17. Glass, K. a, C. W. Kaspar, J. J. Sindelar, A. L. Milkowski, B. M. Lotz, J. Kang, N. G. Faith, E. Enache, a I. Kataoka, and C. Henry. 2012. Validation of Pepperoni process for control of Shiga toxin-producing Escherichia coli. J. Food Prot. 75:838–846. 18. Heir, E., A. L. Holck, M. K. Omer, O. Alvseike, M. Høy, I. Måge, and L. Axelsson. 2010. Reduction of verotoxigenic Escherichia coli by process and recipe optimisation in dry- fermented sausages. Int. J. Food Microbiol. Elsevier B.V. 141:195–202. 19. Holck, A. L., L. Axelsson, T. M. Rode, M. Høy, I. Måge, O. Alvseike, T. M. L’Abée-Lund, M. K. Omer, P. E. Granum, and E. Heir. 2011. Reduction of verotoxigenic Escherichia coli in production of fermented sausages. Meat Sci. Elsevier Ltd 89:286–295. 20. House, B., J. V. Kus, N. Prayitno, R. Mair, L. Que, F. Chingcuanco, V. Gannon, D. G. Cvitkovitch, and D. Barnett Foster. 2009. Acid-stress-induced changes in enterohaemorrhagic Escherichia coli O157 : H7 virulence. Microbiology 155:2907–2918. 21. Hughes, M. ., J. . Kerry, E. . Arendt, P. . Kenneally, P. L. . McSweeney, and E. . O’Neill. 2002. Characterization of proteolysis during the ripening of semi-dry fermented sausages. Meat Sci. 62:205–216. 22. Jiang, Y., J. A. Scheinberg, R. Senevirathne, and C. N. Cutter. 2015. The efficacy of short and repeated high-pressure processing treatments on the reduction of non-O157:H7 shiga-toxin producing Escherichia coli in ground beef patties. Meat Sci. 102:22–26. 23. Kalchayanand, N., T. M. Arthur, J. M. Bosilevac, J. W. Schmidt, R. Wang, S. D. Shackelford, and T. L. Wheeler. 2012. Evaluation of commonly used antimicrobial interventions for fresh beef inoculated with Shiga toxin–producing Escherichia coli serotypes O26, O45, O103, O111, O121, O145, and O157:H7. J. Food Prot. 75:1207–1212. 24. Khan, I., C. N. Tango, S. Miskeen, B. H. Lee, and D.-H. Oh. 2017. Hurdle technology: a novel approach for enhanced food quality and safety – a review. Food Control 73:1426– 1444. 25. Leistner, L. 2000. Basic aspects of food preservation by hurdle technology. Int. J. Food

Microbiol. 55:181–186. 26. Liao, Y.-T., J. C. Brooks, J. N. Martin, A. Echeverry, G. H. Loneragan, and M. M. Brashears. 2015. Antimicrobial interventions for O157:H7 and non-O157 Shiga toxin–producing Escherichia coli on beef tenderized steaks. J. Food Prot. 78:511–517. 27. Lin, J., M. Smith, K. Chapin, H. Baik, G. Bennett, and J. Foster. 1996. Mechanisms of acid resistance in enterohemorrhagic Escherichia coli. Appl. Envir. Microbiol. 62:3094–3100. 28. Lindqvist, R., and M. Lindblad. 2009. Inactivation of Escherichia coli, Listeria monocytogenes and Yersinia enterocolitica in fermented sausages during maturation/storage. Int. J. Food Microbiol. 129:59–67. 29. Lindqvist, R., and M. Lindblad. 2011. Time to growth and inactivation of three STEC outbreak strains under conditions relevant for fermented sausages. Int. J. Food Microbiol. Elsevier B.V. 145:49–56. 30. Luna-Gierke, R. E., P. M. Griffin, L. H. Gould, K. Herman, C. A. Bopp, N. Strockbine, and R. K. Mody. 2014. Outbreaks of non-O157 Shiga toxin-producing Escherichia coli infection: USA. Epidemiol. Infect. 142:2270–2280. 31. Magistà, D., M. Ferrara, M. A. Del Nobile, D. Gammariello, A. Conte, and G. Perrone. 2016. Penicillium salamii strain ITEM 15302: A new promising fungal starter for salami production. Int. J. Food Microbiol. 231:33–41. 32. Marianski, S., and A. Marianski. 2015. The art of making fermented sausagesThird. Outskirts Press, Seminole, FL. 33. Mathusa, E. C., Y. Chen, E. Enache, and L. Hontz. 2010. Non-O157 Shiga toxin-producing Escherichia coli in foods. J. Food Prot. 73:1721–1736. 34. McLeod, A., I. Måge, E. Heir, L. Axelsson, and A. L. Holck. 2016. Effect of relevant environmental stresses on survival of enterohemorrhagic Escherichia coli in dry- fermented sausage. Int. J. Food Microbiol. 229:15–23. 35. Merrikh, H., A. E. Ferrazzoli, A. Bougdour, A. Olivier-Mason, and S. T. Lovett. 2009. A DNA damage response in Escherichia coli involving the alternative sigma factor, RpoS. Proc. Natl. Acad. Sci. 106:611–616. 36. Miszczycha, S. D., F. Perrin, S. Ganet, E. Jamet, F. Tenenhaus-Aziza, M.-C. Montel, and D. Thevenot-Sergentet. 2013. Behavior of different shiga toxin-producing Escherichia coli serotypes in various experimentally contaminated raw-milk cheeses. Appl. Environ. Microbiol. 79:150–158. 37. Montet, M. P., S. Christieans, D. Thevenot, V. Coppet, S. Ganet, M. L. D. Muller, L. Duniere, S. Miszczycha, and C. Vernozy-Rozand. 2009. Fate of acid-resistant and non-acid resistant shiga toxin-producing Escherichia coli strains in experimentally contaminated French fermented raw meat sausages. Int. J. Food Microbiol. 129:264–270. 38. Nissen, H., and A. Holck. 1998. Survival of Escherichia coli O157:H7, Listeria

monocytogenes and Salmonella Kentucky in Norwegian fermented, dry sausage. Food Microbiol. 15:273–279. 39. Pokharel, S., J. C. Brooks, J. N. Martin, A. Echeverry, A. R. Parks, B. Corliss, and M. M. Brashears. 2016. Internalization and thermal susceptibility of shiga toxin-producing Escherichia coli (STEC) in marinated beef products. Meat Sci. 116:213–220. 40. Rivera-Reyes, M., J. A. Campbell, and C. N. Cutter. 2017. Pathogen reductions associated with traditional processing of landjäger. Food Control 73:768–774. 41. Rode, T. M., A. Holck, L. Axelsson, M. Høy, and E. Heir. 2012. Shiga toxigenic Escherichia coli show strain dependent reductions under dry-fermented sausage production and post-processing conditions. Int. J. Food Microbiol. 155:227–233. 42. Sartz, L., B. De Jong, M. Hjertqvist, L. Plym-Forshell, R. Alsterlund, S. Löfdahl, B. Osterman, A. Ståhl, E. Eriksson, H.-B. Hansson, and D. Karpman. 2008. An outbreak of Escherichia coli O157:H7 infection in southern Sweden associated with consumption of fermented sausage; aspects of sausage production that increase the risk of contamination. Epidemiol. Infect. 136:370–380. 43. Schimmer, B., K. Nygard, H.-M. Eriksen, J. Lassen, B.-A. Lindstedt, L. T. Brandal, G. Kapperud, and P. Aavitsland. 2008. Outbreak of haemolytic uraemic syndrome in Norway caused by stx2-positive Escherichia coli O103:H25 traced to cured mutton sausages. BMC Infect. Dis. 8:41. 44. Sebranek, J. G., and J. N. Bacus. 2007. Cured meat products without direct addition of nitrate or nitrite: what are the issues? Meat Sci. 77:136–147. 45. Thomas, R., A. S. R. Anjaneyulu, and N. Kondaiah. 2008. Development of shelf stable pork sausages using hurdle technology and their quality at ambient temperature (37±1°C) storage. Meat Sci. 79:1–12. 46. Thomas, R., A. S. R. Anjaneyulu, and N. Kondaiah. 2010. Quality of hurdle treated pork sausages during refrigerated (4 ± 1°C) storage. J. Food Sci. Technol. 47:266–272. 47. United States Department of Agriculture - Food Safety and Inspection Service. 2013. Food Safety Lessons Learned from the Lebanon Bologna Outbreak-FSIS Compliance Guideline: Lebanon Bologna. http://www.fsis.usda.gov/wps/wcm/connect/d5be2be1- 3c57-45f6-af53 e71393eaaeb6/Compliance_Guideline_Lebanon_Bologna.pdf?MOD=AJPERES 48. United States Department of Agriculture - Food Safety and Inspection Service. 1999. FSIS policy: non-intact raw beef products and E. coli O157:H7. Backgrounder. https://www.fsis.usda.gov/Oa/background/O157policy.htm 49. United States Department of Agriculture - Food Safety and Inspection Service. 2012. Morphologies of representative strains from six non-O157 Shiga toxin- producing Escherichia coli (STEC) grown on modified Rainbow Agar, p. 1–7. In Microbiological Laboratory Guidebook. https://www.fsis.usda.gov/wps/wcm/connect/baef7bd5-7170-

