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The Fate of Escherichia coli 0157:H7 when exposed to sublethal and lethal concentrations of common industrial sanitizers

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The Fate of Escherichia coli 0157:H7 when exposed to sublethal and lethal concentrations of common industrial sanitizers
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Hunt, Kristen Ann
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English
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xii, 86 leaves : ill. ; 29 cm.

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Antibiotics ( jstor )
Bacteria ( jstor )
Chemicals ( jstor )
Chlorine ( jstor )
Escherichia coli ( jstor )
Food ( jstor )
Microbial sensitivity tests ( jstor )
pH ( jstor )
Quaternary ammonium compounds ( jstor )
Sanitizing ( jstor )
Animal Sciences thesis, Ph.D ( lcsh )
Dissertations, Academic -- Animal Sciences -- UF ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis (Ph.D.)--University of Florida, 2003.
Bibliography:
Includes bibliographical references.
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Printout.
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Vita.
Statement of Responsibility:
by Kristen Ann Hunt.

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THE FATE OF Escherichia coli 0157:H7 WHEN EXPOSED TO SUBLETHAL AND
LETHAL CONCENTRATIONS OF COMMON INDUSTRIAL SANITIZERS


















By

KRISTEN ANN HUNT


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2003
































Copyright 2003

by

Kristen Ann Hunt

































I dedicate my dissertation to my husband, Philip, and to my two sons, Aaron and Jared. Their love, pride and guidance helped to keep me disciplined and motivated throughout my studies.















ACKNOWLEDGMENTS

I extend my sincere admiration and gratitude to my committee chairperson,

professor and advisor, and friend, Dr. Roger L. West, for his continual guidance and support. I also wish to express my appreciation to the members of my supervisory committee, Dr. D. Dwain Johnson, Dr. Sally K. Williams, and Dr. Gary E. Rodrick, for their help in the completion of this project. Special thanks are given to Dr. West and the Department of Animal Sciences for financial support throughout my graduate studies at the University of Florida and this project.

Special recognition is given to Deibel Laboratories, Incorporated for the use of

their facilities, laboratory equipment, personnel and supplies. I wish to personally thank Dr. Robert H. Deibel for his support and understanding throughout the completion of this project. I also wish to thank LeaAnne B. Green as well, not only for her assistance but for the constant encouragement, non-judgmental listening, and above all else, her friendship.

In addition, I would like to recognize Larry Eubanks, Byron Davis and Tommy and Brian Estevez for the friendship and humor they provided to me throughout this project. Regards go to fellow graduate students Gabriel Cosenza, Robin Hamm and Ben Warren for their camaraderie throughout our studies. In particular, I wish to thank Ben for his assistance with the statistical analysis of my project.

My husband, Philip Hunt, and my parents, Steve and Colleen Goodfellow, deserve special credit for putting up with me throughout my studies, qualifying exams, and in


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particular, the completion of my dissertation. I sincerely thank them for their love, understanding and support. I thank them all for believing in me and encouraging me to better myself and above all else, I thank them for their patience. And, I thank God for helping me to keep the heart, faith and mind to make my dreams become realities.


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TABLE OF CONTENTS
page

ACKN OW LED GM ENTS ................................................................................................. Iv

LIST OF TABLES ........................................................................................................... viii

LIST OF FIGURES ........................................................................................................ x

ABSTRACT....................................................................................................................... xi

CHAPTER

I INTROD UCTIO N .................................................................................................... 1

2 REVIEW OF LITERATURE .......................................................................................5

Characteristics of Escherichia coli 0 157:H 7 ........................................................... 5
Clinical A spects of Escherichia coli 0 157:H7......................................................... 7
Prevalence of Escherichia coli 0 157:H 7 ................................................................. 9
Selected O utbreaks of Escherichia coli 0 157:H7 .................................................. 10
Resistance and Adaptation Characteristics of Escherichia coli 0157:H7..............13
Acid Resistance and Acid Tolerance................................................................ 13
Antibiotic Resistance....................................................................................... 19
Therm al Tolerance and Adaptation ................................................................ 23
Adaptation and Tolerance of Sanitizing Agents.............................................. 25
Chem ical Sanitizers in the Food Industry.............................................................. 27
Chlorine Sanitizers ...........................................................................................28
Q uaternary Am m onium Sanitizers .................................................................. 29
Peroxyacetic Acid Sanitizers ........................................................................... 30
Iodine Sanitizers ............................................................................................. 31
The Ideal Sanitizer.................................................................................................. 32
Surfaces and Attachm ent ........................................................................................ 33
Recovery of Stressed M icroorganism s .................................................................. 34

3 M ATERIALS AND M ETHOD S .......................................................................... 36

Bacterial Cultures ....................................................................................................36
Sanitizer Solutions .................................................................................................. 40
Determination of Minimum Inhibitory Concentration (MIC)................................40
Zone Inhibition ...................................................................................................... 42
Planktonic Bacteria................................................................................................ 43


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Bacterial Reassessm ent........................................................................................... 44
Solid Substrate ............................................................................................................45
Adhesion of M icrobial Cells.................................................................................. 45
Sanitizer Treatm ents for Adherent Cells ............................................................... 46
M icrobiological Analysis....................................................................................... 46
Statistical Analysis.................................................................................................. 47

4 RESULTS AND DISCUSSION ..............................................................................48

Determ ination of M inim um Inhibitory Concentration ...............................................48
Planktonic Bacteria................................................................................................ 50
Chlorine Com pound .........................................................................................50
Quaternary Am m onium Com pound....................................................................53
Peroxyacetic Acid Com pound......................................................................... 56
Iodine Com pound ........................................................................................... 59
Bacterial Reassessm ent........................................................................................... 60
Adherent Bacteria .................................................................................................. 63
Attachm ent Levels........................................................................................... 63
Sanitizer Treatm ents for Adherent Cells ......................................................... 64

5 SUM M ARY AND CONCLUSION S .................................................................... 71

REFERENCES ..................................................................................................................75

BIOGRAPHICAL SKETCH ......................................................................................... 86




























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LIST OF TABLES


Table page

1 The minimum inhibitory concentrations (MICs) of various sanitizers against
Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 after
incubation at 37'C for 24 hours .......................................................................... 48

2 Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia
coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to
1.0 mg/L of Clorox@ for 5 minutes at 22'C ........................................................ 51

3 Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia
coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 0.5
mg/L of Clorox@ for 5 minutes at 22'C ............................................................ 52

4 Mean log values by day (n= 18) for planktonic bacterial isolates of Escherichia
coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to
10.0 mg/L of Zepamine-ATM for 5 minutes at 220C ..........................................53

5 Mean log values by day (n= 18) for planktonic bacterial isolates of Escherichia
coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to
11.0 mg/L of Zepamine-ATM for 5 minutes at 220C ..........................................55

6 Mean log reduction values by trial (n=18) for Escherichia coli 0157:H7 strain
ATCC 700599 and strain FSIS 063-93 when exposed to 11.0 mg/L of ZepamineA TM for 5 m inutes at 22'C .................................................................................... 56

7 Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia
coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 1.0
mg/L per 4.4 mg/L of Zep-PerosanTM for 5 minutes at 22'C...............................57

8 Mean log reduction values by trial (n=18) for Escherichia coli 0157:H7 strain
ATCC 700599 and strain FSIS 063-93 when exposed to 1.0 mg/L of
peroxyacetic acid per 4.4 mg/L of hydrogen peroxide of Zep-PerosanTM for
5 m inutes at 22'C ................................................................................................. 57

9 Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia
coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 2.0
mg/L per 8.8 mg/L of Zep-PerosanTM for 5 minutes at 22'C...............................58


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10 Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia
coli 0157:H7 strain FSIS 063-93 when exposed to 0.25 mg/L of Zep-I-DineTM
and 0.50 mg/L of Zep-I-Dine for 5 minutes at 22'C...........................................60

11 The minimum inhibitory concentrations for Escherichia coli 0157:H7 strain
ATCC 700599 before and after pretreatment with sub-lethal levels of various
san itiz ers ...................................................................................................................6 1

12 The minimum inhibitory concentrations for Escherichia coli 0157:H7 strain
FSIS 063-93 before and after pretreatment with sub-lethal levels of various
san itiz ers ...................................................................................................................6 2

13 The minimum inhibitory concentrations of various sanitizers for Escherichia
coli 0157:H7 strain FSIS 063-93 planktonic bacterial isolates before and after
pretreatment with sub-lethal levels of Zepamine-ATM ........................................63

14 Mean log values (n=18) for planktonic and adherent bacterial isolates of
Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when
exposed to 1.0 mg/L of Clorox@ for 5 minutes at 22'C.....................................65

15 Mean log values (n= 18) for planktonic and adherent bacterial isolates of
Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when
exposed to 11.0 mg/L of Zepamine-ATM for 5 minutes at 22'C ..........................66

16 Mean log values (n= 18) for planktonic and adherent bacterial isolates of
Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when
exposed to 3.0 mg/L per 13.2mg/L of Zep-PerosanTM for 5 minutes at 22'C .........68

17 Mean log values (n=18) for planktonic and adherent bacterial isolates of
Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when
exposed to 0.5 mg/L of Zep-I-DineTM for 5 minutes at 22'C .............................69


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LIST OF FIGURES


Figure page

1 Absorbance of Escherichia coli 0157:H7, strain ATCC 700599, in Lauryl
Sulfate broth over 24 hours ................................................................................. 38

2 Growth of Escherichia coli 0157:H7, strain ATCC 700599, on Violet Red Bile
A gar over 24 hours............................................................................................... 38

3 Absorbance of Escherichia coli 0157:H7, strain FSIS 063-93, in Lauryl Sulfate
broth over 24 hours ........................................................................................... 39

4 Growth of Escherichia coli 0157:H7, strain FSIS 063-93, on Violet Red Bile
A gar over 24 hours ............................................................................................... 39

5 Escherichia coli 0157:H7 strain ATCC 700599 Standard Curve ......................41

6 Escherichia coli 0157:H7 strain FSIS 063-93 Standard Curve..........................42


x















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

THE FATE OF ESCHERICHIA COLI 0157:H7 WHEN EXPOSED TO SUBLETHAL AND LETHAL CONCENTRATIONS OF COMMON INDUSTRIAL SANITIZERS By

Kristen Ann Hunt

August 2003

Chair: Roger L. West
Major Department: Animal Sciences

This study tested the hypothesis that pre-exposure of Escherichia coli 0157:H7 to sub-lethal levels of industrial sanitizers could affect the survival of cells to subsequent exposure at lethal levels. The susceptibility of planktonic and adherent cells to sanitizing compounds was compared. The ability for an acid tolerant Escherichia coli 0157:H7 strain to provide cross-protection to the cells when exposed to chemical sanitizers was also examined.

E. coli 0157:H7 cells were exposed to a chlorine compound, an iodophor, a quaternary ammonium compound (quat) and a peroxyacetic acid compound (PAA). Results show that at the concentrations utilized in this study, the iodophor provided the greatest reduction in planktonic cells followed by chlorine, then quat and the PAA compound. All sanitizers, except the peroxyacetic acid, were effective (greater than 5 log reduction) against the planktonic Escherichia coli 0157:H7 bacterial isolates. Data from the planktonic stage of testing showed that lack of recovery by plate count method after


xi









sanitizer treatment did not mean that an organism was no longer present. Both strains demonstrated the ability to recover and grow in broth after treatment with all of the sanitizers

All sanitizers tested were significantly less effective against the adherent cells than the planktonic cells. The iodophor compound was the only sanitizer found to be effective against both planktonic and adherent cells. Neither the chlorine compound nor the PAA compound was effective against the adherent cells. The quaternary ammonium compound was only effective against the acid tolerant strain for adherent cells.

Throughout testing, the un-adapted Escherichia coli 0157:H7 strain typically

showed higher survival rates than the known acid tolerant strain. Additionally, with one exception, the bacterial cells that exhibited an increased minimum inhibitory concentration did not demonstrate any increased resistance when exposed to other sanitizers.

While the pretreatment of bacterial cells with a quaternary ammonium and a

peroxyacetic acid compound resulted in survival at higher concentrations, the values were still far below the recommended usage level. This adaptation does however demonstrate the importance of proper cleaning and sanitation procedures to ensure a safe product.


xii














CHAPTER 1
INTRODUCTION

Escherichia coli 0157:H7 is an important foodborne pathogen not only in the

United States but globally (80, 95, 123, 129, 138). Increased knowledge and awareness of this human pathogenic bacterium by the scientific community and the general populace along with better surveillance systems and techniques have contributed to partial control of this organism. Despite the increased recognition and research devoted to E. coli 0157:H7, it remains a public health problem (129). Each year approximately 76 million people experience foodborne illness (84). The Centers for Disease Control (CDC) estimates that 73,000 persons become ill and 61 people die annually in the United States alone as a result of infection from E. coli 0157:H7 (24, 28).

In addition to being a public health problem, Escherichia coli 0157:H7 is a familiar foe to the food industry. Even with governmental regulations and Hazard Analysis Critical Control Point (HACCP) in place, the industry continues to be plagued by this organism (1, 2, 8). Millions of pounds of "contaminated" ground beef are recalled annually (8, 41, 46, 47). Although E. coli 0157:H7 was initially viewed as a threat associated primarily with ground beef, it now shows up in raw vegetables, unpasteurized juice, dairy products, and in the water in which we swim or drink (1, 2, 8, 25, 41, 95). Even foods which were once considered to be "safe" from enteric infections such as drycured salami and fermented sausage have now been associated with E. coli 0157:H7 infections and outbreaks (41, 129). In recent years, outbreaks and sporadic incidences of


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illness attributed to E. coli 0157:H7 have occurred in foods such as coleslaw, iceburg and romaine lettuce, fruit salad, cheese curds, yogurt, and even in cake (25, 41).

Research has shown that Escherichia coli 0157:H7 is an adaptive organism that can survive hostile and harsh environments (8, 38, 39, 41). The organism is a hardy pathogen that has managed to survive and adapt to varied environmental stresses (39). Due to the hardy nature of the organism and its ability to adapt to new environments the need to be able to control or eliminate Escherichia coli 0157:H7 is of critical importance to the food industry (8, 103). Conditions designed to reduce bacterial counts such as refrigeration, freezing, and treatment of the carcass(es) with organic acids may in fact enhance the survival of the organism (132). When bacteria are exposed to certain food processing treatments designed to extend shelf life, sub-lethal injury can result. It is possible that bacteria which have been subjected to heating, refrigeration, freezing, acid, low pH, and even sanitizers may be present in foods. From a food safety standpoint this poses an unacceptable risk as inactivated or sub-lethally injured bacteria may be able to undergo repair and resume growth (120).