46fa-a62a-431772924a6d/MLG_5B_Appendix_2_01.pdf?MOD=AJPERES 50. United States Department of Agriculture - Food Safety and Inspection Service. 2001. Performance standards for the production of processed meat and poultry products; proposed rule, p. 12592–12600. In Federal Register. https://www.federalregister.gov/documents/2001/04/13/01-9196/performance- standards-for-the-production-of-processed-meat-and-poultry-products-notice-of- technical 51. United States Department of Agriculture - Food Safety and Inspection Service. 2012. USDA Targeting six additional strains of E. coli in raw beef trim starting Monday. News Release - Congr. Public Aff. https://www.usda.gov/wps/portal/usda/usdamediafb?contentid=2012/05/0171.xml&pri ntable=true 52. Usaga, J., R. W. Worobo, and O. I. Padilla-Zakour. 2014. Effect of acid adaptation and acid shock on thermal tolerance and survival of Escherichia coli O157:H7 and O111 in apple juice. J. Food Prot. 77:1656–1663.

Table 1. Serogroup and strains used in all experiments. Serogroup ERC # Source Virulence O26 6.1592 test strain Stx1, eae O26 7.3964 unknown unknown O26 8.0176 unknown unknown O26 10.0701 cow Stx1, eae O26 10.1400 cow Stx1, eae O45 Lab strain** unknown unknown O45 050E01736* human unknown O45 M11027674001A* human unknown O45 11.0709 cow Stx1, Stx2 eae O45 14.0151 cow Stx1, eae O103 5.1658 cow Stx1, Stx2 O103 6.1623 test strain Stx1, Stx2 O103 9.0036 test strain Stx1, eae O103 9.0108 cow Stx1, eae O103 90.0393 unknown Stx1, Stx2 eae O111 4.0522 cow unknown O111 5.1636 cow Stx1, eae O111 7.1639 cow unknown O111 10.0705 cow Stx1, Stx2 eae O111 10.0731 test strain unknown O121 Lab strain** unknown unknown O121 7.1732 cow unknown O121 7.1678 cow unknown O121 10.0709 cow unknown O121 05E02072* human unknown O145 4.0907 unknown Stx2, eae O145 6.1598 test strain unknown O145 10.0707 cow Stx1, Stx2 O145 10.0708 cow Stx1 O145 10.2421 test strain Stx2, eae O157 EDL 933** food Stx1, Stx2 O157 PA 2* human Stx2 O157 Sakai* human Stx1, Stx2 O157 6.1593 test strain unknown O157 7.1495 ground beef unknown Note: * denotes strains obtained from Dudley lab and ** denotes strains obtained from Cutter lab (Food Science Department, Penn State).

Table 2. Sausage processing parameters. Processing Step mT* (°F/°C) MT (°F/°C) mH** MH Time (h) Static Cooling 42.8/6 46.4/8 - - 8 Fermentation 75.2/24 78.8/26 0 0 8 Fermentation 75.2/24 78.8/26 55 65 12 Fermentation 71.6/22 75.2/24 60 70 12 Fermentation 68/20 71.6/22 65 75 24 Drying 64.4/18 68/20 68 78 Drying 60.8/16 64.4/18 72 80 Drying 57.2/14 60.8/16 75 82 Up to Maturing/Ripening 53.6/12 57.2/14 75 80 240 Vacuum-Packaged Storage 73.4/23 75.2/24 - - 672 Note: *T represents temperature; **H represents humidity; m and M denote “minimum” and “maximum,” respectively.

Table 3. Average pH measurements of dry, fermented sausages at various points during processing. Processing Steps Time (h) Control O26-O145 O45-O157:H7 O103-O111 O121 Fermentation T0 5.77 ± 0.05 A 5.69 ± 0.01AC 5.73 ± 0.03 AC 5.74 ± 0.01 AC 5.72 ± 0.03 AC Fermentation T96 4.96 ± 0.05 B 4.96 ± 0.06 B 4.95 ± 0.09 B 4.95 ± 0.06 B 4.99 ± 0.05 B Drying T168 5.03 ± 0.08 B 5.03 ± 0.09 BC 5.01 ± 0.10 BC 5.04 ± 0.06 BC 5.00 ± 0.06 B Drying T264 5.16 ± 0.04 B 5.16 ± 0.08 B 5.17 ± 0.07 BC 5.09 ± 0.04 BC 5.09 ± 0.05 B Drying T312 5.14 ± 0.01 B 5.14 ± 0.12 B 5.31 ± 0.05 BC 5.26 ±0.05 BC 5.24 ± 0.03 B Storage 1 T480 5.27 ± 0.04 BC 5.27 ± 0.04 AB 5.31 ± 0.05 BC 5.34 ± 0.08 CD 5.31 ± 0.04 B Storage 2 T648 5.27 ± 0.16 BC 5.27 ± 0.05 B 5.33 ± 0.07 C 5.35 ± 0.12 CD 5.31 ± 0.05 B Storage 3 T816 5.39 ± 0.10 C 5.39 ± 0.05 BC 5.39 ± 0.07 C 5.33 ± 0.15 BD 5.38 ± 0.05 BC Storage 4 T984 5.26 ± 0.08 BC 5.26 ± 0.05 BC 5.36 ± 0.14 C 5.40 ± 0.07 CD 5.31 ± 0.08 B Note: ± denotes SE of least squared means. Different superscript letters indicate significant difference within a column. Values are an average of three replications and in each replication the values are an average three measurements by time point. Significance declared at α < 0.05

Table 4. Average aw measurements of dry, fermented sausages at various points during processing. Processing Steps Time (h) Control O26-O145 O45-O157:H7 O103-O111 O121 Fermentation T0 0.9774 ± 0.00 A 0.9805 ± 0.01 AC 0.9723 ± 0.03 AC 0.9814 ± 0.01 A 0.9793 ± 0.03 AC Fermentation T96 0.9325 ± 0.01 B 0.9414 ± 0.06 B 0.9463 ± 0.09 B 0.9487 ± 0.06 B 0.9466 ± 0.05 B Drying T168 0.9142 ± 0.01 B 0.9285 ± 0.09 B 0.9325 ± 0.10 B 0.9374 ± 0.06 BC 0.9377 ± 0.06 B Drying T264 0.8945 ± 0.01 C 0.8950 ± 0.08 B 0.9056 ± 0.07 B 0.9049 ± 0.04 BC 0.8958 ± 0.05 B Drying T312 0.8334 ± 0.00 D 0.8672 ± 0.12 B 0.8545 ± 0.05 B 0.8672 ± 0.05 BC 0.8630 ± 0.03 B Storage 1 T480 0.8732 ± 0.01 CD 0.8935 ± 0.04 BD 0.8873 ± 0.07 BD 0.8933 ± 0.08 AC 0.8861 ± 0.04 B Storage 2 T648 0.8772 ± 0.01 CD 0.8723 ± 0.05 BD 0.8792 ± 0.07 BD 0.8925 ± 0.12 AC 0.8852 ± 0.05 B Storage 3 T816 0.8910 ± 0.01 C 0.8917 ± 0.05 CD 0.8864 ± 0.07 CD 0.8983 ± 0.12 BC 0.8887 ± 0.05 C Storage 4 T984 0.8773 ± 0.01 CD 0.8827 ± 0.05 CD 0.8845 ± 0.14 CD 0.8862 ± 0.07 AC 0.8827 ± 0.08 B Note: Note: ± denotes SE of least squared means. Different superscript letters indicate significant difference within a column Values are an average of three replications. Significance declared at α < 0.05

Table 5. Log reductions of STEC during production of dry, fermented sausages enumerated on Rainbow Agar O157:H7. Stage Time O26 O45 O103 O111 O121 O145 O157:H7 Initial T0 6.41 ± 0.22A 6.25 ± 0.30A 7.00 ± 0.39A 6.67 ± 0.42A 7.06 ± 0.16A 6.38 ± 0.30A 6.52 ± 0.30A Fermentation T96 6.31 ± 0.16A 6.18 ± 0.31A 6.36 ± 0.23AB 6.05 ± 0.33AB 6.40 ± 0.21AB 6.43 ± 0.29A 6.00 ± 0.30AB LR T0-T96 0.10 0.07 0.31 0.29 0.66 -0.05 0.51 Drying T312 5.35 ± 0.03AC 5.77 ± 0.21AB 5.72 ± 0.21AC 5.41 ± 0.02A 5.30 ± 0.70BC 5.44 ± 0.13AB 5.16 ± 0.10ABC T96- LR T312 0.95 0.41 0.64 0.64 1.10 0.99 0.84 Storage ST4 3.75 ± 0.25D 3.73 ± 0.45C 4.31 ± 0.06C 4.05 ± 0.17C 4.42 ± 0.14C 4.01 ± 0.07C 3.09 ± 0.55E T312- LR ST4 1.60 2.04 1.41 1.36 0.88 1.44 2.07 Total LR T0-ST4 2.66 2.52 2.36 2.29 2.64 2.38 3.42 Note: ± represents SE of the least square means, in parentheses p-values with significance declared at α=0.05. Different superscripts letters indicate significant difference within a serogroup. Total log reductions (LR) were calculated as Initial – ST4. Significance declared at α < 0.05