As the majority of the responsibility for providing a "safe" product now falls on the food industry, it is essential to have and maintain an effective sanitation program (8, 81). Sanitation procedures are implemented in the food industry to aid in the production of a safe product with an acceptable level of quality (93). As defined in Principles of Food Sanitation, in regard to the food industry, sanitation is the "creation and maintenance of hygienic and healthful conditions." In regard to science, sanitation is "to provide wholesome food handled in a clean environment by healthy food handlers, to prevent contamination with microorganisms that cause foodborne illness and to minimize the






3


proliferation of food spoilage microorganisms" (82, p.2). In simpler terms, sanitation is reducing the number of bacteria present on a surface. Sanitation does not mean sterile; in other words there may still be some bacteria present on a surface that has been sanitized.

Three categories of sanitizers exist. They include physical sanitizing, radiation sanitizing, and chemical sanitizing. For this study, the focus will be on chemical sanitizing. The category of chemical sanitizers is extremely broad. It includes chlorine compounds, iodine compounds, bromine compounds, quaternary ammonium compounds, acid sanitizers, acid anionic sanitizers, acid-quaternary sanitizers, hydrogen peroxide compounds, peroxyacetic acids, ozone, glutaraldehydes, and microbicides (82).

Chemical sanitizers are deemed to be effective on food contact surfaces if they demonstrate a five-log reduction in planktonic bacteria (43). Efficacy testing of sanitizers with non-adherent bacteria could be misleading as to the sanitizers' true effectiveness under processing conditions where the bacterial cells may be attached to a variety of surfaces. Research has found that various sanitizers failed to provide an adequate reduction (three-log) in attached bacteria where they had clearly demonstrated the ability to be effective against planktonic bacterial cells (71, 93).

This study tested the hypothesis that pre-exposure of Escherichia coli 0157:H7 to sub-lethal levels of common industrial sanitizers could affect the survival of cells subsequently exposed to lethal levels of the sanitizers. The susceptibility of planktonic cells to sanitizing compounds was compared to the susceptibility of adherent cells when exposed to the same chemical treatment. The chemical sanitizers utilized included a sodium hypochlorite solution (bleach), a peroxyacetic acid compound (Zep-PerosanTM), a quaternary ammonium compound (Zepamine ATM), and an iodine compound (Zep-I-






4


DineTM). Additionally, the survival of E. coli 0157:H7 cells exposed to sub-lethal levels of the sanitizers and the ability to recover and resuscitate said cells was investigated. Data were collected and compared for an un-adapted E. coli 0157:H7 strain as well as for an acid resistant strain.














CHAPTER 2
REVIEW OF LITERATURE

Characteristics of Escherichia coli 0157:H7

Escherichia coli 0157:H7 is a small, gram-negative, non-sporing, straight rod. This pathogenic organism is a facultative anaerobe and can therefore grow in the presence or absence of oxygen (1, 2, 7). It grows rapidly from 30'C to 42'C with generation times ranging from 0.49 hr at 37'C to 0.64 hr at 420C (40). At temperatures of 440C to 45'C, the organism grows poorly (104). Thermal inactivation studies of E. coli 0157:H7 in ground beef demonstrated that the pathogen has no unusual resistance to heat with D values of 270, 45, 24, and 9.6s at 57.2, 60.0, 67.8 and 64.3'C, respectively (40). However, it does appear to survive well in frozen storage at -20'C. Doyle and Schoeni

(40) found that Escherichia coli 0157:H7 could survive in ground beef when frozen at

-80'C and held at -20'C with no major changes in population for up to nine months.

Escherichia coli 0157:H7 is one of hundreds of strains of the bacterium

Escherichia coli. Most E. coli strains are harmless; however, some, such as Escherichia coli 0157:H7, are pathogenic and cause diarrheal illness (24, 41). The strains that cause disease are categorized into specific groups based on virulence properties, mechanisms of pathogenicity, clinical syndromes, and distinct O:H serogroups (41, 95). Escherichia coli 0157:H7 is a member of the enterohemorrhagic E. coli (EHEC) group (1, 2, 41). Additionally, isolates of E. coli are serologically differentiated on the basis of three major surface antigens. The 0 (somatic) antigen, the H (flagella) antigen and the K (capsular)


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antigen enable serotyping of the organism. Currently, at least 174 0, 56 H, and 80 K antigens have been identified (41).

Members of the EHEC group, such as Escherichia coli 0157:H7, are clonal in origin and phenotypically and genotypically very similar (73). The bacterium Escherichia coli 0157:H7 is distinct from other strains in that it is unable to ferment sorbitol in 24 hours and does not produce p-glucuronidase (41, 73, 98). It possesses a 60MDa plasmid, expresses an uncommon 5,000 to 8,000 molecular weight outer membrane protein, and has an attaching and effacing (eae) gene (41, 73, 95). The eae gene is responsible for the production of an attaching and effacing lesion (A/E) that causes the degeneration and effacement of intestinal epithelial cell microvilli, intimate adherence of bacteria to the epithelial cells, and assembly of highly organized cytoskeletal structures in the cells beneath intimately attached bacteria (69). The cytoskeletal structures are composed of components such as actin, talin, ezrin, and a-actinin (65). Once Escherichia coli 0157:H7 adheres to the bowel mucosa, it will grow and secrete an array of extracellular products including potent cytotoxins (73). These cytotoxins are referred to as Shiga toxins, Shiga-like toxins and verotoxins (95).

The mechanism by which E. coli 0157:H7 causes disease is not yet fully

understood, but an important factor in its virulence is the production of verotoxins (38). Escherichia coli 0157:H7 can produce Shiga toxin 1 (Stx 1), Shiga toxin 2 (Stx 2), or both (95). Most isolates of E. coli 0157 produce only Stx 2; occasionally isolates produce both Stx 1 and Stx 2, and rarely isolates are found to produce only Stx 1 (54). Shiga toxin I is homogeneous where Shiga toxin 2 is comprised of numerous variants. The toxins share about 60 percent of their DNA and amino acid homology but are






7


immunologically distinct (73). Both are compound toxins consisting of an A subunit and a pentameric B subunit that are the active and binding portions of the toxins, respectively (38, 73). The hollow ring formed by the B subunit is where the C-terminus end of the A subunit is inserted. The B subunits mediate binding to specific glycosphingolipids, termed globotriosyl ceramides (Gb3 and Gb4), which are receptors on the surface of cells of specific body tissues in eukaryotes (38, 73). The globotriosyl ceramide, Gb3, is abundant in the cortex of the human kidney (38). Both toxins have similar modes of action, which involve blocking protein synthesis by inhibiting elongation factor-idependent aminoacyl binding of t-RNA to ribosomal subunits (38, 73). The toxicity of the two toxins however is dissimilar. Shiga toxin 2 has been found to be 1,000 times more cytotoxic than Shiga toxin I towards human renal microvascular endothelial cells

(73). Although Stx 1 and Stx 2 have similar structures and similar mechanisms of action, the genes for Shiga toxin 1 and Shiga toxin 2 are carried on two separate bacteriophages. Bacteriophages are viruses capable of infecting bacterial cells (1, 2).

Clinical Aspects of Escherichia coli 0157:H7

Illness associated with Escherichia coli 0157:H7 is primarily the result of a

foodborne infection. The disease may also be transmitted via person to person, especially in institutional settings or child care facilities. Escherichia coli 0157:H7 can also be transmitted by consumption of inadequately chlorinated drinking water or by swimming in contaminated lakes and pools (24, 25, 41, 95). Once the bacterium has been ingested, the onset of illness averages from 18 to 36 hours (7). Under the right conditions, anyone can become infected with Escherichia coli 0157:117, but many will be asymptomatic

(41). As with many foodborne infections, the susceptibility of those exposed varies greatly with the very young, the elderly and the immunocompromised being more likely






8


to develop severe illness and serious complications that may lead to chronic sequelae or death (1, 2, 25, 41). Other factors such as the number of bacteria ingested, the amount of toxin produced in the intestine, the degree to which the toxin is absorbed and the sensitivity of an individual's cells to the toxin also contribute to the severity of the disease (1, 2).

An Escherichia coli 0157:H7 infection may involve a variety of symptoms

including mild diarrhea, hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS), and thrombotic thrombocytopenic purpura (TTP) (24, 38). Hemorrhagic colitis consists of sudden and severe abdominal cramping followed by watery diarrhea that progresses to grossly bloody diarrhea. Vomiting may also occur but there is little to no fever with the illness and the condition can last an average of four to ten days (38, 41, 98). Approximately 5 to 7 percent of persons infected will suffer from HUS. Hemolytic uremic syndrome generally affects children and has a 3 to 5 percent mortality rate (24). Hemolytic uremic syndrome causes red blood cells within the capillaries of the kidneys and other organs to clot, resulting in the accumulation of waste products in the blood, kidney failure, heart failure, blindness, seizures, strokes, coma and death (24, 38). One third of those with hemolytic uremic syndrome will have abnormal kidney function for many years, some will require long term dialysis, and 8 percent of HUS patients will have lifelong complications such as high blood pressure, seizures, blindness, paralysis or the effects of having part of their bowel removed (24). Rarely, a person will develop thrombotic thrombocytopenic purpura. This largely affects adults and histologically resembles HUS except that the central nervous system is principally involved and there is






9


a high mortality rate. In such cases, blood clots develop in the brain causing neurological abnormalities and death (38, 41, 98).

The infectious dose of the microorganism is unknown (41). It is believed to be very low with the Centers for Disease Control estimating that as little as 10 organisms would be able to cause illness (24). Analysis of frozen ground beef patties involved in an outbreak revealed that as little as 0.3 cells per gram to 15 cells per gram caused illness. Similarly, analysis of salami associated with a foodborne outbreak showed that 0.3 to 0.4 cells of Escherichia coli 0157:H7 per gram of salami were able to cause illness (41). Additional evidence of a low infectious dose is the capability of the disease to be transmitted from person to person as well as in water (38, 41).

Prevalence of Escherichia coli 0157:H7

Studies performed by the Centers for Disease Control have established a yearly baseline of Escherichia coli 0157:H7 of an average of 2.4 cases per 100,000 persons

(28). Cases in Canada reported over a five year period spanning from 1990 to 1994 ranged from 3.0 to 5.3 per 100,000 (138). In the United Kingdom there were considerable regional variations in the isolation rates within individual countries. In Northern Ireland, there have been fewer than 3 isolates per year since 1989. In contrast, the mean annual isolation rate in England and Wales in 1994 was 0.80 per 100,000 with Scotland experiencing substantially higher incidence. The mean annual isolation rate in Scotland was found to be 2.4 per 100,000 inhabitants. The reason for this variation is unknown (123). Epidemiologic data from Argentina show that this country has the highest rate in the world. With data regarding only children from 6 to 48 months in age, the incidence of infection annually is approximately 22 per 100,000 in Buenos Aires alone. The incidence of disease related to Escherichia coli 0157:H7 in this country






10


appears to be 7 to 10 times greater than data reported from "high-risk" areas in the world

(80). Data reported do not seem to indicate any pattern. The incidence of infection, even in the United States, is sporadic. In the United States the numbers of incidence recorded annually have roughly remained the same with slight increases or decreases occurring each year. The reported cases were 2.7, 2.3, 2.8, 2.1, 2.9, and 2.1 per 100,000 persons from 1996 to 2001, respectively (28).

Selected Outbreaks of Escherichia coli O157:H7

Escherichia coli 0157:H7 has been isolated from samples of foods linked to human illness (54). While E. coli 0157:H7 is typically associated with foods of bovine origin, it has been found in a variety of food vehicles including ground beef, raw milk, yogurt, roast beef, salad dressing, cantaloupe, coleslaw, cake, mayonnaise, orange juice, apple cider, and water, both in un-chlorinated municipal water and swimming water (28, 41, 137).

The first documented outbreak of Escherichia coli 0157:H7 occurred in Oregon in 1982, with 26 cases and 19 persons requiring hospitalization (41, 137). All patients had bloody diarrhea and severe abdominal pain with the age of those infected ranging from 8 to 76 years. This outbreak was associated with eating undercooked hamburgers from fast food restaurants of a particular chain. Only 3 months later, a second outbreak occurred with the same fast food chain being implicated again. This time the incidence occurred in Michigan with 21 cases and 14 persons being hospitalized. The range in age spanned from 4 years to 58 years. Escherichia coli 0157:H7 was isolated from the patients and frozen ground beef patties (109).

A large outbreak of E. coli 0157:H7 associated with contaminated municipal

drinking water occurred in Missouri between December of 1989 and January of 1990. Of






I I


the 243 persons affected, 86 had bloody diarrhea, 32 were hospitalized and 4 died. The four persons where the infection resulted in death were women 79 years of age or older. The outbreak occurred after two large water mains broke as a result of cold weather and before chlorination of the water supply (128).

In the fall of 1991 an outbreak of Escherichia coli 0157:H7 occurred in

Massachusetts involving 23 cases of which 6 required hospitalization. The Centers for Disease Control and the State of Massachusetts public health officials found that freshpressed apple cider made at one mill was significantly associated with the illness (16, 137). Escherichia coli 0157:H7 was not isolated from the apple cider made by the implicated processor, but it was revealed in inoculated studies that the pathogen could survive in apple cider for twenty days at 8'C (141).

The largest outbreak documented in the United States occurred in 1993 and

involved four Western states, Washington, Idaho, California and Nevada. The incident resulted from eating undercooked hamburgers from a single fast-food chain. In all, 731 cases were identified: 629 in Washington, 13 in Idaho, 57 in Nevada, and 34 in California. Of the 629 cases in Washington, 48 were found to be the result of person-toperson transmission. The age of patients ranged from 4 months to 88 years and before the outbreak was over, a total of 178 persons were hospitalized, 56 developed hemolytic uremic syndrome (HUS), and 4 children died (41, 133, 137).

In 1994, an unusual outbreak of E. coli 0157:H7 occurred in Washington and California that involved 19 cases. The food associated with the incident was dry fermented salami. Among the 15 confirmed cases in Washington, 3 patients developed HUS and of the 4 cases in California, 2 patients developed HUS. The age of the persons






12


affected ranged from 6 to 77 years of age (23). Dry cured salami is not cooked but is fermented and dried. Inoculated studies have revealed that Escherichia coli 0157:H7 can survive the fermentation, drying and storage processes involved in the production of fermented sausage (52).

More recently, the first reported outbreak of E. coli 0157:H7 in the United States involving the direct transmission of the pathogen from farm animals to humans occurred during the spring and fall of 2000 in Pennsylvania and Washington. The outbreak resulted in 56 cases of illness of which 19 required hospitalization. The persons affected were preschool to school age children. The farm implicated had no separate area designated for the interaction between visitors and farm animals and the wash areas were not supplied with soap (26).