Table 6. Means of the log reduction rate of acid-adapted STEC during production of dry, fermented sausages. Serogroup Fermentation Drying Storage Overall O26 0.11 ± 0.08A 0.51 ± 0.17 A 0.32 ± 0.08 A 0.40 ± 0.05 A O45 0.10 ± 0.07 A 0.35 ± 0.22 A 0.43 ± 0.07 A 0.42 ± 0.09 A O103 0.31 ± 0.37 A 0.71 ± 0.26 A 0.19 ± 0.11 A 0.33 ± 0.04 A O111 0.29 ± 0.09 A 0.61 ± 0.11 A 0.19 ± 0.11 A 0.33 ± 0.04 A O121 0.66 ± 0.31 A 0.51 ± 0.55 A 0.24 ± 0.05 A 0.37 ± 0.05 A O145 + 0.05 ± 0.15 A 0.56 ± 0.28 A 0.28 ± 0.08 A 0.39 ± 0.05 A O157 0.52 ± 0.23 A 0.44 ± 0.20 A 0.50 ± 0.04 A 0.48 ± 0.09 A Note: Different letters in a column indicate significant difference between serogroups compared to O157:H7 (p=0.01). + signifies a positive slope indicating growth instead of reduction. Significance declared at α=0.01

Chapter 4

Conclusions and Future Research

4.1 Conclusions

The purpose of the present study was to evaluate the differences between E. coli O157:H7 and non-O157:H7 Shiga toxin-producing E. coli (STEC) O26, O45, O103, O121, and O145 subjected to stressful conditions relevant to the processing of dried, fermented sausages, both in vitro and in situ.

In the first set of in vitro experiments, STEC cocktails were left untreated (control) or exposed to mild acidic conditions (pH 4.5), followed by treatments with low water activity (aw

0.88 and aw 0.75) for 4 days at 24°C in tryptic soy broth (TSB) and evaluated for survival. In a second in vitro experiment, control and acid-adapted STEC cocktails were subjected to desiccation (28 days at 24°C) on sterile paper disks and remaining populations determined.

In acidified TSB (pH 4.5), control serogroups O45, O103, O111 and O121 were significantly different from E. coli O157:H7. In all cases, E. coli O157:H7 survived better than the non-O157:H7

STEC. When analyzing acid-adapted cells, all non-O157:H7 STEC were significantly different, with

E. coli O157:H7 surviving better than all non-O157:H7 STEC. Although the non-O157:H7 STEC behaved differently, they appear to be less resistant to acidic conditions than E. coli O157:H7.

When each serogroup was analyzed separately, to observe the effect of acid-adaptation on survival, no significant differences were observed between control and acid-adapted cells.

Results from experiments using TSB with modified water activity (aw; 0.88 and 0.78;

TSB88 or TSB78) indicate that there was no significant difference between the survival of E. coli

O157:H7 and any of the non-O157:H7 STEC, regardless of acid-adaptation. Similarly, there were no significant differences between the control and the acid-adapted cells when each serogroup

93 was analyzed separately. Log reduction rates indicate that all serogroups were reduced equally and no significant differences existed between E. coli O157:H7 and the non-O157:H7 STEC.

However, in TSB88, the log reduction rate of acid-adapted E. coli O121 was significantly lower than the non-adapted control. Collectively, these results suggest that acid-adaptation had no significant effect on improving the survival of any of the serogroups, when compared to their respective non-adapted, control counterparts when subjected to reduced water activity conditions. In summary, results from experiments conducted using in vitro models (modified TSB and dry filter paper) suggest that E. coli O157:H7 is better able to survive than the non-O157:H7, regardless of acid-adaptation. Furthermore, prior acid-adaptation did not confer STEC protection from subsequent stressors.

In a third experiment, conducted in situ with acidified ground beef slurries, non-adapted and acid-adapted STEC were subjected to mild acidic conditions (pH 4.5) or low aw (0.88 or 0.75), stored for 4 days at 24°C, and survival was determined. Results demonstrated that there were no significant differences between E. coli O157:H7 and the non-O157:H7, regardless of condition

(control or acid-adapted). Furthermore, there was no significant difference between acid- adapted and control cells of each serogroup. Similarly, in ground beef slurries with adjusted aw, no significant differences were observed between E. coli O157:H7 and the non-O157:H7 STEC.

Likewise, there were no differences between control and acid-adapted cells of each serogroup tested. These results suggest that non-O157:H7 STEC serogroups are equally or more susceptible than E. coli O157:H7 when exposed to the stressors tested in this research.

In the final experiment, the survival of acid-adapted E. coli O157:H7 and the “big six” non-

O157:H7 STEC were evaluated during the production of a dry, fermented sausage (DFS). Results

94 indicated that all STEC were able to survive during fermentation, drying and subsequent vacuum packaging. Although pathogens were recovered throughout the study to the last sampling day, the inoculua used were artificially high and are unlikely to represent a real-world processing scenario.

Together, these results suggest that the non-O157:H7 STEC have a similar susceptibility to conditions of lowered pH and aw when compared to E. coli O157:H7 populations. Therefore, interventions used to control E. coli O157:H7 should be equally effective in controlling non-

O157:H7 STEC during conditions similar to those used in this experiment. Despite these findings, it is prudent to include non-O157:H7 STEC in future validation experiments, since these pathogens are subject to the USDA-FSIS “zero tolerance” policy (7). Other researchers have demonstrated that multiple serogroups should be used when evaluating certain interventions or during validation studies. For example, E. coli O121 has been shown to survive better than other serogroups in very low moisture environments, whereas E. coli O145 has been shown to survive a range of cooking temperatures in meat products (1, 5). Moreover, including these pathogens in such studies strengthens the results.

Finally, the information obtained in this study may be of interest to researchers or to food industry professionals who conduct process control validations or challenge studies with E. coli

O157:H7 and non-O157:H7 STEC, as well as the regulatory officials that must assess these types of processes for safety.

4.2 Future research Future research should focus on targeting more strains of each serogroup in order to obtain more insight into their behavior and strengthen the current results. Testing more non-

O157:H7 STEC serogroups and strains in situations mimicking real-life processing conditions will add to the validity and increase confidence that conditions which control E. coli O157:H7 will also control non-O157:H7 STEC.

In the present study, competition that may have risen from co-culturing serogroups was not assessed. Therefore, it would be interesting to know if some serogroups exhibit competition and are able to survive better than others when mixed in cocktails in experimentally-inoculated food systems. Such information could provide a better understanding of the virulence and pathogenicity of particular serogroups. Additionally, this type of research could help processors decide which strains or serogroups to use in cocktails and which ones to use individually for process validations.

Many food processes rely on more than one sub-lethal hurdle levels to control a pathogen since a single intervention may not be sufficient. It is important for researchers, processors, and/or regulators seeking to validate interventions for these pathogens, to determine which hurdles should be used first, albeit in situations where the order of interventions can be modified without affecting the quality or integrity of the product. Such approaches might reveal what intensities may be needed for each intervention in order to control the most resistant serogroup, if any. In order to control these particular serogroups and achieve higher reductions, validations may need to employ additional hurdles or a pre-intervention step, such as a freeze-thaw cycle

(3), an acid treatment of trim prior to mixing of ingredients (6), or increasing the intensity, time, or degree of the final thermal treatment (2, 4).

Finally, additional interventions, such as modified atmosphere packaging, high pressure processing, or antimicrobial packaging could be employed as additional hurdles against these pathogens. The information obtained in the present and future studies could help researchers, regulators, and industry personnel to expand upon the current knowledge that addresses the behavior of non-O157:H7 STEC during interventions and/or conditions encountered during food processing.

References

1. Corliss, B., J. C. Brooks, J. N. Martin, A. Echeverry, A. R. Parks, S. Pokharel, and M. M. Brashears. 2015. The influence of beef quality characteristics on the internalization and thermal susceptibility of Shiga toxin-producing Escherichia coli (STEC) in blade-tenderized beef steaks. Meat Sci. 110:85–92. 2. Elhadidy, M., A. Álvarez-Ordóñez, and A. lvarez-Ordez. 2016. Diversity of survival patterns among Escherichia coli O157: H7 genotypes subjected to food-related stress conditions. Front. Microbiol. 7:1–10. 3. Holck, A. L., L. Axelsson, T. M. Rode, M. Høy, I. Måge, O. Alvseike, T. M. L’Abée-Lund, M. K. Omer, P. E. Granum, and E. Heir. 2011. Reduction of verotoxigenic Escherichia coli in production of fermented sausages. Meat Sci. 89:286–295. 4. Omer, M. K., O. Alvseike, A. Holck, L. Axelsson, M. Prieto, E. Skjerve, and E. Heir. 2010. Application of high pressure processing to reduce verotoxigenic E. coli in two types of dry-fermented sausage. Meat Sci. 86:1005–1009. 5. Pokharel, S., J. C. Brooks, J. N. Martin, A. Echeverry, A. R. Parks, B. Corliss, and M. M. Brashears. 2016. Internalization and thermal susceptibility of shiga toxin-producing Escherichia coli (STEC) in marinated beef products. Meat Sci. 116:213–220. 6. Rivera-Reyes, M., J. A. Campbell, and C. N. Cutter. 2017. Pathogen reductions associated with traditional processing of landjäger. Food Control 73:768–774. 7. United States Department of Agriculture - Food Safety and Inspection Service. 2012. USDA Targeting six additional strains of E. coli in raw beef trim starting Monday. News Release - Congr. Public Aff. https://www.usda.gov/wps/portal/usda/usdamediafb?contentid=2012/05/0171.xml&pri ntable=true

Appendix A

Pathogen Reductions Associated with Traditional Processing of Landjäger

Note: This manuscript has been published. Rivera-Reyes, M., J. A. Campbell, and C. N. Cutter. 2016. Pathogen Reductions Associated with Traditional Processing of Landjäger. Food Control 73:768-774.

Abstract To determine if traditional processing would reduce Escherichia coli O157:H7 (EC), Listeria monocytogenes (LM), and Salmonella spp. (S) in landjäger, beef and pork trim (95% lean) and pork fat was experimentally-inoculated to obtain a pathogen population of ca. 8 log10CFU/g. The trim was spray-treated with 4.5 % lactic acid (30 min, 25°C), ground, mixed with seasonings, and a starter culture added. Sausages were stuffed, pressed, fermented (to pH 4.8), cold smoked, and dried to a water activity of 0.88. Sausages were vacuum packaged, stored (20 days, ca. 23°C), and evaluated for microbial populations, pH, and water activity. Lactic acid treatment of beef trim reduced EC, S, and LM 0.23, 0.42, and 0.22 log10 CFU/g, respectively. Subsequent fermentation and drying reduced EC, LM, and S 3.94, 4.11, and 4.29 log10 CFU/g respectively. Average log reductions of 7.83, 6.19, and 7.21 log10 CFU/g were observed for EC, LM and S, respectively, for the duration of the study. This study demonstrates that traditional processing of landjäger may result in a ca. 5 log reduction of foodborne pathogens.

100

Introduction

Fermented, dry or semi-dry sausages are ready-to-eat (RTE), shelf-stable products that are produced by fermenting a seasoned (sugar, salt and various spices), raw-meat batter to a pH of

≤ 5.3 and a final moisture-to-protein (M:P) ratio of ≤ 3.1:1.0 (1, 12). The fermentation can be carried out by microflora naturally present in the raw meat or by the direct addition of a commercial starter culture. Following fermentation, sausages may be smoked to enhance the development of desired flavor, color, and aroma. Finally, sausages are dried to remove up to 15% moisture for semi-dry sausages, or up to 50% moisture for dry sausages (1).

Traditionally, fermented dry and semi-dry sausages have been regarded as safe products, due to their low pH, low water activity (aw), salt content, and the presence of competing microflora. Combined, these attributes are expected to limit survival and growth of pathogenic bacteria (2, 3, 4, 6, 7). However, several cases of foodborne illnesses have been associated with these types of products. In 1994, Escherichia coli O157:H7 was responsible for an outbreak associated with dry-cured salami. More recently, Lebanon bologna was linked to an outbreak of

E. coli O157:H7 (6). These outbreaks led the USDA-Food Safety and Inspection Service (USDA-

FSIS) to reconsider the safety of these products and establish guidelines to ensure their safety

(USDA-FSIS, 2001).

The USDA-FSIS guidelines provide five alternatives to thermal treatments for these types of RTE sausages, one of which is to scientifically validate an alternative process to demonstrate required lethality for pathogens of interest, for example a 5 log CFU/g reduction of E. coli

O157:H7, if the sausage formulation contains beef products (USDA-FSIS, 2001). Other pathogens

101 of concern in fermented, RTE sausages include Salmonella spp. and Listeria monocytogenes.

Regulations for pathogen reductions and limits for allowable number of cells in the final product differ by type of pathogen, food product and regulating agency (FDA vs USDA). The USDA-FSIS has issued a “zero tolerance” policy for E. coli O157:H7 in raw and intact beef products. In addition, L. monocytogenes also has a zero tolerance in RTE foods, due in part to its high mortality rate (USDA-FSIS, 2014).

Several studies have evaluated the survival of E. coli O157:H7, L. monocytogenes, and

Salmonella spp. in various fermented sausages. Nissen & Holck (1998) evaluated the effect of storage temperature on pathogen survival and noted that after 5 months of refrigerated storage, both E. coli O157:H7 and L. monocytogenes could still be recovered, whereas Salmonella

Kentucky was unable to be recovered. Furthermore, they concluded that storage at 20°C might be preferable for fermented sausages since none of the pathogens could be recovered after 5 months of storage at this temperature (8). Ellajosyula et al. (1998) examined the effect of fermentation pH, heating temperature, and time on the reduction of E. coli O157:H7 and S.

Typhimurium. They concluded that fermentation or heating alone was unable to obtain reductions of >2 log units, but the combination of both, fermenting to a final pH of 4.7 and gradual heating to 120°F in 10.5h, achieved reductions of >7 log units for both pathogens (4).

Ducic et al. (2016) demonstrated a 1.3 log CFU/g reduction for E. coli O157:H7 in beef sausage, a 1.4 log CFU/g reduction for S. Typhimurium in pork sausage, and 0.8 and 0.5 log CFU/g reductions for L. monocytogenes in pork and beef sausage, respectively (3). Similarly, Porto-Fett et al. (2008) demonstrated that fermentation and drying alone reduced E. coli O157:H7 by 0.03 and 1.11 log10 CFU/g in sausages fermented to pH 5.3 and 4.8, respectively, while S. Typhimurium

102 and L. monocytogenes were reduced by 1.52 and 3.51 log10 CFU/g and 0.07 and 0.74 log10 CFU/g, respectively (9).

Despite different experimental conditions (fermentation pH, starter cultures used or final aw), studies have demonstrated that pathogens, although they can be reduced significantly by a combination of processes, may be present in the final product unless an adequate heat treatment is applied (5, 6, 7, 9).

Landjäger is a fermented, semi-dry sausage, originating from southern Germany. It is traditionally produced through fermentation at 24°C (75.2°F) and drying (18-14°C in 88 to 76% relative humidity) (10). Since landjäger is typically produced without a heat step, the safety and stability of the product is attributed to the low pH (≤ 5.0) and low aw (≤0.90) to control pathogenic bacteria. For sausage processors who do not include a thermal treatment as part of their process, due to the potential unfavorable impact on product quality, alternative processes must be employed in order to ensure food safety. Therefore, processors must demonstrate pathogen reductions through validation studies that satisfy USDA-FSIS requirements for food safety (13).

The objectives of the present research were to determine if a traditional landjäger process would result in a ca. 5 log10 CFU/g reduction of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella spp. The information from this study may be of interest to researchers, processors, and regulatory officials who are interested in determining the safety of landjäger and other similarly-produced, semi-dry fermented RTE sausages.

103

Materials and Methods

Bacterial Strains

Three isolates of E. coli O157:H7, including ATCC 43895, Sakai, and PA-2; four strains of

Salmonella enterica subsp. enterica, including serovars Typhimurium (ATCC 14028), Panama

(ATCC 7378; human isolate), Derby (ATCC 6960; tank water and pork pies) and Montevideo; four isolates of Listeria monocytogenes, including Scott A, serotype 1/2a isolate FSL R2-603 (outbreak linked to deli meats in 2000), serotype 4b isolate H3396 (outbreak associated with hotdogs in

1998) and L. monocytogenes Pirie (ATCC 19115; human isolate) were obtained from the Food

Microbiology Culture Collection (Department of Food Science, Pennsylvania State University). All cultures were stored at -80°C in tryptic soy broth (TSB) (Difco, Detroit, MI) supplemented with

20% glycerol (Sigma Aldrich, St. Louis, MO) to ensure viability. These frozen stock cultures were revived by transferring 0.1 ml of each frozen culture to 10 ml of fresh TSB and incubated aerobically at 37°C for 24h. A loopful of the overnight culture was streaked onto Tryptic Soy agar

(TSA) plates and incubated at 37°C for 24 h to obtain isolated colonies. An overnight culture was prepared from isolated colonies of each pathogen that were transferred to 10 ml of fresh TSB and incubated at 37°C for 24 h. The inoculum for the experiments was prepared by transferring

1 ml of the of the previous overnight culture into 100 ml of fresh TSB and incubated overnight at

37°C for 24 h to obtain ca. 9 log10 CFU/ml. Ten ml of the cultures were transferred to 500 ml of

TSB for another 24 h at 37°C prior the experiment. To obtain a concentrated inoculum, a modified procedure was used (Richard & Cutter, 2011). Equal volumes (500 ml) of each pathogen culture were centrifuged at 11,000 x g for 5 min in sterile centrifuge bottles, washed once with a volume of buffered peptone water (BPW), resuspended in fresh BPW at one tenth of the original TSB

104 volume, and mixed together in the bottles prior to inoculation. Using this methodology, single- pathogen cocktail populations were found to be ca. 9.5, 8.7, and 9.1 log10 CFU/ml for E. coli

O157:H7, Salmonella spp., and L. monocytogenes respectively.