One of the largest product recalls in the history of the United States resulted

following a multi-state outbreak of Escherichia coli 0157:H7 in 2002. The Colorado Department of Public Health and Environment identified the outbreak among Colorado residents. To date, six other states have also been linked to the outbreak involving 28 cases of which 7 required hospitalization. The outbreak has been linked to the consumption of contaminated ground beef and ground beef products recalled by the Con Agra Beef Company. The recall, which originally involved 354,200 pounds of ground beef, preceded the outbreak. The recall was initiated due to detection of E. coli 0157:H7 during routine microbiological testing by the United States Department of Agriculture (USDA). Following the detection of this multi-state outbreak and the initiation of an inplant inspection of the Con Agra Beef Company by USDA, the nationwide recall of






13


354,200 pounds was expanded to a recall of 18.6 million pounds of fresh and frozen ground beef and ground beef trimmings (27).

As a direct result of these outbreaks and the many others that have occurred, changes in food processing procedures and regulatory actions have been implemented in an attempt to prevent further outbreaks from occurring. Ultimately, we are still "searching for solutions" (8).

Resistance and Adaptation Characteristics of Escherichia coli 0157:H7

Escherichia coli 0157:H7 is an organism capable of adapting to new and hostile environments in order to survive (8). Some have suggested that Escherichia coli 0157:H7 cells can enter into a viable but non-culturable (VBNC) state and survive for long periods of time. Regardless of the existence of a VBNC state, the organism has proven to be a hardy pathogen that has managed to survive and adapt to varied environmental stresses (39). The hurdle approach to food processing may not be sufficient to eliminate or reduce to an acceptable level this pathogenic organism. In fact, such treatments may in fact enhance the survival of the organism (120, 132). Additionally, concern has recently been raised that pathogenic microorganisms like Escherichia coli 0157:H7 can develop resistance to the antimicrobials and sanitizing agents used in food processing and manufacturing (33). Acid Resistance and Acid Tolerance

Unlike many foodborne pathogens, E. coli 0157:H7 has proven to be uniquely

tolerant to acidic environments (31, 38, 41, 117, 132). Although it will not flourish at pH values below 5.5, it is able to survive at pH values as low as 2.0 (36). Escherichia coli 0157:H7 can also survive extended storage at low pH values and at low temperatures. Glass and others (52) showed that E. coli 0157:H7 can survive the fermentation, drying






14


and storage process in fermented sausage with a pH of 4.5 at 4'C for up to two months with only a slight reduction in cell populations. In inoculated studies, Escherichia coli 0157:H7 survived in ketchup with a pH of 3.6 to 3.9 at 5'C for up to seven weeks (140). Zhao and others (141) reported that E. coli 0157:H7 survived for up to thirty-one days in unpasteurized apple cider with a pH of 3.6 to 4.0 at 80C.

Resistance of Escherichia coli 0157:H7 to acidic conditions may be the result of a genetically induced acid response system (51). Acid resistance, also termed acid tolerance response (ATR), and acid tolerance, also termed acid shock response (ASR), are considered to be important determinants in Escherichia coli 0157:H7's virulence, and contribute to its ability to survive and cause infection (21, 51). As defined by Foster (49), acid resistance (ATR) occurs when a microorganism is exposed for an extended period of time to moderately acidic conditions, for example, a pH of 5.0, triggering a response which enables it to be able to withstand pH values of < 2.5. The ATR requires the induction of protein synthesis to provide protection against acid stress. In ATR, certain outer membrane components of E. coli are genetically modified to protect against internal cell acidification by preventing the passage of hydrogen ions into the cell (112). Conversely, acid tolerance (ASR) occurs when a microorganism exhibits enhanced survival when exposed to pH values between 2.5 to 4.0 after no exposure or only a brief exposure to moderately acidic conditions (49). Both responses appear to result in the production of proteins responsible for the prevention of or the recovery from damage to cells (105, 106). Heyde and Portalier (59) found that a shift from pH of 6.9 to 4.3 induced at least sixteen polypeptides, seven of which were identified as acid shock proteins.






15


Garren and others (51) investigated the survival of Escherichia coli 0157:H7 and non-0157:H7 isolates due to induced acid tolerance (ATR) or acid shock responses (ASR) when exposed to lactic acid. Both treatment groups were incubated at 25'C and 32'C for twenty one days to determine if E. coli isolates could demonstrate a sustained ATR or ASR. Temperature, pH, strain of E. coli, and phase of growth were all important variables in the survival of both acid tolerance and acid shock treated isolates of Escherichia coli. Highest survival rates for all isolates occurred at a pH of 4.0 at 250C while no detectable survivors were found at a pH of 3.5 at 32'C. Pathogenic isolates (0157:H7) outperformed non-pathogenic strains.

In cases where a difference occurred, acid shocked cells had approximately a twolog higher survival rate than acid tolerance (acid adapted) cells, likely due to the growth phase of the cells. Law (73) stated that stationary-phase bacteria are 1,000 times more resistant to acid than exponentially growing organisms and do not need prior exposure to low pH to exhibit resistance. Benjamin and Datta (14) stated that there is an altered gene expression during stationary-phase. The rpo-regulated proteins which may provide resistance to chemical and physical stresses are associated with the stationary-phase. In addition, many physiological changes that are regulated by the rpoS gene product of this phase have been linked to increased acid resistance in enteric bacteria (29,48, 53, 59, 72, 76).

Garren and others (51) reported that acid shocked bacteria in foods could survive over a sustained period of time at lower temperatures provided that the contaminating bacteria are in stationary-phase. These results are important to the meat-processing industry as acidic dips and washes are utilized in an attempt to control microbial growth






16


on carcasses, and equally important to the general food industry as acidulants are used to extend shelf life and improve flavor (35, 121). Such treatments may induce ASR and cause the proteins needed for protection against this stress to be produced (51). Berry and Cutter (15) found that acid adapted Escherichia coli 0157:H7 negatively influenced the effectiveness of acetic acid washes in reducing the numbers of this organism on carcasses. Additionally, foods which rely on acidic pH to inactivate pathogens, such as fermented sausage, mayonnaise, yogurt, and apple juice, are known vehicles of infection with Escherichia coli 0157:H7 (85).

Buchanan and Edelson (21) studied the effect of acidulant identity on the pHdependent stationary-phase acid resistance response of enterohemorrhagic Escherichia coli cells grown in BHI (Brain Heart Infusion) broth at 37'C. The study utilized four acids, 0.5% citric, malic, lactic, and acetic, which were adjusted to a pH of 3.0 using hydrochloric acid. The results were compared to data utilizing only hydrochloric acid (HCI). Hydrochloric acid was found to be the least damaging to cells while lactic acid was the most detrimental. In general, acetic acid had a greater effect than citric or malic acid.

Buchanan and Edelson (21) found, as did Garren and others (51), that exponentially growing cells were more sensitive than those in stationary-phase. The exponential-phase cells were sensitive to all four acids with a four-log reduction in two hours whereas the stationary-phase cells decreased by less then one-log with all acids except for lactic acid. Additionally, the acid-adapted stationary-phase showed even further increased survival with only a two-log reduction when exposed to lactic acid. It again appears that maximum survival of Escherichia coli in strongly acidic environments is associated with






17


the stationary-phase acid habituation. Researchers (13, 20, 29, 76) suggested that both the constitutive rpoS gene-regulated and the inducible pH-dependent acid resistance systems are active in this acid-adapted stationary-phase.

The mechanism of acid tolerance has been speculated but not yet fully elucidated. Doyle and others (41) suggested that it is associated with protein(s) that may appear as a result of pre-exposure to acid conditions. Stress proteins have been found to enhance the ability of an organism to withstand a number of challenges such as hydrogen peroxide, acid and alkaline pH, heat and osmolarity (79).

Lin and others (78) proposed that there are several acid resistance systems that are involved in the acid tolerance of pathogenic Escherichia coli and each system is needed to survive the different acid stress environments of the stomach (pH 1 to 3) and the intestine (pH 4.5 to 7 with high concentrations of volatile fatty acids). Lin and others

(77) identified three distinct low-pH induced acid survival systems for Escherichia coli. They are an acid-induced oxidative system that requires rpoS, an acid-induced argininedependent system and a glutamate-dependent system (77). RpoS, an alternative sigma factor (o) that is involved in regulating the expression of a variety of stress response genes, is only partially involved in the later two systems (58, 72, 78, 96). The acid induced system is expressed in oxidatively metabolizing bacteria grown in complex media but will protect cells in minimal medium to pH 2.5. This system is not apparent in fermentatively metabolizing cells (77, 78). The arginine-dependent system involves an arginine decarboxylase system that utilizes adi, an arginine decarboxylase gene, and its regulators, cysB and adiY to provide acid resistance. This system will only function if






18


arginine is present (78). Similarly, the glutamate-dependent system will only function in the presence of glutamate (78).

When testing the three acid resistance systems against extreme acid exposure at a pH < 2.0, mild acid exposure at a pH of 4.0 utilizing benzoic acid, and mild acid exposure at a pH of 4.4 utilizing a volatile fatty acid (VFA) cocktail comprised of acetic, propionic and butyric acid at levels approximate to those in the intestine, Lin and others

(78) found that the involvement and effectiveness of each system varied. At extreme acid shock the oxidative system was ineffective with < 1 percent survival of enterohemorrhagic Escherichia coli strains. The arginine-dependent system had 10 to 50 percent survival at pH 2.5 and limited survival at pH 2.0. The glutamate-dependent system was effective at both a pH of 2.0 and 2.5 with 80 to 100 percent survival of EHEC isolates. In the weakly acidic benzoic acid solution, the glutamate system again was most effective with the oxidative system being modestly effective and the arginine-dependent system being ineffective. When exposed to the VFA cocktail, both the arginine and glutamate-dependent systems were very effective for at least 7 hours; however, the oxidative system was only mildly effective for 3 hours and ineffective at 7 hours. Lin and others (78) suggested that the arginine and glutamate-dependent systems functioned to maintain a less acidic intracellular pH in extremely acidic environments, while the oxidative system minimizes the actual damage to macromolecules.

Generally, it has been considered that all stages and components involved in stress tolerance induction are intracellular (94, 126). Rowbury and Goodson (113) proposed that stress responses may in fact be associated with the appearance in medium of extracellular agents that are essential for habituation. They termed such agents






19


"extracellular induction components" (EIC). Rowbury and Goodson (113) suggested that a heat-stable protein (EIC') is present in the media and is converted by mildly acidic pH (4.5 to 6.0) to an EIC, also a protein, that induces acid tolerance in Escherichia coli. While there is little information of the conversion of EIC' to EIC, Rowbury and Goodson (113) found that it doesn't involve proteolytic removal of the fragment because a mixture of protease inhibitors did not stop the conversion. EIC' is not significantly effected by chloramphenicol, not destroyed at 75'C or by exposure to pH 2.0 or pH 11.5. It is however reversibly activated to EIC at mildly acidic pH values (4.5 to 6.0). The benefit of an extracellular induction component would be that it might provide earlier warning of impending lethal stress.

Antibiotic Resistance

Escherichia coli 0157:H7 has further shown its ability to adapt to environmental conditions and stress with a new trend towards antibiotic resistance (41). Early research by Ratnam and others (107) revealed that E. coli 0157:H7 isolates were sensitive to most antibiotics. Ratnam and others (107) found that only 2.9 percent of 174 Escherichia coli 0157:H7 isolates were resistant to an antibiotic. Similarly, Kim and others (67) found that in 1987 all of the isolates that they tested were susceptible to the antibiotics tested. However, when they performed the study again in 1991, it was discovered that several of the strains had adapted and developed a resistance to streptomycin, sulfisoxazole, and tetracycline (67). Meng and others (86) found antibiotic resistance in E. coli 0157:H7 and E. coli 0157:NM strains isolated from animals, humans, and food. They found twenty-four percent of the isolates tested to be resistant to at least one antibiotic, nineteen percent of the isolated strains to be resistant to three or more antibiotics and two of the






20


isolated strains were resistant to six antibiotics including ampicillin, kanamycin, sulfisoxazole, streptomycin, tetracycline, and ticamillan.

Researchers today feel they have a relatively good insight into the mechanism(s) by which bacteria, such as Escherichia coli 0157:H7, have become resistant to antibiotics. As bacteria have evolved, they have developed diverse mechanisms to transmit resistance traits not only to members within their species but also to other species (66). The genetic traits that code for antibiotic resistance are located either in the chromosomes of the bacteria, in plasmids or transposons (86). Plasmids and transposons are extrachromosomal elements that a bacterium may possess and if a bacterium possesses either of these they may code for antibiotic resistance or may serve other functions (66, 86). Both plasmids and transposons consist of tiny circular DNA that is about 1 percent the size of a chromosome (66, 74).

There are three primary mechanisms of antibiotic resistance. First, resistance can be a natural attribute of an organism, such as an impermeable outer membrane that resists penetration (18, 97, 136). Gram-negative bacteria have a thick lipopolysaccharide layer that can act as a barrier to limit the diffusion of antibiotic molecules into the cell (18). Gram-negative bacteria are more resistant to lipophilic and amphiphilic inhibitors such as dyes, detergents, and antibiotics due to their outer membrane (97, 136). Additionally, gram-positive bacteria have lipophilic substances in their cell walls that retard penetration of hydrophilic, cationic and antimicrobial compounds (18). Second, resistance can be spontaneously acquired (18). Single point mutations to drugs can occur. In the lab, resistance to Naladixic Acid and Rifampin occurred spontaneously (66). Finally, resistance can be acquired through genetic exchange (4, 5, 66, 89).






21


Although all three modes of resistance are important, resistance through genetic exchange is the clinically relevant form of antibiotic resistance (114). As antibiotics utilized for therapeutic purposes generally have specific target sites in microbial cells, they have greater potential to result in mutations and in the development of acquired resistance (18, 116). Some of the methods of resistance that occur genetically include the inactivation of the antibiotic, an alteration of the target, synthesis of an alternate pathway, and efflux of the antibiotic (75). Genetic exchange of drug resistance has been documented through both epidemiological observations (11, 130) and experimental models (37). Additionally, Mizan and others (89) found that the transfer of genetic elements such as plasmids between microflora and enterohemorrhagic Escherichia coli appeared to occur readily in rumen fluid. Genetic exchange has been found to occur in three ways, transformation, transduction, and conjugation (4, 5, 18, 135).