Inoculation and Processing of Trim

Trim (5 kg [11 lbs]; 4°C), containing pork boneless ham (1.8 kg [4 lbs]; 95% lean), beef knuckles (1.8 kg [4 lbs]), and pork back fat (1.6 kg [3 lbs]) was obtained from the Penn State Meat

Laboratory (University Park, PA). Control (non-inoculated) and treatment trim were placed in autoclaved metal bins and inoculated either with sterile buffered peptone water (BPW; control) or the pathogen cocktail (250 ml). Trim pieces were turned and mixed in the bin to ensure even coverage. Inoculated trim was left undisturbed for 30 min to allow for pathogen attachment.

Control and inoculated trim was then transferred and placed on sterile plastic trays and treated by spraying the inoculated surfaces with a solution of 4.5% lactic acid (25°C, pH 2.5; Birko, US) using a handheld tank sprayer (Leader Plus, Hudson, US) for 45 s. Trim was turned and sprayed again for 45 s to ensure adequate coverage, placed inside a cooler, and left to stand for another

30 minutes. After inoculation, the surface pH of the trim was measured using a surface pH probe

(Model A57184, Beckman Coulter, US). The control and experimentally-inoculated trim were fine ground once through a 4.5 mm plate using a benchtop grinder (Universal Mincer, Jupiter, GE) and the resulting batter was seasoned with salt (NaCl; 2.93% wt/wt), red wine (1.63% wt/wt), dextrose (0.60% wt/wt), black pepper (0.32% wt/wt), granulated garlic (0.16% wt/wt), ground caraway (0.16% wt/wt), ground coriander (0.09% wt/wt), curing salt (6.25% NaNO2; 156 ppm), and sodium erythorbate (546 ppm). The mixture was hand massaged using sterile gloves until all ingredients were mixed uniformly into the batter. A commercial, freeze-dried starter culture

105

(Safepro® B-LC-007; CHR Hansen, Denmark) containing Pediococcus acidilactici, Staphylococcus carnosus, Staphylococcus xylosus, Lactobacillus sakei, and Pediococcus pentosaceus was revived following the manufacturer’s recommendations. The revived starter culture was added to the meat batter, ca. 1.1 g per 5 kg (11 lbs) of meat to obtain ca. 7.2 log10 CFU/g.

Sausage Preparation

The control and inoculated batter were hand-stuffed (The Sausage Maker, Buffalo, NY) into 32/35 mm hog casings (Quality Casings, Hebron, KY). Sausages, ca. 20 cm (8 in) long and weighing ca. 100 grams each, were linked in pairs and placed on a press (ca. 55 cm x 55 cm). The press was held together with 24” clutch lock bar clamps (Irwin Tools, Huntersville, NC) and then transferred to the smokehouse (Vortron model TR2-850, Beloit, WI), fermented at an average temperature of 23.3°C (74°F) and a relative humidity (RH) of 61% (U12 Stainless Steel Temp

Logger; Onset HOBO Dataloggers, Bourne, MA) to a target pH of 4.8 (Testo pH2 model 206,

Sparta, NJ). After fermentation (72 h), sausages were smoked (Hickory sawdust; The Frantz Co,

Milwaukee, WI) for a period of 2 h at 30°C (87°F, RH 70%). After smoking, sausages were dried for 3 d at 21.6°C (average 71°F, RH 60%) to a target aw of 0.88 (AquaLab 4 TE; Decagon, Pullman,

WA). After drying, sausages were removed from the smokehouse, allowed to acclimate to ambient temperature (ca. 23°C), transferred to the laboratory, and vacuum packaged in 3-mil standard barrier vacuum bags (15.2 cm x 20.3 cm; OTR approx. 0.5-0.83 cc/100in2/24hr; Prime

Source, Kansas City, MO) using an Ultravac UV-250 (Koch Equipment; Kansas City, MO, USA).

Microbial Analysis

106

At each sampling time (see Table 1), two sausages each, from the control and inoculated product, were aseptically removed for microbial analysis, pH, and aw measurements. For microbial analysis, sausages were placed in sterile, filtered stomacher bags (BagFilter,

InterScience; St. Norm, France), weighed, and diluted 1:10 in sterile buffered peptone water

(BPW, Difco). Samples were stomached for 2 min at speed setting 3 (JumboMix® 3500 VP,

InterScience). The resulting stomachate of each sausage was serially diluted in 9 ml of BPW and aliquots of 0.1 ml were spread plated in duplicate, onto selective agars to achieve a detection limit of ca. 0.5 log10 CFU/g. When necessary, a 1 ml aliquot (0.5 ml in quadruplicate) was directly plated from the stomachate onto selective agars. Xylose lysine deoxycholate agar (XLD; Remel,

Lenexa, KS), cefixime tellurite sorbitol MacConkey agar (CT-SMAC; Remel, Lenexa, KS) and modified Oxford agar with antibiotic supplement (MOX; Remel, Lenexa, KS) were used for the enumeration of Salmonella spp. (S), E. coli O157:H7 (EC), and L. monocytogenes (LM), respectively. Plates were incubated aerobically at 37°C for 24h (EC and S) or 48h (LM), after which typical looking colonies were enumerated manually. When no bacteria were detected during standard plating, a value of 1 CFU/g was used to allow for conversion to log10 CFU/g. Enrichments were performed simultaneously during processing as follows. When counts on the plates were below the detection level (0.5 log10 CFU/g), enrichments were used to determine presence of EC,

S, and LM. Gram-negative (GN) broth (Difco, US) was used to enrich for EC, while lactose broth

(LB) (Himedia, IN) and Rappaport-Vassiliadis (RV; Remel, Lenexa, KS) broth and University of

Vermont Medium (UVM; Difco) and Fraser broth (Difco) were used as primary and secondary enrichments, for S and LM respectively. Enrichments were performed as previously described.

Briefly, 1 ml aliquots of stomachate were transferred to pre-enrichments (9 ml) broths (GN, LB,

107 and UVM for EC, S, and LM, respectively), followed by selective enrichment in RV broth and Fraser broth for S and LM, respectively (Scheinberg, Valderrama, & Cutter, 2013). Enriched samples were incubated aerobically at 37°C, transferred into secondary enrichments the following day, and incubated as previously described. Enrichments were plated on selective agars and further confirmed by latex agglutination (Oxoid, UK). In each of the four independent replicates, two control (un-inoculated) and two of the inoculated sausages were sampled at each sampling interval. Bacterial numbers were expressed as log10 CFU/g.

Statistical Analysis

Bacterial populations (averaged duplicates) were converted to log10 CFU/g to allow for analysis and transformation of data in order to meet the assumptions of the statistical test. Log reductions were calculated by subtracting final log counts (B) from initial log counts (A): Log reduction (LR) = log10 (A) – log10 (B). Resulting data were analyzed using the SAS statistical program version 9.4 (SAS Institute, Cary, NC). The mixed procedure was used to analyze the effect of pH, aw, pathogen type, replicate number, and time on log counts. The Kenward-Roger method was used to determine degrees of freedom as it adjusts for bias, improves the F statistic and is especially useful with small sample sizes (Schaalje, McBride, & Fellingham, 2002). Fixed variables were pathogen type, replicate number, and the interactions between pathogen and time, and pathogen and replicate number. Time was included as a repeated effect with pathogen type by replicate number as the subject. Least squares means and standard errors were determined for all fixed effects. The regression procedure was used to analyze the effect of pH and aw on pathogen log reductions. Significance was declared at p<0.05.

Results

108

The inoculation level in the trim was ca. 8.50, 7.81, and 7.85 log10 CFU/g of product for

EC, S, and LM respectively. Initial pH of the trim was on average, 6.10 and 6.08 for control and treatment trim, respectively. Surface pH of trim after lactic acid treatment was 5.52 and 5.44 for control and treatment trim, respectively. Reductions obtained by lactic acid treatment of the trim after inoculation with the pathogen cocktail and prior to grinding were not statistically significant.