Transformation is used to move DNA between bacteria, plants, and animals. In

transformation, DNA is removed from donor cells and added to recipient cells that are in a competent state (one that is capable of binding the DNA). The recipient cells either take the donor DNA into their cytoplasm where it may exchange into the recipient DNA or if it is a plasmid, it will replicate (135). Transformation involves many techniques such as the use of calcium chloride solutions, salt solutions, coated beads, and electricity depending upon if the organism is bacteria, a prokaryotic cell or a eukaryotic cell (4, 5)

Transduction involves the mediation of viruses called bacteriophages. The

bacteriophages actually transport the DNA. Scientists who were studying conjugation discovered transduction accidentally in 1952. Gene transfer occurred even when the two






22


bacterial membranes were separated and DNase did not inhibit the transfer. This process came to be known as transduction (5).

Transfer involving bacteriophages can be advantageous and efficient. In contrast to other methods, intimate contact between bacteria is not required. Additionally, bacteriophages can carry large blocks of deoxyribonucleic acid (DNA) and can survive harsh conditions that eliminate bacterial populations. Thus, DNA important to a population can be preserved until a host is re-introduced into an environmental niche

(87).

Conjugation is basically the ability of the bacterial cells to transfer DNA between cells that are in physical contact (135). It occurs between members of the same species, members of closely related species, and even from bacteria to prokaryotes and from bacteria to some eukaryotic cells (4, 5). In order for conjugation to occur, the donor cells must carry a unique plasmid that contains a set of genes that makes the transfer possible (4, 5, 89).

A novel system, believed to play a role in the acquisition and dissemination of antibiotic resistant genes in bacteria that exhibit multiple resistances, has also been identified (56). The system is referred to as bacterial integrons (139). Hall and Stokes

(57) defined integrons as mobile DNA elements with a specific structure consisting of two conserved segments flanking a central region containing gene cassettes that usually code for resistance to specific antimicrobials. Several classes of integrons have been identified to date with the majority of those identified belonging to class 1 type (63). Hall and Stokes (57) found that class 1 type integrons consist of a 5' conserved region that encodes a site-specific recombinase (integrase) and strong promoter(s) that ensures






23


the expression of the integrated cassettes. Additionally, the 3' conserved region in class type 1 integrons carries the genes qacAE and sul-1, and an open reading frame of unknown function. Hall and Stokes (57) determined that qacAE specifies resistance to antiseptics and disinfectants and that sul-1 confers sulfonamide resistance.

Zhao and others (139) conducted a study to characterize antimicrobial susceptibility patterns among Shiga toxin-producing Escherichia coli (STEC), including Escherichia coli 0157:H7, isolated from cattle, ground beef, and humans and to determine if resistant phenotypes observed could be attributed to integron-mediated resistant gene cassettes. They found that of the 50 isolates tested, seventy-eight percent exhibited resistance to two or more antimicrobials. Multiple resistances to streptomycin, sulfamethoxazole, and tetracycline were observed most often. Integrons were found in STEC isolates, including Escherichia coli 0157:H7 and were demonstrated to be transferable via conjugation to other strains. However, Zhao and others (139) also identified isolates that displayed multiple antibiotic resistances that did not contain any gene cassettes. Therefore, while integrons may play an important and active role in multiple resistances, other mechanisms also contribute to the antibiotic resistance phenotypes. Thermal Tolerance and Adaptation

Characteristically, the optimum growth temperature for Escherichia coli 0157:H7 is 37'C. Although the organism has been associated with milk and cooked ground beef patties, it does not appear to possess any unusual resistance to heat (41, 98). Mongold and others (91) found that thermotolerant mutants were more likely to occur in isolates that had previously been adapted to 41-42C but that such mutants could also result out of isolates previously adapted to only 32'C. Additionally, it was found that such mutants afforded little advantage over other strains at lethal temperatures. Research into the






24


upper thermal limits of E. coli 0157:H7 suggested that some strains can survive at temperatures between 49-52'C (99, 120). Another study found that certain strains could survive at temperatures as high as 55'C relevant to prior storage and holding conditions

(62). Corry (32) suggested that the composition of the food may in fact provide protection for bacteria at elevated temperatures. A study by Splittstoesser and others (124) found that high concentrations of solutes in apple juice did afford protection to Escherichia coli 0157:H7 in regard to thermotolerance. The organism was still found to be relatively heat sensitive with D-values at 52, 55, and 58'C of 12, 5.0 and 1.0 minutes respectively.

Arsene and others (9) investigated the heat shock response of Escherichia coli that allows cells to adapt to environmental and metabolic changes and to survive the stress conditions. It was found that an upshift from 30 to 42'C resulted in the rapid induction of synthesis of more than 20 heat shock proteins (HSPs). Major heat shock proteins are molecular chaperones and proteases. The two major chaperone systems of E. coli, determined by their abundance (15-20 percent of total protein at 46'C), are the DnaK and GroE systems. These systems play a vital role in preventing aggregation and refolding proteins.

Studies investigating the minimal temperature for E. coli 0157:H7 have shown that the organism is capable of growth at temperatures as low as 8'C and can produce verotoxins at 100C (99, 100). Additionally, Jackson and others (62) demonstrated the organism's ability to remain viable when stored at -18'C for up to fifteen days and Semanchek and Golden (120) found viable cultures of E. coli 0157:H7 after seven months of storage at -20'C. Knudsen and others (68) found that Escherichia coli






25


0157:H7 survived without significant decline on cut strawberries held at 5'C and that frozen inoculated strawberries evaluated 19 months after the initial sampling date still had populations of Escherichia coli 0157:H7 present. Populations were highest (>log 4.6 cfu/sample) in strawberries with added sucrose. Populations without added sucrose could only be detected upon enrichment of the sample.

Barkocy-Gallagher and others (12) evaluated isolates of Escherichia coli 0157:H7 at -20, 1, 4, and 7'C in ground beef samples to see if genomic differences could account for differing abilities to survive at the various temperatures. They found that no one strain or genomic cluster was more successful at survival in persisting low temperatures. All strains evaluated showed limited growth at temperatures of 4'C and 7'C and while small losses in cell numbers occurred at both 10C and -20'C, it is evident from this study that freezing cannot be expected to eliminate Escherichia coli 0157:H7 at least in ground beef.

When assessing the ability of Escherichia coli 0157:H7 strains to resist heating or freezing, care must be taken by the food industry as variations in the temperature ranges capable of supporting or sustaining the bacteria may occur. These variations may be due to storage conditions, the growth phase of the organism, product formulations, sampling techniques, and the particular serotype of the strain (62, 120). Adaptation and Tolerance of Sanitizing Agents

As stated previously, chemical sanitizers are considered to be effective on food contact surfaces if they demonstrate a five-log reduction in planktonic bacteria (43). In order to obtain maximum benefits sanitizing should be performed after cleaning as the use of the sanitizer(s) leads to the inactivation of cells which may result in biofilm formation (61, 82). The continued adherence of the inactivated cells and cell fragments






26


may potentially create a suitable environment for future bacteria. The creation of a biofilm can lead to the enhancement and survival of future cells (22, 43, 61, 82). It has been documented that microorganisms of a biofilm matrix on food contact surfaces display more resistance to toxic compounds than their single-celled counterparts in suspension (64, 71).

Unlike antibiotics, biocides do not have a specific target site. Biocides usually

exert their cytotoxic effects through multiple non-specific targets (18). Therefore, while a single mutation or chemical transformation of a cellular target can provide antibiotic resistances in bacteria, biocidal action is rarely affected by such an event. Resistance to such compounds can occur when a cell develops reduced permeability, like a mucopolysaccharide outer layer within a biofilm (18). Additionally, there is some evidence that a plasmid could be responsible for changes in the cell envelope which increases the resistance to biocides (114, 115).

Attachment to surfaces can also have an impact on bacterial resistance to

disinfection as a planktonic organism is susceptible to a disinfectant from all sides and angles but an attached organism is only susceptible from one side (18). Research conducted by Farrell and others (43) illustrated that enumeration by plate count techniques alone is insufficient for indicators of sanitizer efficacy. They found that low numbers of typical and/or injured E. coli 0157:H7 cells could remain on contact surfaces after sanitation with chlorine and peroxyacetic acid. Ronner and Wong (110) found similar results when studying the effects of hypochlorite sanitizers on Listeria monocytogenes and Salmonella typhimurium. Additionally, Mosteller and Bishop (93) found that various sanitizers failed to provide an adequate reduction (three-log) in






27


attached bacteria where they had clearly demonstrated the ability to be effective against the planktonic bacteria. Krysinski and others (71) found similar results.

Chemical Sanitizers in the Food Industry

Sanitizing agents have been used to reduce microorganisms on processing equipment for nearly 100 years. The selection of a sanitizer is usually based on economics, equipment type and its ability to be cleaned, and clean-up scheduling (33). Chemical sanitizers available for use in the food industry vary in chemical composition and activity. In general, the more concentrated a sanitizer, the more rapid and effective its action (7). The individual characteristics of a chemical sanitizer must be known and fully understood so that the most appropriate sanitizer can be selected. For a sanitizer to be effective when combined with cleaning compounds the temperature of the cleaning solution should be < 55'C and the soil should be light. The efficacy of chemical sanitizers is affected by the following physical-chemical factors: exposure time, temperature, concentration, pH, equipment cleanliness, water hardness, and bacterial attachment (82).

As defined by the Environmental Protection Agency, sanitizers are "pesticide products that are intended to disinfect or sanitize, reducing or mitigating growth or development of microbiological organisms including bacteria, fungi or viruses on inanimate surfaces in the household, institutional, and/or commercial environment" (40 Code of Federal Regulations [CFR] 455.10) (30). Sanitizers utilized by the food industry include chlorine and chlorine derivatives, iodine derivatives, quaternary ammonium compounds, acid-anionic sanitizers, hydrogen peroxide, peroxyacetic acid, and acidified sodium chlorite (21 CFR 178.1010) (30). Generally, sanitizers are utilized to inactivate target microorganisms on the food contact surfaces of cleaned food processing and food






28


service equipment. More recently, these compounds have been used for the inactivation of bacteria on raw, unprocessed food products such as meat and poultry carcasses as well as fruits and vegetables (33). The usage level of each sanitizer on food products, food contact surfaces and plant equipment is regulated by the United States Department of Agriculture (USDA). As per the Food and Drug Administration (FDA), (21 CFR 178.1010) (30), the maximum permitted use levels on food-contact surfaces without rinsing for chlorine is 200 parts per million (ppm), 25 ppm for iodophors and 200 ppm for quaternary ammonium compounds. The allowable level for peroxyacetic acid was not found.

Chlorine Sanitizers

Chlorine sanitizers are commonly used in the food industry. They are likely the most commonly utilized surface sanitizer as they have proven to be effective on a broad spectrum of microorganisms and are relatively inexpensive (82). As little as 0.6 to 13 parts per million (ppm) of free available chlorine is able to inactivate 90 percent of most planktonic bacteria within 10 seconds (82). Further illustration of chlorines ability to act rapidly was demonstrated by Kotula and others (70) when they showed that cultures of Clostridium perfringens, Escherichia coli, Proteus vulgaris, Kocuria varians, Salmonella spp., and Pseudomonas spp. were destroyed by 3 ppm of free available chlorine in 15 seconds. When added to water, chlorine immediately oxidizes all inorganic and organic compounds in the solution. Chlorine sanitizers damage proteins essential for enzymatic activity and diminish bacterial cell function until the bacterial cells ultimately die. Although these compounds are generally said to be lethal to the widest variety of microorganisms, their ability to destroy microorganisms, required concentration, and residual properties vary with the specific sanitizer used. Additionally, the effectiveness






29


is affected to varying degrees by temperature, pH, organic residues, and water hardness. Chlorine sanitizers include calcium and sodium hypochlorite (household bleach), chloramines, and chlorine dioxides. These sanitizers are most effective at a slightly acidic pH and at temperatures below 120 degrees Fahrenheit. They have limited residual activity and can be very corrosive to metals. Also, they deteriorate when stored at temperatures above 60'C or in the presence of light. While they are relatively inexpensive, they dissipate rapidly during storage and can produce a poisonous gas if mixed with a more acidic compound. Organic residues neutralize the hypochlorites whereas the chloramines are less sensitive to the residues, are more shelf stable and are active over a broader pH range (6.0 - 9.5). Finally, the chlorine dioxides, unlike the two previously mentioned, are not corrosive, not effected by organic residues, and can be used at very low concentrations (1 - 5 ppm). The chlorine dioxides however, are fairly expensive in comparison (7, 61, 82).

Quaternary Ammonium Sanitizers

Quaternary Ammonium sanitizers are often referred to as "quats". Quats are

ammonium compounds in which four organic groups are linked to a nitrogen atom that produces a positively charged ion (cation) (82). They form a bacteriostatic film that inhibits bacterial growth. They also are very effective on porous surfaces because of their penetration ability. For these reasons, they are widely used on floors, walls, equipment and furnishings of meat and poultry plants (82). Additionally, quaternary ammonium sanitizers are non-corrosive, have definite residual activity, and are more resistant to inactivation by organic material (7, 42, 82). In general, quats are colorless, odorless, stable against temperature fluctuation, non-toxic and non-irritating to skin (82). Quaternary ammonium compounds also work well against gram-positive bacteria,






30


especially Listeria monocytogenes, but are less effective against gram-negative bacteria (7, 82). The mechanism of germicidal activity of quaternary ammonium compounds is not fully understood. It is believed that the surface-active nature of the compounds surrounds and covers the cells outer membrane, causing a failure of the wall, resulting in the leakage of internal organs and enzyme inhibition (82). McDonnell and Russell (83) suggest that after the quaternary ammonium compound compromises the bacterial cell walls it reacts with the cytoplasmic membrane to produce membrane disorganization, leakage of the intracellular material, and degradation of proteins and nucleic acids. Some disadvantages to using quaternaries are that they must be used in higher concentrations (generally 200 - 400 ppm), are slower acting and require a contact time of several minutes for total effectiveness. Also, anionic surfactants like soaps and synthetic detergents neutralize the sanitizer. Calcium, iron, and aluminum ions also react with quaternaries to lower their efficacy. Finally, hard water may reduce the activity of the quaternary ammonium compounds but this can be overcome with use of higher concentrations (7, 42, 50, 61, 82).

Peroxyacetic Acid Sanitizers

Peroxyacetic acid sanitizers are antimicrobial compounds that act as both an

oxidizer and an acid. They consist of hydrogen peroxide and peroxyacetic acid and are relatively new to the food industry (81). Peroxyacetic acids are considered to be effective against bacteria, yeasts, molds, and fungal and bacterial spores (10). They are fast acting and very effective against biofilm formation (81, 111). It is believed that peroxyacetic acids denature proteins and enzymes and increase cell wall permeability by disrupting sulfhydryl (-SH) and disulfide (S-S) bonds. Additionally, they are less affected by organic matter than other sanitizers (83). They are environmentally safe, as the






31


compounds break down into oxygen and water. Peroxyacetic acids do not react with proteins to produce toxic or carcinogenic compounds (7). Peroxyacetic acid compounds are effective over a broad pH range as well as a broad temperature range, as low as 40 degrees Fahrenheit. Additionally, they are non-foaming and lethal to a broad spectrum of microorganisms (6, 7, 81). Some disadvantages to using peroxyacetic acids are that they are corrosive to non-stainless steel metals and copper alloys and lose their effectiveness in the presence of some metals and organic materials (81). Additionally, the odor released by such compounds can be irritating to the nose and throat (7). Iodine Sanitizers

lodophors are antimicrobial compounds containing iodine and a surfactant (92). They are deemed to be effective at killing 99.999% of planktonic bacterial cells at a concentration as low as 6.25 mg/L in only 30 seconds (82). The mode of antibacterial action of iodine has not been studied in detail. It is believed that diatomic iodine is the major antimicrobial agent, which disrupts bonds that hold cell protein together and inhibits protein synthesis (82). They are generally inexpensive although they can be more expensive than the chlorine compounds. They are very rapid acting; however, they are ineffective at alkaline pH values. Iodine compounds are most effective at a pH of 2.5 to 3.5. They are also rapidly inactivated by contact with organic matter (6, 7, 82). Additionally, iodine loss may occur during the storage of the iodophor compounds (42). Iodine sanitizers maintain more residual activity than the chlorine compounds and are not as corrosive. Unlike chlorine compounds, iodophors are usually not used as surface sanitizers and instead are primarily used as employee skin sanitizers in food-processing plants. lodophors, but not iodine itself, are generally mild and non-irritating to the skin. They have residual bactericidal activity on the skin that can last for up to an hour, making






32


them ideal for sanitizing workers' hands (7, 42, 82). A potential disadvantage is that they do tend to cause discoloration of some materials (7). Iodine is vaporized at temperatures of > 50'C and is inefficient at low temperatures. Iodine sanitizers may also cause off flavors in foods. Consequently, the use of iodophors on food contact surfaces is limited

(42). Additionally, iodine sanitizers are incompatible with wastewater treatment systems

(7).

The Ideal Sanitizer

According to Marriott (82), for a sanitizer to be desirable and useful it should possess several properties. First, an ideal sanitizer should have uniform microbial destruction properties and broad spectrum activity against vegetative bacteria, yeasts and molds in order to kill rapidly. Water hardness and pH values should not alter its effectiveness. Additionally, it should maintain its effectiveness in the presence of organic matter, detergents, and soap residues. It should offer good cleaning properties, be nontoxic and non-irritating, and have an acceptable odor or no odor. The ideal sanitizer should be easy to use, for example it should be water soluble in all proportions and be easy to measure in use solution. It should also be stable in both concentrated and use dilution forms, inexpensive and readily available.

Obviously, no one sanitizer can possess all of the ideal properties. The chemical selected as a sanitizer depends on the type of processing plant and the product being produced (42). Sanitizers selected should produce a 99.999% kill of 75 to 125 million Escherichia coli and Staphylococcus aureus within 30 seconds after application at 20'C to be deemed effective (82).






33


Surfaces and Attachment

It has been shown that even when cleaning and sanitation procedures are followed and are consistent with good manufacturing practices that microorganisms can still remain on all food processing surfaces (34, 111). Even with the inactivation of bacterial cells, a desired result of sanitation, the cells or fragments of the cells may remain attached. This would create a conditioning layer that could enhance future attachment of other bacteria (22). It is therefore important to examine the surfaces associated with bacterial attachment in the food processing industry as bacterial attachment is the first step in biofilm formation (34).

The ability for microorganisms to attach seems to be affected by nutrient conditions and environmental factors such as temperature and atmosphere (34). Additionally, it has been suggested that low-nutrient systems may enhance that ability for an organism to attach (19). Sasahara and Zottola (119) also found that pure cultures may show reduced adherence capabilities in comparison to mixed populations. Researchers have also shown that bacterial cells in early or late log phase attached at twice the rate of those in stationary phase. Similarly, bacterial cells in the log phase were found to have greater adherence when compared to the stationary or death phase (45, 125).

Stainless steel is one of the most common surfaces found in food processing

facilities (60). To the unaided eye the surface appears to be smooth, but when viewed under a microscope it is in fact found to be very rough. The flaws could potentially harbor spoilage or pathogenic organisms as bacteria tend to accumulate in scratches and other irregularities on metal surfaces (127).

Buna-N rubber and Teflon@ are other materials that may be found in a food

processing plant, especially in a dairy processing facility. Such materials are commonly






34


used for gaskets (93, 110). These surfaces represent those that are most difficult to sanitize. As with stainless steel, these gasket materials appear to be smooth. They are in fact covered with minute holes and cracks which may provide adherent cells protection from antimicrobial agents and surfactants (93).

Other surfaces that have been investigated as potential food contact surfaces

include a high density polyethylene plastic, polyvinyl chloride plastic and cement (64, 131). Joseph and others (64) found that organisms had a greater propensity to attach to plastic (107) followed by cement (106) and steel (105). Such cells in a food processing facility may not be removed by routine cleaning and sanitation procedures and therefore could be a source of contamination of foods coming into contact with such surfaces (34, 64, 111).

Recovery of Stressed Microorganisms

Both viable and injured microorganisms that have been exposed to stress such as chemical shock may still remain on the surface or food that has been treated (34, 111). Therefore, it is essential for recovery methodology to be accurate. The use of a selective media may prohibit the recovery of sub-lethally injured bacteria (122). Silk and Donnelly (122) found that an incubation step prior to the plating of Escherichia coli 0157:H7 with a selective media increased the level of recovery. However, these levels were still below recovery levels obtained when using a non-selective media.

Additionally, researchers have suggested that plate count methodology does not accurately represent the bacterial population present of a surface (3, 93). Andrade and others (3) stated that plate count methodology underestimates the number of cells on a surface since some cells may not be removed. They found the impedance method to recover 25 times more cells than the plate count method. Similarly, Mosteller and Bishop






35


(93) found recovery of bacteria with the plate count method to be less representative of the actual numbers present than the impedance method. The impedance method, which measures metabolic growth of individual cells, detects both reversibly and irreversibly attached cells (3, 93). Mosteller and Bishop (93) found direct epifluorescent filter technology (DEFT) to be superior in recovery of bacteria to both the impedance and plate count methodology. The DEFT method enumerates both reversibly and irreversibly attached bacteria as well as single cells and clumps of bacteria (93).

Rossoni and Gaylarde (111) have suggested that one way to resolve the issue of recovery counts is to utilize non-parametric statistics.














CHAPTER 3
MATERIALS AND METHODS

This study was conducted in two phases. Phase one of the study involved the use of four commonly used food-processing sanitizers, a chlorine compound, a quaternary ammonium compound, an iodine compound and a peroxyacetic acid compound in conjunction with planktonic cells from two distinct Escherichia coli 0157:H7 strains, one unadapted and one known to be acid tolerant, in three trials, each of which lasted for three days. Phase two consisted of the use of the same four sanitizers in conjunction with adherent bacterial cells from the two Escherichia coli 0157:H7 strains utilized in phase one. Phase two was also performed in three trials, with each trial lasting one day. All trials were conducted at Deibel Laboratory in Gainesville, Florida. The protocols for the three trials for phase one and the protocol for the three trials of phase two were identical.


Bacterial Cultures

One of the Escherichia coli 0157:H7 strains utilized in this study was obtained

from the American Type Culture Collection (ATCC). The strain is designated as ATCC 700599. It was isolated from a salami product in 1994 and is known to be acid tolerant. This strain is identified as a biohazard type I as it is not known to have caused human illness. Upon receiving the freeze-dried isolate, the bacterium was resuscitated in Nutrient Broth (Difco Laboratories, Detroit, Michigan) at 37'C under aerobic conditions. The stock culture was then maintained through monthly transfers on slants of Trypticase soy agar (TSA) (Difco) supplemented with 0.5 percent yeast extract (TSAYE) (Difco)


36






37


and stored at 2.5'C. The other Escherichia coli 0157:H7 strain used throughout this study was obtained from Dr. Robert Deibel of Deibel Laboratories, Incorporated. The strain is designated as FSIS 063-93. It was isolated from a meat product in 1993. This culture was also maintained through monthly transfers on slants of Trypticase soy agar supplemented with 0.5 percent yeast extract (TSAYE) (Difco). Before use, the cultures were grown in Trypticase soy broth (TSB) (Difco) supplemented with 0.5 percent yeast extract (TSBYE) (Difco) overnight (at least 12 hours) at 37'C. Ten-fold dilutions were performed using 0.1 percent peptone solution from Bacto Peptone (Difco).

Growth characteristics were established for both strains of Escherichia coli

0157:H7 by performing 24 hour growth curves in Lauryl Sulfate broth at 37'C. Aliquots of the incubating cultures were removed after 1, 3, 5, 8, 12, 15, 18, 21, and 24 hours respectively. The Optical Density (OD) of each aliquot was determined on a Spectrophotometer 20 (Milton Roy Company, Item 333172, USA) and portions of each aliquot were plated in triplicate on Violet Red Bile Agar (VRBA) (Difco). Both the absorbance curve and the growth curve for E. coli 0157:H7, strain ATCC 700599, showed a rapid increase during the first five hours and then tend to plateau (Figure 1 and Figure 2). The absorbance curve and growth curve for E. coli 0157:H7, strain FSIS 06393, also exhibited a rapid increase during the first five hours but fluctuated more than the other strain over the next nineteen hours (Figure 3 and Figure 4).








38


0.8


A 7 -----


06 E 05

U')
OU 04

0

03



02



01



0


f-


5


10


15
Time (hours)


20


25


30


0


Figure 1. Absorbance of Escherichia coli 0157:H7, strain ATCC 700599, in Lauryl

Sulfate broth over 24 hours


9



85



8



75







65



6





55 5-


0


5


10


15
Time (hours)


20


25


30


Figure 2. Growth of Escherichia coli

Agar over 24 hours


0157:H7, strain ATCC 700599, on Violet Red Bile


-


---


-

I i

- - ----







- - - - - - --- - - - -- ------ ------------ - -- -- --- - - - - - -- - - - - - -



- - - --- - - -- - --- -- - ------- -


.7









39


0.7








06
0 5 - --- -- - ---- - -






04

0 2


(0 02











01
0 5 10 15 20 25 30
Time (hours)


Figure 3. Absorbance of Escherichia coli 0157:H7, strain FSIS 063-93, in Lauryl Sulfate

broth over 24 hours



9 - ------ -




8.5



8




75





0


65 6 5.5 5
0 5 10 15 20 25 30

Time (hours)


Figure 4. Growth of Escherichia coli 0157:H7, strain FSIS 063-93, on Violet Red Bile

Agar over 24 hours






40


Sanitizer Solutions

Escherichia coli 0157:H7 cells were exposed to four commonly utilized industrial plant sanitizers which included a Chlorine compound (Clorox@ regular bleach), a Peroxyacetic Acid compound (Zep PerosanTM), a Quaternary Ammonium compound (Zepamine-ATM), and an Iodine compound (Zep-I-DineTM). The active ingredient for Clorox is 6.0% Sodium Hypochlorite. The active ingredients of Zep Perosan are 5.1% Peroxyacetic Acid and 21.7% Hydrogen Peroxide. The active ingredients of Zepamine A are 5.0% n-Alkyl Dimethyl benzyl ammonium chloride and 5.0% n-Alkyl Dimethyl ethylbenzyl ammonium chloride. The active ingredient for Zep-I-Dine is 1.75% iodinet from the alpha (p-nonylphenyl)-omega-hydroxypoly (oxythylene)-iodine complex. All dilutions of the sanitizing agents were prepared in 99 ml quantities in 250 ml sterile dilution bottles on each day of testing. The sanitizers were diluted with autoclaved distilled water to obtain the necessary concentration.

Determination of Minimum Inhibitory Concentration (MIC)

The minimum inhibitory concentration for each sanitizer was determined by a method similar to that utilized by Pickett and Murano (102). The MIC (defined as the lowest concentration of a sanitizer that will prohibit growth of Escherichia coli 0157:H7 cultures) was determined by inoculating 106 E. coli 0157:H7 cells per ml into sterile test tubes containing serial dilutions of the sanitizing solutions and then incubating at 370 C for 48 hours. Once an initial MIC was determined, further dilutions were made between that dilution and the next lower dilution to more precisely approach the actual MIC. Growth was measured at 580 nm on a Spectrophotometer 20 after 24 and 48 hours. All tests were performed in triplicate. The average of the lowest concentration of each sanitizer showing a bacterial population with an OD value of < 0.01 was designated as






41


the minimum inhibitory concentration. A standard curve relating bacterial counts obtained by standard plate count on Violet Red Bile agar with the optical densities was determined and utilized throughout the study to provide an estimate of the bacterial population present in suspension (Figure 5 and Figure 6). The actual number of bacteria present in the cell suspension was determined more precisely by plate count values.

Once the minimum inhibitory concentration was determined, the sub-lethal and lethal concentrations were determined. The sub-lethal level is the concentration of sanitizer corresponding to one dilution lower than the minimum inhibitory concentration. The lethal level is the concentration of sanitizer corresponding to one dilution greater than the minimum inhibitory concentration.

0.6






0.4 - -


0.3 -
y 0.1641x - 0.963
02
=0.8033
(02


0. 1 - _ _ -


0
5 55 6 65 7 75 8 85 9
Log Count (CFU/mI)
Figure 5. Escherichia coli 0157:H7 strain ATCC 700599 Standard Curve






42


4y =0.1585x - 0. 9492
o~ 03


R2 0.9422




0
5 55 6 65 7 75 8 8.5 9
Log Count (CFU/mi)

Figure 6. Escherichia coli 0157:H7 strain FSIS 063-93 Standard Curve Zone Inhibition

Zone inhibition tests were performed to determine the bacteriostatic activity of the sanitizer solutions. Tests were performed utilizing the Kirby-Bauer technique (88). In this method, seeded agar plates are utilized. The seeded agar plates were created by dipping sterile swabs into each of the Escherichia coli 0157:H7 cultures, which were grown overnight at 37'C to a level of 108 cfu/ml, and streaking the bacteria onto MeullerH-inton (Difco) agar plates. Plates were cross-streaked in at least three different directions to cover the agar surface. Using aseptic technique, blank test discs (Difco, Detroit, Michigan, B31039) were dipped for 30 seconds into various concentrations of the sanitizing agents. The impregnated filter discs were then applied immediately to the seeded agar surface of the Meuller-Hinton agar plates. The plates were incubated upright


n r I






43


at 37'C for 48 hours. After incubation, the plates were examined and clear zones were measured in millimeters from the edge of the discs.