Log reductions observed for 4.5% lactic acid spray treatment of pathogens were on average, 0.23,

0.42, and 0.22 log10 CFU/g for EC, S and LM, respectively.

Using the methodologies described above, landjäger processing resulted in a consistent reduction of internal pH to values of ≤ 5.0 after fermentation. Fermentation was stopped once sausages reached target pH, with the average internal pH ranging from 4.9 to 5.0, for control and treatment sausages, respectively. After fermentation, sausages were dried to an average aw of ≤

0.88, after which sausages were vacuum packaged. As expected, there were no significant differences (p > 0.05) in pH or aw between control sausages and treatment sausages throughout the process, indicating that the addition of a pathogen cocktail did not affect the growth and performance of the starter culture (Figures 1 and 2).

Data on the behavior of E. coli O157:H7, Salmonella spp., and L. monocytogenes during the processing of landjäger sausage is presented in Table 1. In general, reductions for all three pathogens were similar, as demonstrated in Figure 3. Interestingly, there were some notable differences as to when reductions became statistically significant (p < 0.05) for each pathogen.

Reductions for EC were significant after the process of fermentation was concluded; however, for S, significant reductions occurred up to 24 h of fermentation, while reductions for LM only became significant after smoking had occurred (Table 1).

109

The process of fermentation alone resulted in reductions of 2.92, 1.57, and 3.64 log10

CFU/g for EC, LM, and S, respectively; whereas, drying provided additional reductions of 1.02,

2.54, and 0.65 log10 CFU/g, respectively. Subsequent log reductions for EC after fermentation, were not significant (p >0.05) until day 12 of vacuum packaged storage at 23°C. Reductions were significant from smoking at 96 h to day 16 (552 h) of vacuum packaged storage at 23°C. By day

20 of storage, EC was no longer recoverable (negative by enrichment). In contrast, S was not reduced significantly after fermentation. By day 12 of vacuum packaged storage, S was no longer recoverable (confirmed negative by enrichment). LM reductions after smoking (96 h) were no longer significant (p > 0.05) and it continued to be recoverable until the last day of vacuum packaged storage.

The regression model showed that both pH and aw contributed to log reductions for EC, whereas S and LM were mostly affected by aw. These results are in contrast with those reported elsewhere. Researchers found that aw was more significant than pH in reducing EC, while pH was more significant than aw in reducing LM (Hwang et al., 2009). It is somewhat unexpected that aw impacted LM survivability more than pH, given that reductions during storage were not considered statistically significant.

Results obtained in this study indicate that traditional processing of landjäger sausage, coupled with vacuum packaged storage at ambient temperature can result in a safe product.

Moreover, initial trim treatment with lactic acid might contribute to the observed pathogen reductions. Overall, total log reductions by the end of the storage period were over 5 log10 CFU/g for each pathogen and only LM could be recovered in very low amounts or was only positive in landjäger samples following enrichment.

110

Discussion In this study, lactic acid treatment (4.5%) of trim resulted in lower reductions of the pathogens, when compared to those reported elsewhere (Wolf et al., 2012; Fouladkhah et al.,

2012; Harris, Miller, Loneragan, & Brashears, 2006). For example, Wolf et al. (2012) demonstrated a 0.5 to 1 log10 CFU/g reduction in pathogens experimentally inoculated onto trim.

These findings are not unexpected, given that Wolf et al. (2012) treated trim by submersion in a

4.4% lactic acid solution, which may have provided better coverage. Similar reductions were observed by Fouladkhah et al. (2012) in which counts of E. coli O157:H7 were reduced by 0.5 and

1 log10 CFU/g following immersion of experimentally-inoculated beef in a 5% lactic acid solution at 25°C and 55°C, respectively. Ellebracht et al. (1999) inoculated beef trimmings with EC and S, treated the trim with hot water (95°C), alone or in combination with a 2% lactic acid solution, and ground the treated beef. Results demonstrated that hot water treatment alone resulted in 0.9 and 0.7 log10 CFU/g of EC and S, respectively, while treatments with hot water and 2% lactic acid resulted in reductions of 1.5 and 1.8 log10 CFU/g, respectively (Ellebracht, Castillo, Lucia, Miller,

& Acuff, 1999). More recently, Blagojevic et al. (2015) treated beef trimming intended for dry fermented sausage production by submersion in a 4% hot lactic acid solution. These treatments resulted in reductions of 3.9 and 3.6 log10 CFU/g for EC and S, respectively, in the final product; however reductions for LM were insignificant (Blagojevic et al., 2015) In this study, a room temperature solution of lactic acid was used. It has been suggested that application of lactic acid to beef trim at warmer temperatures might improve pathogen reductions on trim. Therefore, the lower reductions obtained by the lactic acid treatment in the present study could in part, be due to its lower temperature upon application.

111

Although pathogen reductions with the lactic acid treatment were lower than expected, small reductions from several hurdle techniques, could contribute to the overall log reduction goal. It must be noted that an artificially high inoculum level was purposely used in this study in order to maintain high enough counts to be able to observe the effect of sausage processing on the survival of E. coli O157:H7, Salmonella spp., and L. monocytogenes. This level of contamination is not expected to reflect a real processing scenario.

Previous research exploring the efficacy of fermentation and drying to reduce pathogen loads during sausage production to the levels required by USDA-FSIS standards has demonstrated that this process alone is unlikely to achieve such reductions (Hwang et al., 2009; Porto-Fett et al., 2008). Results have been variable across studies and may be influenced by sausage fermentation temperature, final pH, final water activity, initial pathogen loads, storage temperature and overall experimental design (i.e. surface inoculation versus batter inoculation).

To date, no research has demonstrated a 5 log reduction for E. coli O157:H7 in semi-dry sausages.

Porto-Fett et al. (2008) studied the viability of multi-strain mixtures of L. monocytogenes, S.

Typhimurium, and E. coli O157:H7 inoculated into soudjuk-style fermented, semi-dry sausages.

The sausages fermented to a final pH of 4.8, exhibited reductions of 0.74, 3.51, and 1.11 log10

CFU/g for L. monocytogenes, Salmonella spp., and E. coli O157:H7, respectively. These reductions are consistent with what others have reported previously. Ducic et al. (2016) reported reductions of 0.8 and 0.5 log10 CFU/g reductions for L. monocytogenes in pork and beef sausages, respectively. In addition, the authors reported reductions of 1.4 log10 CFU/g in pork sausages and

1.3 log10 CFU/g in beef sausages for Salmonella spp. and E. coli O157:H7, respectively (Ducic et al., 2016).

112

The results observed in the present study demonstrated higher reductions after fermentation and drying for all three pathogens than those previously reported, although they did not achieve a 5 log reduction. It has been previously noted that maturation

(fermentation/drying) time, especially for sausages that do not undergo a heat treatment, is instrumental in reducing pathogenic bacteria, and that shorter maturation times could result in insufficient pathogen reductions (Lindqvist & Lindblad, 2009). In addition, the temperature at which sausages are stored has been shown to influence pathogen survival. In a study by Quinto et al. (2014) the greatest reduction in VTEC (STEC) were observed at higher temperatures. After

66 days of storage, STEC could not be recovered from sausages stored at 25°C, while sausages stored at 16°C and 12°C had reductions of less than 2 log CFU/g (Quinto et al., 2014). This trend had previously been reported on by Nissen and Holck. (1998). They monitored the survival E. coli

O157:H7 on a Norwegian fermented sausage. After 5 months of storage at 4°C and 20°C E. coli

O157:H7 was still recoverable in low numbers at 4°C but had fallen below the detection limit on sausages stored at 20°C. The authors concluded that from a food safety standpoint it was recommended these type of sausages be stored at room temperature (Nissen & Holck, 1998).

Other studies have also noted that higher storage temperatures are preferred over refrigerated temperatures for reducing pathogens (Lindqvist & Lindblad, 2009; Porto-Fett et al., 2008).

The reductions observed in this study are probably the result of a combination of various hurdle techniques applied throughout processing. First, the use a commercial starter culture ensured a controlled and consistent fermentation, which resulted in a rapid drop in pH to <5.0.

Secondly, the commercial starter culture used in this study also served as a bio-protectant, due its ability to produce pediocin (a type of bacteriocin) which has known anti-listerial properties.

113

Researchers have shown that L. monocytogenes can be inhibited by competing lactic acid bacteria that are added as a starter culture, in part due to the production of organic acids and/or bacteriocins by the organisms (Nieto-Lozano et al., 2010; Raimondi, Popovic, Amaretti, Di Gioia,

& Rossi, 2014). The choice of starter cultures has previously been shown to affect the survival of pathogenic bacteria. In a study by Lahti et al. (2001) two different starter cultures were evaluated for their ability to reduce E. coli O157:H7 and L. monocytogenes in dry sausage. In sausages inoculated with a high pathogen inoculum, starter culture B was more effective than starter culture A, reducing EC by 4.84 and 2.79 log10 CFU/g, respectively by the end of storage period.