In this method, as the substance diffuses from the filter discs into the agar, the

concentration decreases as a function of the square of the distance of diffusion, until at some point it is no longer effective at inhibiting microbial growth. The effectiveness of a particular antimicrobial agent is determined by the diameter of the clear zones produced. The clear areas are growth-inhibited zones that appear where the sanitizers or other antimicrobial agents were able to prevent bacterial growth (88). It was determined that this method was not suitable for the purposes of this study. The diffusion discs were only able to hold approximately 10 microliters of the sanitizing agents and such small amounts were rapidly oxidized by the media itself. Only concentrations much greater than the minimum inhibitory concentration were able to produce a clear zone. Therefore, the results for this method are not meaningful and are not included.

Planktonic Bacteria

Planktonic cells from growth flasks were harvested by centrifugation at 12,400 rpm in a Micro Centrifuge (Fisher Scientific, Model 235C, Pittsburgh, Pennsylvania) at room temperature, approximately 22'C, for 15 minutes. Cell pellets were then resuspended in the same volume of 0.5% saline solution, as a wash, and re-centrifuged for an additional 15 minutes also at 12,400 rpm. Cell pellets were then resuspended in 0.5% saline solution to a final concentration of approximately 108 colony forming units (cfu)/milliliter (ml). One milliliter of the cell suspension was then added to 9 milliliters of a sanitizing solution at a concentration lower than the previously determined minimum inhibitory concentration. The bacterial cells were exposed to the sanitizer solutions at room temperature for 5 minutes. One milliliter of this cell-sanitizer mixture was then






44


immediately removed and added to 9 milliliters of neutralizer (D/E Neutralizing broth, Difco). The mixture was allowed to set in the neutralizer solution for 30 seconds. Then an aliquot of the mixture was removed, diluted as necessary, and plated on Violet Red Bile agar in triplicate. The spread plate method was utilized for all samples and will be explained in detail in the microbiological analysis section. Concentration levels for all stages were verified by direct plating. All plates were incubated upright at 37'C overnight and then results were recorded. A milliliter of the cell-sanitizer-neutralizer solution was also removed and placed into a tube of Lauryl Sulfate broth and incubated at 37'C for 24 hours. This allowed for growth of uninjured cells and also enabled potentially injured cells to recover and grow. The cells of this tube were then utilized for day 2 and day 3 of this project. This procedure was repeated for each of the four sanitizers in three trials with each trial lasting for three consecutive days of sanitizer exposure at a previously determined sub-lethal level for the planktonic cells. Numbers of removed cells were calculated as cfu/ml and the results for all sanitizer experiments were the average of the three trials.

Cultures that exhibited survival at the initial concentration of a sanitizer(s) underwent subsequent experimentation following the above procedure at a higher concentration. Cultures that were unable to survive at the initial concentration of a sanitizer(s) underwent subsequent experimentation at a lower concentration also following the same protocol as outlined above.

Bacterial Reassessment

The bacteria isolates that survived the planktonic stage of the study were harvested and maintained on a culture slant (TSAYE) with monthly transfers as previously described. Upon completion of the planktonic testing phase, these surviving bacterial






45


isolates were reassessed for their minimum inhibitory concentration, sub-lethal and lethal concentration levels. For any culture that showed an increase for any sanitizer treatment, this culture was then tested against other sanitizers to determine if the resistance (i.e. the increase in the minimum inhibitory concentration, sub-lethal and lethal concentration) could then be increased against other sanitizers by cross-protection.

Solid Substrate

Stainless steel plates (1 mm thick), type 304 with a mill finish (Thompson Sheet Metal, Gainesville, Florida), were utilized for the sanitizer challenge experiments. They were cut into coupons, size 2.54 x 7.62 centimeters. All coupons were degreased and cleaned with Dawn@ dishwashing detergent, washed with 70% Isopropyl Alcohol (LabChem Inc., product no. LC15760-2, Pittsburgh, Pennsylvania) and then rinsed thoroughly with distilled water. All coupons were then air dried before autoclaving in sealed pouches (Fisher Scientific, product no. 01-812-54, Pittsburgh, Pennsylvania).

Adhesion of Microbial Cells

Escherichia coli 0157:H7, strain ATCC 700599 and strain FSIS 063-93, were

cultured overnight in TSB at 37'C. These cultures were then centrifuged, washed in the same volume of 0.5% saline solution, re-centrifuged, and then resuspended in 0.5% saline solution to give a final concentration of 108 cfu/ml. These concentrations were estimated by spectrophotometry (Spectrophotometer 20), using a standard curve of optical density at 580 nm against colony forming units. Concentrations were also verified through direct plating. Sterile stainless steel coupons were then suspended in the bacterial solutions and incubated at room temperature, approximately 22'C, without shaking, for 4 hours. Static incubation was used to better mimic the conditions in a processing facility and also because Blanchard and others (17) stated that attachment formed in this manner may be






46


more resistant to sanitation. Coupons were then removed with sterile forceps, rinsed with distilled water for roughly 1 minute to remove poorly adhering cells and allowed to dry under a hood for about 20 minutes. Several of the dry coupons were swabbed (a 6.45 centimeter square area) to get an approximation of the level of bacteria that was able to attach. The remaining dry coupons were then aseptically placed into a sterile wire staining rack and the rack was then aseptically placed into a sterile staining dish and treated with the sanitizing agents or control distilled water as described below.

Sanitizer Treatments for Adherent Cells

The stainless steel coupons with adherent cells were submerged at room

temperature, approximately 22'C, for 5 minutes in the sanitizer solution or in distilled water for a control. The coupons were then removed and rinsed in sterile distilled water for the removal of loosely attached cells. A 6.45 centimeter square area of the coupons, which was previously marked with a permanent pen, was then swabbed for 30 seconds. The swabs were then put into the appropriate neutralizing solution, vortexed, and diluted in 0.5% peptone and plated on VRBA (Difco) for enumeration of cells. Each of the four sanitizers was utilized and each experiment was performed in triplicate.

Microbiological Analysis

The inoculum level of the Escherichia coli 0157:H7 bacterial cultures were

verified by direct plating. Spread plating was utilized for both phase one and phase two of this study. For each initial dilution plated, a volume of 0.5 ml was dispensed onto prepoured Violet Red Bile agar plates. Subsequent dilutions, which utilized a volume of 0.1 ml, were also dispensed onto pre-poured VRBA plates. A 60 mm sterile plastic spreader (Fisher Scientific, product no. 05-541-11, United Kingdom) was then used to evenly distribute the sample over the plate as the plate was spun (134). All samples were plated






47


in triplicate. The VRBA plates were incubated upright at 37'C for 48 hours. Plates with 30 to 300 colonies were counted and recorded.

Statistical Analysis

Plate count data were converted to log values for analysis. Data from the

planktonic stage of the experiment were analyzed with the mixed model program (PROC MIXED) of SAS (SAS Institute, Cary, North Carolina) (118). Comparisons between the strains of Escherichia coli 0157:H7, day of treatment, sanitizer, concentration, nested within each sanitizer, and trial were made using the Ismeans statement of SAS. Treatment effects and differences were considered to be significant when P <0.05.

Data from the adherent stage of testing were also analyzed by SAS. The general linear model (PROC GLM) was utilized; however, the data was again analyzed using a mixed model with the random component being trials nested within the interaction of the other three variables (strain, sanitizer, attached). The /test option statement was used at the end of the statement to provide the appropriate f-tests. Comparisons were again made using the lsmeans statement. Treatment effects and differences were considered to be significant when P < 0.05.














CHAPTER 4
RESULTS AND DISCUSSION

Determination of Minimum Inhibitory Concentration

Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 exhibited numerically different minimum inhibitory concentration (MIC) levels except with the Zep-PerosanTM treatment (Table 1). Escherichia coli 0157:H7 strain FSIS 063-93 demonstrated a higher minimum inhibitory concentration level when exposed to Clorox@, Zep-I-DineTM, and Zepamine-ATM than the ATCC 700599 strain (Table 1). Table 1. The minimum inhibitory concentrations (MICs) of various sanitizers against
Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 after
incubation at 37'C for 24 hours
E. coli 0157:H7 E. coli 0157:H7
FSIS 063-93 ATCC 700599
Sanitizer Minimum Inhibitory Concentration
Clorox@ 3.25 mg/L 1.0 mg/L
Zep-I-DineTM 1.5 mg/L -*
Zepamine-ATM 13.0 mg/L 12.5 mg/L
Zep-PerosanTM 3.5 mg/L 3.5 mg/L
*No minimum inhibitory concentration was found

The two bacterial strains did not perform as expected. It was hypothesized that the ATCC 700599 strain, which is known to be acid tolerant, would outperform the FSIS 063-93 strain. Literature reviewed suggested that acid tolerant bacteria often exhibit increased resistance to other stresses such as heat, irradiation, and other antimicrobial agents (21). In this study, strain ATCC 700599, did not exhibit any increased resistance over strain FSIS 063-93 and did not appear to offer any cross-protection to sanitation treatment. Perhaps the pH of the sanitizing solutions was not sufficient to induce the stress response system(s) which would afford strain ATCC 700599 greater protection


48






49


than the FSIS 063-93 strain (78, 113). With the exception of the peroxyacetic acid, the pH of the sanitizing solutions at the concentrations utilized in this study was essentially neutral.

Marriott (82) stated that as little as 0.6 mg/L to 13 mg/L of chlorine is able to inactivate 90% of most planktonic bacteria. The minimum inhibitory concentration values obtained for both strains for the Clorox@ treatment were within this range. Marriott (82) also stated that 6.25 mg/L of an iodine solution is sufficient to reduce a planktonic bacterial population by 99.999%. The minimum inhibitory concentration for strain FSIS 063-93 fell well below this value and no MIC could be found for the ATCC strain. At a value of only 0.25 mg/L of the iodine sanitizing solution the ATCC 700599 strain was unable to survive. Pickett and Murano (102) found that the minimum inhibitory concentration for quaternary ammonium against Listeria monocytogenes was 5 mg/L. In this study the ATCC 700599 strain and the FSIS 063-93 strain produced a MIC of 12.5 mg/L and 13.0 mg/L, respectively. As quaternary ammonium compounds have been found to be very effective against gram positive bacteria but less effective against gram negative bacteria, it is not surprising that the MIC for the two Escherichia coli 0157:117 strains was higher than that reported by Pickett and Murano (102). GuerinMechin and others (55) found the MIC for Pseudomonas aeruginosa, a gram negative bacterium, to fall within the range of 10 to 20 mg/L. This is comparable to the data found in this study for both Escherichia coli 0157:H7 strains. No comparable data was available for the MIC of the peroxyacetic acid solution.






50


Planktonic Bacteria

Chlorine Compound

A chlorine concentration of 1.0 mg/L killed approximately 107 cfu/ml of

Escherichia coli 0157:H7 strain FSIS 063-93 after a 5 minute exposure and more than 107 cfu/ml of Escherichia coli 0157:H7 strain ATCC 700599 (Table 2). After the chemical shock of 1 mg/L for 5 minutes, a milliliter of the cell-sanitizer-neutralizer solution was removed and placed into a 9 ml tube of Lauryl Sulfate broth and incubated at 37'C for 24 hours to allow for potential recovery of the exposed cells. This procedure was repeated for three consecutive days. On day 2 and day 3, the FSIS 063-93 strain demonstrated a greater than 108 log reduction while the ATCC 700599 strain showed a mean log reduction of 6.62 and 2.14 for day 2 and day 3 respectively (Table 2). The lower mean log reduction values for the ATCC strain 700599 are a result of a failure of the bacterial cells to fully recover to a 108 cfu/ml level in the Lauryl Sulfate broth after the initial chemical exposure on day 1 (Table 2). Additionally, there were significant differences between the ATCC 700599 strain and the FSIS 063-93 strain for day 1, day 2 and day 3 respectively. This is also attributed to the fact that after the initial chemical shock on day 1, the ATCC 700599 strain was not able to recover to a 108 cfu/ml level in the Lauryl Sulfate broth while the FSIS 063-93 strain underwent full recovery in the broth solution (Table 2). Both Escherichia coli 0157:H7 strains exhibited slightly greater than a 1 log survival after treatment on day 1 and both demonstrated less than a I log survival after repeated treatment on day 2 and day 3 (Table 2).






51


Table 2. Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia
coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to
1.0 mg/L of Clorox@ for 5 minutes at 22'C
Day E. coli 0157:H7 E. coli 0157:H7
FSIS 063-93 (cfu/ml) ATCC 700599 (cfu/ml)
Before After Log kill Before After Log kill
treatment treatment treatment treatment
1 8.46 1.54 6.92X 8.94 1.14 7.80a,y
2 8.75 0.27 8.48'x 7.26 0.64 6.62
3 8.50 0.00 8.50ax 2.40 0.26 2.14
a,b,c Different letters in the same column indicate a significant difference among means at the P < 0.05 level.
XY Different letters in the same row indicate a significant difference between FSIS and ATCC means at the P <0.05 level.

A similar pattern was observed for both E. coli 0157:H7 strains when the

concentration of chlorine was reduced to 0.5 mg/L. The FSIS 063-93 strain showed a greater than 106 log reduction on day 1 with a greater than 108 log reduction on day 2 and day 3 (Table 3). The ATCC 700599 strain showed a greater than 107 log reduction at this concentration of chlorine for day 1 and had a 3.87 and 1.99 mean log reduction value on day 2 and day 3, respectively (Table 3). The lower mean log reduction values on day 2 and day 3 for the ATCC 700599 strain can again be attributed to the lack of ability to recover to a level of 108 cfu/ml in the Lauryl Sulfate broth after the chemical shock on day 1 (Table 3). There was no significant difference between the ATCC 700599 strain and the FSIS 063-93 strain for day 1, P=0.37, however, significant differences were observed between the two strains for day 2 and day 3. This is also attributed to the fact that after the initial chemical shock on day 1, the ATCC 700599 strain was not able to recover to a 108 cfu/ml level in the Lauryl Sulfate broth while the FSIS 063-93 strain underwent full recovery in the broth solution (Table 3).






52


Table 3. Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia
coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to
0.5 mg/L of Clorox@ for 5 minutes at 22'C
Day E. coli Ol57:H7 E. coli 0157:H7
FSIS 063-93 (cfu/ml) ATCC 700599 (cfu/ml)
Before After Log kill Before After Log kill
treatment treatment treatment treatment
1 8.79 2.03 6.76'x 8.61 1.55 7.06a,x
2 8.70 0.65 8.05ax 4.23 0.36 3.87 'I
3 8.32 0.00 8.32a,x 2.22 0.23 1.99
a"bc Different letters in the same column indicate a significant difference among means at the P <0.05 level.
XY Different letters in the same row indicate a significant difference between FSIS and ATCC means at the P <0.05 level.