However, in the same high inoculum sausages, the reduction rate of LM was faster with starter culture A. Counts fell below 2.0 log10 CFU/g or were undetectable with starter culture A, whereas counts were between 2.51 and 2.45 log10 CFU/g with starter culture B by the end of the storage period. The greater reduction obtained for EC with starter culture B while significant remains unexplained but the higher reductions obtained for LM with starter culture A was attributed to the presence of bacteriocins. Therefore, the authors concluded that there the choice of starter culture can significantly affect the survival of pathogens in fermented dry sausages (Lahti,

Johansson, Honkanen-Buzalski, Hill, & Nurmi, 2001).

The use of a starter culture for fermentation is recommended over a natural inoculation, since it provides a consistent and controlled fermentation. In contrast, Ducic et al. (2016) used natural microflora present in the meat for the purpose of fermentation. The use of natural microflora is discouraged as it can lead to inconsistent fermentations and negative sensory attributes of the final product. In addition, throughout the process and storage time, lactic acid bacteria continued to survive in high amounts (ca. 6 log; data not shown), which could have

114 outcompeted injured pathogenic bacteria that had survived the process. The reduction of available water to the desired target (aw ≤0.88) is essential to prevent the growth of bacteria during long term storage.

Reductions for Salmonella spp. after fermentation and drying in this study, were higher than reductions observed for E. coli O157:H7 and L. monocytogenes. Ellajosyula et al. (1998) found that Salmonella spp. were more susceptible to fermentation pH and high temperature treatments. The authors also noted that S. Typhimurium was as susceptible as E. coli O157:H7 in fermented and heat-treated Lebanon bologna (Ellajosyula et al., 1998). L. monocytogenes may survive better than E. coli O157 and S. Typhimurium in fermented and dried sausages, which is not unusual, given that the pathogen is a Gram-positive bacterium (Ducic et al., 2016; Ellajosyula et al., 1998; Hwang et al., 2009; Lindqvist & Lindblad, 2009; Nissen & Holck, 1998).

In this study, landjäger processing, including treatment of trim with a 4.5% lactic acid solution and subsequent fermentation, smoking, drying, and vacuum packaged storage at 23°C were evaluated for reducing pathogen populations. EC and S were reduced to undetectable levels by the end of vacuum packaged storage (day 20), while LM continued to be recovered, albeit in very low amounts or only by enrichment. In conclusion, the proposed landjäger sausage process could help achieve higher reductions of EC, LM, and S when combined with strict process controls for pH and aw. It is important to note that some EC outbreaks associated with fermented sausages, such as the 2011 Lebanon bologna outbreak, have occurred due either to improper processing techniques or the use of supporting scientific research that was not reflective of the plant’s processing conditions (Sartz et al., 2008; USDA-FSIS, 2013). Therefore, when producing fermented sausages that do not have a cooking step as part of processing, it is imperative that

115 other pathogen control measures, such as a low pH or low aw are being achieved consistently. In addition, it is important to begin with good quality raw materials that have low microbial loads.

This research could help processors establish and/or validate parameters (pH and aw) for the manufacture of semi-dry, fermented sausages using very similar conditions. More research is needed, regarding the potential of pre-treating trim with antimicrobial agents and its effects on the subsequent reductions of pathogens during fermented sausage processing. Additionally, research is needed to determine the effects of sub-lethal hurdle interventions throughout processing on the survival of injured cells, which could remain viable in the product during storage.

Acknowledgments

This project was funded in part by the Department of Food Science, the Department of Animal

Science at Penn State University, and a USDA National Needs Fellowship.

116

References

American Meat Institute Foundation. (1997). Good Manufacturing Practices for Fermented Dry and Semi-Dry Sausage Products. Barbuti, S., & Parolari, G. (2002). Validation of manufacturing process to control pathogenic bacteria in typical dry fermented products. Meat Science, 62(3), 323–329. Blagojevic, B., Antic, D., Adzic, B., Tasic, T., Ikonic, P., & Buncic, S. (2015). Decontamination of incoming beef trimmings with hot lactic acid solution to improve microbial safety of resulting dry fermented sausages – A pilot study. Food Control, 54, 144–149. Ducic, M., Klisara, N., Markov, S., Blagojevic, B., Vidakovic, A., & Buncic, S. (2016). The fate and pasteurization-based inactivation of Escherichia coli O157, Salmonella Typhimurium and Listeria monocytogenes in dry, fermented sausages. Food Control, 59, 400–406. Ellajosyula, K. R., Doores, S., Mills, E. W., Wilson, R. A., Anantheswaran, R. C., & Knabel, S. J. (1998). Destruction of Escherichia coli O157:H7 and Salmonella Typhymurium in Lebanon bologna by interaction of fermentation pH, heating temperature, and time. Journal of Food Protection, 61(2), 152–7. Ellebracht, E. A., Castillo, A., Lucia, L. M., Miller, R. K., & Acuff, G. R. (1999). Reduction of Pathogens Using Hot Water and Lactic Acid on Beef Trimmings. Journal of Food Science, 64(6), 1094–1099. Fouladkhah, A., Geornaras, I., Yang, H., Belk, K. E., Nightingale, K. K., Woerner, D. R., Smith, G. C., & Sofos, J. N. (2012). Sensitivity of Shiga Toxin–Producing Escherichia coli, MultidrugResistant Salmonella,and Antibiotic-Susceptible Salmonella to Lactic Acid on Inoculated Beef Trimmings. Journal of Food Protection, 75(10), 1751–1758. Harris, K., Miller, M. F., Loneragan, G. H., & Brashears, M. M. (2006). Validation of the use of organic acids and acidified sodium chlorite to reduce Escherichia coli O157 and Salmonella Typhimurium in beef trim and ground beef in a simulated processing environment. Journal of Food Protection, 69(8), 1802–1807. Holck, A. L., Axelsson, L., Rode, T. M., Høy, M., Måge, I., Alvseike, O., L’Abée-Lund, T. M., Omer, M. K., Granum, P. E., & Heir, E. (2011). Reduction of verotoxigenic Escherichia coli in production of fermented sausages. Meat Science, 89(3), 286–295. Hwang, C. A., Porto-Fett, A. C. S., Juneja, V. K., Ingham, S. C., Ingham, B. H., & Luchansky, J. B. (2009). Modeling the survival of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium during fermentation, drying, and storage of soudjouk-style fermented sausage. International Journal of Food Microbiology, 129(3), 244–252. Lahti, E., Johansson, T., Honkanen-Buzalski, T., Hill, P., & Nurmi, E. (2001). Survival and detection of Escherichia coli O157:H7 and Listeria monocytogenes during the manufacture of dry sausage using two different starter cultures. Food Microbiology, 18(1), 75–85. http://doi.org/10.1006/fmic.2000.0373

117

Lindqvist, R., & Lindblad, M. (2009). Inactivation of Escherichia coli, Listeria monocytogenes and Yersinia enterocolitica in fermented sausages during maturation/storage. International Journal of Food Microbiology, 129(1), 59–67. Nieto-Lozano, J. C., Reguera-Useros, J. I., Pelez-Martinez, M. del C., Sacristin-Perez-Minayo, G., Gutierrez-Fernandez, A. J., & la Torre, A. H. de. (2010). The effect of the pediocin PA-1 produced by Pediococcus acidilactici against Listeria monocytogenes and Clostridium perfringens in Spanish dry-fermented sausages and frankfurters. Food Control, 21(5), 679– 685. Nissen, H., & Holck, A. (1998). Survival of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella Kentucky in Norwegian fermented, dry sausage. Food Microbiology, 15(3), 273–279. Porto-Fett, A. C. S., Hwang, C. A., Call, J. E., Juneja, V. K., Ingham, S. C., Ingham, B. H., & Luchansky, J. B. (2008). Viability of multi-strain mixtures of Listeria monocytogenes, Salmonella Typhimurium, or Escherichia coli O157:H7 inoculated into the batter or onto the surface of a soudjouk-style fermented semi-dry sausage. Food Microbiology, 25(6), 793–801. Quinto, E. J., Arinder, P., Axelsson, L., Heir, E., Holck, A., Lindqvist, R., Lindblad, M., Andreou, P., Lauzon, H. L., Marteinsson, V. T., & Pin, C. (2014). Predicting the Concentration of Verotoxin-Producing Escherichia coli Bacteria during Processing and Storage of Fermented Raw-Meat Sausages. Applied and Environmental Microbiology, 80(9), 2715–2727. http://doi.org/10.1128/AEM.03791-13 Raimondi, S., Popovic, M., Amaretti, A., Di Gioia, D., & Rossi, M. (2014). Anti-Listeria starters: in vitro selection and production plant evaluation. Journal of Food Protection, 77(5), 837–42. Richard, A. E., & Cutter, C. N. (2011). Validation of a Pickling Process for Controling Pathogens Associated With Hard-Cooked Eggs. Journal of Food Safety, 31(3), 417–423. Sartz, L., De Jong, B., Hjertqvist, M., Plym-Forshell, L., Alsterlund, R., Löfdahl, S., Osterman, B., Ståhl, A., Eriksson, E., Hansson, H.-B., & Karpman, D. (2008). An outbreak of Escherichia coli O157:H7 infection in southern Sweden associated with consumption of fermented sausage; aspects of sausage production that increase the risk of contamination. Epidemiology and Infection, 136(3), 370–80. Schaalje, G. B., McBride, J. B., & Fellingham, G. W. (2002). Adequacy of Approximations to Distributions of Test Statistics in Complex Mixed Linear Models on JSTOR. Journal of Agricultural, Biological, and Environmental Statistics, Vol. 7(No. 4), 512–524. Scheinberg, J. A., Valderrama, W. B., & Cutter, C. N. (2013). The effects of a pickling process on the reduction of Escherichia coli O157: H7, Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus inoculated onto hard-cooked eggs. Journal of Food Safety, 33(4), 413–417. Toldrá, F., Hui, Y. H., Astiasarán, I., Sebranek, J. G., & Talon, R. (2014). Handbook of Fermented

118

Meat and Poultry (2nd ed.). United States Department of Agriculture - Food Safety and Inspection Service. (2013). Food Safety Lessons Learned from the Lebanon Bologna Outbreak-FSIS Compliance Guideline: Lebanon Bologna. USDA-FSIS. (2001). Performance Standards for the Production of Processed Meat and Poultry Products; Proposed Rule. In Federal Register (Vol. 66, pp. 12592–12600). USDA-FSIS. (2005). Food Standards and Labeling Policy Book. USDA-FSIS. (2014). Controlling Listeria monocytogenes in Post-lethality Exposed Ready-to-Eat Meat and Poultry Products.

119

Table 1. Populations (log10 CFU/g) and log reductions of E. coli O157:H7, L. monocytogenes, and Salmonella spp. through landjäger processing. E. coli O157:H7 L. monocytogenes Salmonella spp. 4.5% Lactic Acid (LA) Spray Treatment Prior LA (Initial) 8.50 ± 0.37A 7.85 ± 0.34A 7.81 ± 0.54A Post LA 8.27 ± 0.40A 7.63 ± 0.32A 7.39 ± 0.45A Log Reduction 0.23 ± 0.29 0.22 ± 0.04 0.42 ± 0.37 Fermentation through drying and storage Prior Fermentation (0h) 7.40 ± 0.10Aa 6.70 ± 0.12Aa 6.31 ± 0.51Aa After Fermentation (72h) 4.48 ± 0.59Ab 5.13 ± 0.38Aa 2.67 ± 0.84Ab After Smoking (96h) 4.55 ± 0.27Ab 3.62 ± 0.41Ab 1.79 ± 0.96Ab After Drying (168h/d0) 3.46 ± 0.40 Ab 2.59 ± 0.88 Ab 2.02 ± 1.00 Ab Log reduction* 5.04 log10 CFU/g 5.26 log10 CFU/g 5.79 log10 CFU/g Final Storage (648h/d20) 0.67 ± 0.23 Ac 1.66 ± 0.11 Ac 0.60 ± 0.23 Ab Total Reduction** 7.83 log10 CFU/g 6.19 log10 CFU/g 7.21 log10 CFU/g Note: ± denotes the standard errors of the mean, log reductions calculated by subtracting final log counts from initial log counts. Log reductions with different upper case letter superscripts in the same row and different lowercase letters in the same column are significantly different (p <0.05).*Log reduction calculated as Prior LA(Initial) log count – After drying log count. **Total log reductions calculated as Prior LA(Initial)-Final Storage(648h/d20).

120

Figure 1

6.5

5.5 pH

4.5

4 0 24 48 72 96 168 216 264 360 456 552 648 Time (hours)

Control pH TMT pH

Note: Control = non-inoculated sausages; TMT = sausages inoculated with pathogen cocktail. Control sausages serve as a point of comparison for a normal fermentation, indicating that the addition of high amounts of pathogens did not adversely affect the process.

121

Figure 2

0.95

0.9

0.85

0.8

WaterActivity 0.75

0.7

0.65 0 24 48 72 96 168 216 264 360 456 552 648 Time (hours)

Control aw TMT aw

Note: Control = non-inoculated sausages; TMT = sausages inoculated with pathogen cocktail. Control sausages serve as a point of comparison for a normal process, indicating that the addition of high amounts of pathogens did not adversely affect the process.

122

Figure 3

LogCounts (CFU/g) 2

0 0 24 72 96 168 216 264 360 456 552 648 Time (hours)

EC LM S

Note: Behavior and reduction trend of EC, LM and S cocktail in inoculated sausages throughout the sausage production and storage time (beginning of fermentation to last day of storage).

123

Appendix B

124

Table B1: Populations (log10 CFU/g) of STEC during processing of dry, fermented sausage. O26-O145 O103-O111 O121 O45-O157 Processing Time TSA SMAC TSA SMAC TSA SMAC TSA SMAC Steps Fermentation T0 7.55 ± 0.21AC 6.77 ± 0.62A 7.33 ± 0.43A 6.83 ± 0.50A 7.30 ± 0.25A 6.82 ± 0.36A 7.51 ± 0.31A 6.88 ± 0.62A Fermentation T96 7.83 ± 0.53AB 6.54 ± 0.61A 7.63 ± 0.32A 6.53 ± 0.43AB 7.85 ± 0.32A 6.23 ± 0.43A 7.76 ± 0.47A 6.23 ± 0.61A Drying T168 8.00 ± 0.63A 6.04 ± 0.26A 7.79 ± 0.61A 6.05 ± 0.13A 7.75 ± 0.61A 6.00 ± 0.13A 7.69 ± 0.51A 6.03 ± 0.26A Drying T264 7.68 ± 0.81A 5.61 ± 0.12A 7.78 ±0.38A 5.13 ± 0.76AB 7.72 ± 0.58A 5.78 ± 0.76A 8.03 ± 0.34A 5.58 ± 0.12A Drying T312 7.04 ± 0.02A 5.52 ± 0.57A 7.64 ±0.54A 5.55 ± 0.15A 7.29 ± 0.34A 5.56 ± 0.15A 7.80 ± 0.19A 5.44 ± 0.57A Storage ST1 6.81 ± 0.68ABC 4.63 ± 0.92AB 6.80 ± 0.45AB 4.63 ± 0.58AC 7.06 ± 0.50AB 4.77 ± 0.55AB 6.67 ± 0.36AB 4.09 ± 0.85AB Storage ST2 6.48 ± 0.35BC 4.13 ± 0.75B 6.48 ± 0.33AB 4.41 ± 0.27AC 6.65 ± 0.11AB 4.26 ± 0.42AB 5.73 ± 0.20B 3.89 ± 0.23B Storage ST3 6.30 ± 0.37BC 3.97 ± 0.50B 6.23 ± 0.32B 3.48 ± 0.47BC 6.33 ± 0.31B 4.00 ± 0.41B 5.67 ± 0.22B 3.42 ± 1.11B Storage ST4 6.27 ± 0.08C 2.87 ± 0.42B 6.13 ± 0.09 B 3.26 ± 0.48C 6.23 ± 0.19B 3.31 ± 0.44B 5.88 ± 0.43B 2.60 ± 1.04B Note: ± denotes SE of LSM. Different superscript letters within a particular a serogroup indicate significant differences. Significance was declared at α≤0.05.

125

VITA Minerva Rivera

Education Doctor of Philosophy (PhD) in Food Science The Pennsylvania State University, University Park, PA, USA Graduated 2017

Master of Science (MS) in Food Science University of Puerto Rico-Mayagüez, Mayagüez, P.R. Graduated 2012

Bachelor of Science (BS) in Animal Science Cum Laude University of Puerto Rico-Mayagüez, Mayagüez, P.R. Graduated 2006

Publications Minerva Rivera-Reyes, Jonathan A. Campbell, Catherine N. Cutter, Pathogen reductions associated with traditional processing of landjäger, Food Control, Volume 73, Part B, March 2017, Pages 768-774. Honors and Awards 2nd Place Penn State Gamma Sigma Delta (2016) Graduate Research Expo – Biological Sciences 3rd Place IFTSA & MARS Competition (2015)- Product Development Team 3rd Place International Association of Food Protection New Developing Scientist Award (2015) – Research Poster Presentation Abstract: Rivera, M., Campbell, J., and Cutter, C. N (2015) Pathogen Reductions Associated with Traditional Processing of Landjäger: A Pilot Study Edith and William B. Rosskam, II Memorial Scholarship in Food Science (2014) Consumer Food Safety Education Travel Conference Award (2014). USDA National Needs Fellowship (2013) New Product Development Proposal & Presentation Award (2008). Undergraduate Summer Research Scientific Poster Presentation Award (2004)

126