Statistical analysis revealed that there were no trial differences for either strain at either concentration level of chlorine. Additionally, neither Escherichia coli 0157:H7 strain was able to be recovered on the Violet Red Bile agar after repeated exposure of chlorine at a concentration of 1.0 mg/L or 0.5 mg/L. The FSIS 063-93 was able to grow in the Lauryl Sulfate broth at 37'C after repeated exposure at 1.0 mg/L (Table 2) and at

0.5 mg/L (Table 3). This would indicate the presence of sub-lethally injured bacteria (21, 122).

The results obtained in this study are similar to those of Joseph and others (64). They found that planktonic Salmonella cells were unable to survive a five minute exposure to chlorine at a concentration of 10 mg/L. In general, Zhao and others (142) found similar results to this study. They tested seven strains of Escherichia coli 0157:H7 and found that six of the seven were susceptible to chemical shock of chlorine at a concentration of only 0.25 mg/L after 1 minute of exposure. They did find one unusual strain that they labeled as E. coli 0157:H7 G which was able to withstand chemical exposure of chlorine at a level of 2 mg/L. It appears that there may be an innate






53


difference among E. coli 0157:1-17 isolates in regard to their ability to tolerate chlorine. A similar finding was cited by Mokgatla and others (90) when testing Salmonella strains isolated from a poultry abattoir. They reported that one of the isolates was able to survive chemical exposure to hypochlorous acid at a level of 72 mg/L and could grow in the presence of a chlorine concentration considered to be antibacterial. Quaternary Ammonium Compound

An initial chemical shock of Zepamine-ATM at a concentration of 10 mg/L resulted in a greater than 105 log reduction for both Escherichia coli 0157:H7 strains utilized in this study (Table 4). Subsequent exposure resulted in a mean log reduction of 7.39 and

8.49 on day 2 and day 3 respectively for the FSIS 063-93 strain. In comparison, the repeated exposure to the quaternary ammonium compound resulted in a mean log reduction value of 5.94 and 7.44 on day 2 and day 3 respectively for the ATCC 700599 strain (Table 4). While both strains were able to recover to a level of 108 cfu/ml in the Lauryl Sulfate broth after the chemical exposure, it appears that the ATCC 700599 strain had the ability to adapt and survive as determined by plate count data (cfu/ml) on Violet Red Bile agar (Table 4).

Table 4. Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia
coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to
10.0 mg/L of Zepamine-ATM for 5 minutes at 22'C
Day E. coli Ol57:H7 E. coli 0157:H7
FSIS 063-93 (cfu/ml) ATCC 700599 (cfu/ml)
Before After Log kill Before After Log kill
treatment treatment treatment treatment
1 8.46 3.29 5.17cx 8.61 2.95 5.66 '*
2 8.78 1.39 7.39b'x 8.46 2.52 5.94by
3 8.52 0.03 8.49ax 8.52 1.08 7.44ay
a,b,c Different letters in the same column indicate a significant difference among means at the P < 0.05 level.
X.Y Different letters in the same row indicate a significant difference between FSIS and ATCC means at the P <0.05 level.






54


In addition to exhibiting significant differences by day within each strain (Table 4), there were also significant differences by day between the two strains. Analysis of the data revealed no significant difference between the two strains at day 1 (P=0.23) however, there was significant differences between the two strains for day 2 and day 3. This is likely due to the fact that the ATCC 700599 strain had less injured cells and therefore exhibited higher colony counts on the VRBA plates for both day 2 and day 3 (Table 4). Perhaps, the E. coli 0157:H7 FSIS 063-93 strain had a greater number of injured cells and therefore was not able to grow as well on the selective media (21, 122). Additionally, there were no significant differences between trials for this concentration of Zepamine-ATM.

Although Escherichia coli 0157:H7 strain FSIS 063-93 exhibited minimal to no survival after three days of repeated exposure to 10 mg/L of Zepamine-ATM (Table 4) on the VRBA, it was decided that both strains, not just the ATCC 700599 strain, would be subjected to 11 mg/L, a higher concentration, of the sanitizer. The results of the exposure of the two strains to the higher level of the sanitizer did not conform to expectations. For reasons that remain unclear, the two strains demonstrated a role reversal at the higher level of sanitizer. At a concentration of 11 mg/L of the quaternary ammonium compound, Zepamine-A, the E. coli 0157:H7 FSIS 063-93 strain showed a higher level of survival than it did at 10 mg/L. The E. coli 0157:H7 ATCC 700599 strain showed a decrease in its survival rate (Table 5). Perhaps at this increased level of exposure, strain FSIS 063-93 underwent habituation. This phenomenon occurs when enterobacteria, especially Escherichia coli spp., respond to low doses of chemical or physical stresses by inducing responses which allow mildly stressed organisms to subsequently resist higher,






55


potentially lethal doses of the same stress (113). There were significant differences within each strain for day of exposure (Table 5). Additionally, there were significant differences between strains in reference to day of exposure. Table 5. Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia
coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to
11.0 mg/L of Zepamine-ATM for 5 minutes at 22'C
Day E. coli 0157:H7 E. coli 0157:H7
FSIS 063-93 (cfu/ml) ATCC 700599 (cfu/ml)
Before After Log kill Before After Log kill
treatment treatment treatment treatment
1 8.87 3.38 5.49C'X 8.91 2.11 6.80
2 8.63 2.86 5.77bx 8.57 1.28 7.29
3 8.52 0.71 7.81a,x 8.49 0.07 8.42a,y
a,b,c Different letters in the same column indicate a significant difference among means at the P <0.05 level.
xy Different letters in the same row indicate a significant difference between FSIS and ATCC means at the P < 0.05 level.

Trial was also a source of variation for mean log reduction values for both of the Escherichia coli 0157:H7 strains when exposed to Zepamine-ATM at a concentration of 11 mg/L (Table 6). Trial 2 was significantly different from both trial 1 and trial 3 for strain ATCC 700599. Strain FSIS 063-93 in trial 2 was also significantly different from trial 1 and trial 3. The difference in trial 2 for both strains is due to higher survival rates on the VRBA plates (Table 5). Possible reasons for this variation may be fluctuation changes in room temperature during trials or fluctuations in incubation temperatures which could lead to variations in bacterial growth. Another explanation is experimental error.






56


Table 6. Mean log reduction values by trial (n=18) for Escherichia coli 0157:H7 strain
ATCC 700599 and strain FSIS 063-93 when exposed to 11.0 mg/L of
Zepamine-ATM for 5 minutes at 22'C
Trial E. coli 0157:H7 E. coli 0157:H7
ATCC 700599 (cfu/ml) FSIS 063-93 (cfu/ml)
1 7.72a 6.70a
2 7.10b 5.92b
3 7.68a 6.45a
a,b Different letters in the same column indicate a significant difference among means at the P < 0.05 level.

Review of the plate count data reveal a downward trend in reference to survival on Violet Red Bile agar after repeated exposure at the sub-lethal sanitizer concentration of 10 mg/L (Table 4) and 11 mg/L (Table 5) for both bacterial strains. These results are similar to that reported by Pickett and Murano (102). They found that sub-lethal exposure of Listeria monocytogenes to quaternary ammonia failed to result in any acquired resistance to subsequent exposure of the same sanitizer. In contrast, GuerinMechin and others (55) found that repeated exposure of sub-lethal levels of quaternary ammonium compounds to Pseudomonas aeruginosa resulted in increased survival of the bacteria upon exposure to subsequently higher levels of the sanitizer. Peroxyacetic Acid Compound

A concentration of 1.0 mg/L of peroxyacetic acid per 4.4 mg/L of hydrogen

peroxide of Zep-PerosanTM only killed approximately 102 cfu/ml of both E. coli 0157:H7 strains after initial exposure to the sanitizer for 5 minutes at 220C. Further exposure to the sanitizer resulted in roughly a three log reduction for day 2 and day 3 for each strain (Table 7). Significant differences exist within both strains for day of exposure (Table 7) however, no significant differences between strains were found for day of exposure.






57


Table 7. Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia
coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to
1.0 mg/L per 4.4 mg/L of Zep-PerosanTM for 5 minutes at 22'C
Day E. coli Ol57:H7 E. coli Ol57:H7
FSIS 063-93 (cfu/ml) ATCC 700599 (cfu/ml)
Before After Log kill Before After Log kill
treatment treatment treatment treatment
1 8.46 6.47 1.99b 8.61 6.45 2.16b
2 8.69 5.51 3.18a 8.43 5.52 2.91ab
3 8.52 4.84 3.68a 8.51 4.97 3.54a
abc Different letters in the same column indicate a significant difference among means at the P < 0.05 level.

Trial was also a source of variation for mean log reduction values for both of the

Escherichia coli 0157:H7 strains when exposed to Zep-PerosanTM at a concentration of

1.0 mg/L of peroxyacetic acid per 4.4 mg/L of hydrogen peroxide (Table 8). Trial 1 was

significantly different from both trial 2 and trial 3 for both Escherichia coli 0157:H7

strains. The difference in trial 1 for both strains is due to lower survival rates on the

VRBA plates (Table 7). Possible explanations for this decrease in survival rate of trial 1

could be an error in the initial sanitizer concentration, an error during dilutions or perhaps

a temperature fluctuation of the incubator.

Table 8. Mean log reduction values by trial (n=18) for Escherichia coli 0157:H7 strain
ATCC 700599 and strain FSIS 063-93 when exposed to 1.0 mg/L of
peroxyacetic acid per 4.4 mg/L of hydrogen peroxide of Zep-PerosanTM for 5
minutes at 22'C
Trial E. coli 0157:H7 E. coli 0157:H7
ATCC 700599 (cfu/ml) FSIS 063-93 (cfu/ml)
1 4.00a 4.17a
2 2.24b 2.16b
3 2.36b 2.53b
a,b Different letters in the same column indicate a significant difference among means at the P < 0.05 level.


As only a small log reduction was found when the two strains of Escherichia coli

0157:H7 were exposed to 1.0 mg/L of peroxyacetic acid per 4.4 mg/L of hydrogen






58


peroxide of Zep-PerosanTM, the concentration was increased to 2.0 mg/L of peroxyacetic acid per 8.8 mg/L of hydrogen peroxide of Zep-PerosanTM and the experiment was repeated. At this higher concentration, very little difference was observed in the mean log reduction values of either strain of Escherichia coli 0157:H7 (Table 9). The FSIS 063-93 strain averaged a 102 cfu/ml reduction for all three days of exposure and the ATCC 700599 strain exhibited a 102 cfu/ml reduction upon initial exposure to the sanitizer and then had approximately a 103 cfu/ml log reduction for day 2 and day 3 of exposure (Table 9). E. coli 0157:H7 strain FSIS 06-93 showed a slightly higher level of growth based on plate count data from VRBA for day 2 and day 3 (Table 9). Significant differences for day of exposure were found for the ATCC 700599 bacterial strain but not for the FSIS 063-93 strain (Table 9). No significant difference was demonstrated between the strains for the initial chemical shock however, there were significant differences between the two Escherichia coli 0157:H7 strains for treatment and log kill on day 2 and day 3. There were no differences observed for trials at a concentration of

2.0 mg/L of peroxyacetic acid per 8.8 mg/L of hydrogen peroxide of Zep-PerosanTM. Table 9. Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia
coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to
2.0 mg/L per 8.8 mg/L of Zep-PerosanTM for 5 minutes at 22'C

Day E. coli 0157:H7 E. coli Ol57:H7
FSIS 063-93 (cfu/ml) ATCC 700599 (cfu/ml)
Before After Log kill Before After Log kill
treatment treatment treatment treatment
1 8.87 6.59 2.28a,x 8.91 6.58 2.33 '*
2 8.81 6.50 2.31 ax 8.76 5.52 3.24a,y
3 8.68 6.30 2.38a,x 8.69 5.42 3.27a,y
a,b,c Different letters in the same column indicate a significant difference among means at the P < 0.05 level.
"Y Different letters in the same row indicate a significant difference between FSIS and ATCC means at the P < 0.05 level.






59


The data suggested that Zep-Perosan, a peroxyacetic acid sanitizer, was not

effective against either of the planktonic E. coli 0157:H7 strains when utilized at such low concentrations. In contrast, Mosteller and Bishop (93) found peroxyacetic acid to be capable of producing a greater than 9 log reduction in planktonic bacterial isolates from Pseudomonasfluorescens, Yersinia enterocolitica, and Listeria monocytogenes after only a thirty second exposure. However, the study conducted by Mosteller and Bishop (93) treated the bacterial cells at the recommended in use concentration of 200 mg/L (4% peroxyacetic acid and 25% hydrogen peroxide) and this study utilized values just below the minimum inhibitory concentration.

The data suggested that Zep-Perosan, a peroxyacetic acid sanitizer, was not

effective against either of the planktonic E. coli 0157:H7 strains when utilized at such low concentrations. In contrast, Mosteller and Bishop (93) found peroxyacetic acid to be capable of producing a greater than 9 log reduction in planktonic bacterial isolates from Pseudomonasfluorescens, Yersinia enterocolitica, and Listeria monocytogenes after only a thirty second exposure. However, the study conducted by Mosteller and Bishop (93) treated the bacterial cells at the recommended in use concentration of 200 mg/L (4% peroxyacetic acid and 25% hydrogen peroxide) and this study utilized values just below the minimum inhibitory concentration.

Iodine Compound

As no minimum inhibitory concentration for the Escherichia coli 0157:H7 ATCC 700599 strain was determined, only the FSIS 063-93 strain was treated with the iodine sanitizer during the planktonic phase of this study. The FSIS 063-93 strain was not able to grow on the Violet Red Bile agar plates after repeated exposure at either concentration. Additionally, the bacterial isolates were unable to recover to a detectable level in the






60


Lauryl Sulfate broth after day 2 (Table 10). Significant differences were found between each day of treatment at both concentration levels but when comparing the log kills for each concentration there was only a significant difference at day 2 (Table 10). No trial differences occurred at either concentration of the iodine sanitizer. Table 10. Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia
coli 0157:H7 strain FSIS 063-93 when exposed to 0.25 mg/L of Zep-I-DineTM
and 0.50 mg/L of Zep-I-Dine for 5 minutes at 22'C
Day Concentration of Concentration of
0.25 mg/L (cfu/ml) 0.50 mg/L (cfu/ml)
Before After Log kill Before After Log kill
treatment treatment treatment treatment
1 8.79 2.57 6.12' 8.46 2.51 5.95b
2 8.37 0.77 7.60a 8.74 0.08 8.66a
3 0.00 0.00 0.00c 0.00 0.00 0.00C
a,b,c Different letters in the same column indicate a significant difference among means at the P < 0.05 level.

The results found in this study are similar to those of Ronner and Wong (110).

These researchers found that planktonic cells of Listeria monocytogenes and Salmonella typhimurium were reduced by 7 to 8 logs when treated with 25 mg/L of iodine for 10 minutes. Joseph and others (64) also showed that planktonic bacteria were unable to survive treatment with an iodine sanitizer. They found that Salmonella weltevreden and Salmonella FCM 40 were completely killed, a 6 log reduction, when exposed to 10 mg/L of iodine for 5 minutes. Finally, Marriott (82) stated that iodine sanitizers could reduce planktonic bacterial cell populations by 99.999% within 30 seconds when treated with a concentration level of 6.25 mg/L.

Bacterial Reassessment

No Escherichia coli 0157:H7 bacterial isolates for strain ATCC 700599 survived the planktonic stage of testing when treated with Clorox@ at 0.5 mg/L or at 1.0 mg/L. Additionally, no survival occurred when the ATCC 700599 strain was treated with Zep-I-






61


DineTM at 0.25 mg/L or at 0.50 mg/L. Repeated sub-lethal exposure to Zep-PerosanTM and Zepamine-ATM did result in an increase in the minimum inhibitory concentration for this strain of Escherichia coli 0157:H7. When repeatedly exposed to sub-lethal levels of Zep-PerosanTM, a peroxyacetic acid sanitizer, the minimum inhibitory concentration of E. coli 0157:H7 strain ATCC 700599 increased by 142.8%. Similarly, pretreatment of the ATCC 700599 bacterial isolates with sub-lethal levels of Zepamine-ATM resulted in a 68% increase of the minimum inhibitory concentration (Table 11). Table 11. The minimum inhibitory concentrations for Escherichia coli 0157:H7 strain
ATCC 700599 before and after pretreatment with sub-lethal levels of various
sanitizers
Minimum Inhibitory Concentration
Sanitizer Untreated Treated
Clorox@ 1.0 mg/L 1.0 mg/L
Zep-I-DineTM *
Zepamine-ATM 12.5 mg/L 21.0 mg/L
Zep-PerosanTM 3.5 mg/L 8.5 mg/L
*No minimum inhibitory concentration was found

The bacterial isolates for E. coli 0157:H7 strain ATCC 700599 that demonstrated an increase in their minimum inhibitory concentrations when treated with sub-lethal levels of Zep-PerosanTM and Zepamine-ATM were then further tested against the other sanitizers utilized in this study. The purpose for this was to determine if this increased resistance would provide cross protection against the other sanitizers and result in a similar increase in the minimum inhibitory concentrations for them. No increase was found in any isolates for Clorox@ or Zep-I-DineTM. The isolates did maintain their increased minimum inhibitory concentration for Zepamine-ATM and Zep-PerosanTM. Guerin-Mechin and others (55) observed similar results when treating Pseudomonas aeruginosa, also a gram-negative bacteria, with quaternary ammonium compounds. They found that the minimum bactericidal concentrations for five different subcultures of






62


Pseudomonas aeruginosa could be substantially increased upon pre-exposure to sublethal levels with two quaternary ammonium sanitizers. Additionally, it was determined that the increased levels of resistance to these sanitizers did not offer any cross protection when the subcultures were subsequently treated with other sanitizing compounds. In contrast, Pickett and Murano (102) found that exposure of Listeria monocytogenes to sub-lethal levels of sanitizers, including a chlorine based compound, an iodine base compound and a quaternary ammonium compound, did not affect the minimum inhibitory concentrations.

The results for Escherichia coli 0157:H7 strain FSIS 063-93 were similar to those of strain ATCC 700599. The pretreatment of the bacterial cells with sub-lethal levels of Clorox@ and Zep-I-DineTM did not result in an increase in the minimum inhibitory concentration for either sanitizer. When previously exposed to sub-lethal levels of Zepamine-ATM and Zep-PerosanTM a 61.5% increase and a 185.7% increase in the minimum inhibitory concentration occurred respectively (Table 12). Results obtained for the FSIS 063-93 strain are similar to that of Guerin-Mechin and others (55). As previously stated, they determined that the minimum inhibitory concentration for a sanitizer, specifically quaternary ammonium compounds, could be increased by the pretreatment of the bacterial cultures with sub-lethal levels of the sanitizer. Table 12. The minimum inhibitory concentrations for Escherichia coli 0157:H7 strain
FSIS 063-93 before and after pretreatment with sub-lethal levels of various
sanitizers
Minimum Inhibitory Concentration
Sanitizer Untreated Treated
Clorox@ 3.25 mg/L 3.25 mg/L
Zep-I-DineTM 1.5 mg/L 1.5 mg/L
Zepamine-ATM 13.0 mg/L 21.0 mg/L
Zep-PerosanTM 3.5 mg/L 10.0 mg/L






63


Additionally, when the bacterial isolates that were exposed to sub-lethal levels of Zep-PerosanTM were again tested against Clorox�, Zep-I-DineTM and Zepamine-ATM, no cross protection occurred as no increase in the minimum inhibitory concentrations resulted. The cells did maintain their previous increase in minimum inhibitory concentration for the quaternary ammonium sanitizer and the peroxyacetic acid sanitizer. In contrast to the ATCC 700599 strain, the FSIS 063-93 strain did exhibit crossprotection when pretreated with sub-lethal levels of Zepamine-ATM, the quaternary ammonium sanitizer. The repeated sub-lethal chemical shock with Zepamine-ATM on E. coli 0157:H7 strain FSIS 063-93 resulted in a decrease in the minimum inhibitory concentration for the chlorine compound, the maintenance of the increased minimum inhibitory concentrations for the quaternary ammonium sanitizer and the peroxyacetic acid sanitizer, and an increase of 186.7% for the minimum inhibitory concentration for the iodophor compound (Table 13).

Table 13. The minimum inhibitory concentrations of various sanitizers for Escherichia
coli 0157:H7 strain FSIS 063-93 planktonic bacterial isolates before and after
pretreatment with sub-lethal levels of Zepamine-ATM
Minimum Inhibitory Concentration
Sanitizer Untreated Treated
Clorox@ 3.25 mg/L 2.2 mg/L
Zep-I-DineTM 1.5 mg/L 4.3 mg/L
Zepamine-ATM 13.0 mg/L 21.0 mg/L
Zep-PerosanTM 3.5 mg/L 10.7 mg/L


Adherent Bacteria

Attachment Levels

Microbial attachment and the development of biofilms are known to occur on many surfaces and in many different environments (34, 64, 93). In this study it was determined that both Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 attached






64


to the stainless steel chips after a 4 hour static suspension at a level of approximately 104 cfu/cm2. Dewanti and Wong (34) found similar results. When stainless steel chips were inoculated with Escherichia coli 0157:H7 at a level of 106 cfu/ml, they found the bacteria were able to adhere to the surface at a level from 103 to 105 cfu/cm2 after a 1 hour incubation at room temperature. Similar results were obtained by Hood and Zottola (60) who found that Salmonella typhimurium, Listeria monocytogenes, Pseudomonasfragi, Pseudomonasfluorescens, and Escherichia coli 0157:H7 bacterial cells were all able to attach to stainless steel within 1 hour in various test growth media. In particular, they determined that depending upon the test media, Escherichia coli 0157:H7 cells were able to attach at levels of 103 to 105 cfu/cm2 . Farrell and others (43) also observed a similar attachment level for Escherichia coli 0157:H7 bacteria on stainless steel. Their research showed that after only a 5 minute incubation period, E. coli 0157:H7 cells were able to attach to the surface at a level of 103 to 104 cfu/cm2 Sanitizer Treatments for Adherent Cells

As previously stated, chemical sanitizers are considered to be effective on food

contact surfaces if they demonstrate a five-log reduction in planktonic bacteria (43) and a greater than three-log reduction for adherent cells (93). In this study the treatment of Escherichia coli 0157:H7 bacterial isolates with 1.0 mg/L of Clorox@ was effective against planktonic cells but failed to provide an adequate reduction in adherent E. coli 0157:H7 cells (Table 14).

There were no significant differences between the strains for log kill for adherent cells however significant differences existed between the adherent cells and the planktonic cells (Table 14). The chlorine treatment of the adherent bacterial cells provided slightly less than a two-log reduction for the FSIS 063-93 strain and slightly






65


greater than a two-log reduction for the ATCC 700599 strain. In this case, the use of the chlorine sanitizer was no more effective than the use of water alone. Treatment of the stainless steel coupons with water also provided a 1 to 2 log decrease in the bacterial counts for both E. coli 0157:H7 strains.

Table 14. Mean log values (n=18) for planktonic and adherent bacterial isolates of
Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when
exposed to 1.0 mg/L of Clorox@ for 5 minutes at 22'C
Strain Cell type Mean log values (log cfu/ml or cfu/chip)
Before treatment After treatment Log kill
FSIS Planktonic 8.46 1.54 6.92b
FSIS Attached 5.47 3.77 1.70c
ATCC Planktonic 8.94 1.14 7.80a
ATCC Attached 5.56 3.48 2.08c
a,b,c Different letters in the same column indicate a significant difference among means at the P < 0.05 level.

Researchers have found similar results when treating adherent pathogenic bacteria with a chlorine sanitizer. Restaino and others (108) found that chlorine at 100 mg/L did not provide a significant difference from water in reducing the bacterial population of adherent Staphylococcus aureus cells on Formica surfaces. Both the chlorine sanitizer and water provided only a two-log decrease in the bacterial isolates. In another study, Joseph and others (64) found that at a concentration of 10 mg/L, Cl2 was able to reduce greater than 105 log of planktonic Salmonella spp. after a 5 minute contact time. The same sanitizer was only able to reduce the adherent bacterial cells by I log after a 25 minute exposure. Additionally, when Joseph and others (64) increased the C12 concentration up to 50 mg/L, adherent bacteria were still only reduced by 2 logs after 25 minutes. Andrade and others (3) also found that adherent bacterial cells were more resistant than non-adherent cells when treated with chlorine. In contrast, Farrell and others (43) found a three-log reduction or higher when adherent Escherichia coli






66


0157:H7 cells on stainless steel were treated with chlorine. However, they treated the cells at a level of 200 mg/L and when the stainless steel coupons were enriched after sanitizer treatment 63 to 88 % of the chips were found to be positive for Escherichia coli 0157:H7. This indicates that injured organisms remained on the surface after treatment and were able to recover upon enrichment. This phenomenon also occurred in the current study when planktonic E. coli 0157:H7 cells were exposed to chemical shock with Clorox@ (Table 2 and Table 3).

As shown by Table 15, significant differences existed between both strain and cell type in regards to log kills attained. The quaternary ammonium compound was found to be effective for both planktonic and adherent cells for the ATCC 700599 strain. In contrast, the sanitizer was effective against only the planktonic cells of the FSIS 063-93 strain. Treatment of adherent Escherichia coli 0157:H7 FSIS 063-93 cells resulted in less than a 1 log reduction.

Table 15. Mean log values (n=18) for planktonic and adherent bacterial isolates of
Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when
exposed to 11.0 mg/L of Zepamine-ATM for 5 minutes at 22'C
Strain Cell type Mean log values (log cfu/ml or cfu/chip)
Before treatment After treatment Log kill
FSIS Planktonic 8.87 3.38 5.49'
FSIS Attached 5.58 4.88 0.70d
ATCC Planktonic 8.91 2.11 6.80a
ATCC Attached 5.67 1.62 4.05c
abc Different letters in the same column indicate a significant difference among means at the P <0.05 level.

Results found by Mosteller and Bishop (93) supports the current research. They found that quaternary ammonia at 200 mg/L killed more than 5 logs of planktonic bacterial cells of Pseudomonasfluorescens, Yersinia enterocolitica, and Listeria monocytogenes in just 30 seconds. Nonetheless, the same sanitizer was unable to provide






67


a three log reduction in the same bacterial isolates when the cells were attached to rubber or Teflon@. Additionally, Restaino and others (108) showed quaternary ammonium at 150 mg/L to be ineffective against adherent Staphylococcus aureus cells both in the presence and absence of organic material. After a 60 minute exposure time, they found a

2 and 2.5 log reduction, respectively. Similarly, Trachoo and Frank (131) found that a quaternary ammonium compound at a level of 50 mg/L was able to inactivate Campylobacterjejuni cells with no biofilms present in 45 seconds. With biofilms, the same sanitizer even at an increased level of 200 mg/L did not inactivate the bacterial cells. Peng and others (101) also found planktonic cells to be the most susceptible to sanitizers, followed by attached, single cells and then cells present in a biofilm. They determined that 100 mg/L of quaternary ammonia resulted in a greater than five-log reduction in planktonic Bacillus cereus cells within 15 seconds. However, neither 100 mg/L or 200 mg/L of the same sanitizer was effective against the cells in a biofilm.

Results of this study found Zep-PerosanTM, a peroxyacetic acid, to be somewhat ineffective against both planktonic and adherent Escherichia coli 0157:H7 cells at the concentrations utilized. Both strains showed a three-log reduction for planktonic cells with no significant differences between the two strains. The strains did show a significant difference for the adherent cells (Table 16). The FSIS 063-93 strain showed less than a one-log decrease for the adherent cells while the ATCC 700599 strain showed a two-log reduction.

As in this study, Mosteller and Bishop (93) found that peroxyacetic acid was not effective against adherent bacterial cells. Unlike current research, they concluded that at a level of 200 mg/L peroxyacetic acid was effective at reducing planktonic bacterial cells






68


by more than 5 logs. Trachoo and Frank (131) also found peroxyacetic acid to be quite effective against planktonic bacterial cells. They found that at 50 mg/L (27.5 % of hydrogen peroxide and 5.8 % of peracetic acid) the sanitizer effectively eliminated all planktonic cells of Campylobacterjejuni, also a gram negative bacteria, within 45 seconds. Even at this higher concentration, the sanitizer while effective against planktonic cells, was not able to inactivate the bacterial cells within a biofilm matrix after a 3 minute exposure.

Table 16. Mean log values (n=18) for planktonic and adherent bacterial isolates of
Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when
exposed to 3.0 mg/L per 13.2mg/L of Zep-PerosanTM for 5 minutes at 220C Strain Cell type Mean log values (log cfu/ml or cfu/chip)
Before treatment After treatment Log kill
FSIS Planktonic 8.49 5.27 3.22a
FSIS Attached 5.49 5.36 0.13c
ATCC Planktonic 8.59 5.04 3.55a
ATCC Attached 5.59 3.29 2.30b
a,b,c Different letters in the same column indicate a significant difference among means at the P < 0.05 level.

As in this study, Mosteller and Bishop (93) found that peroxyacetic acid was not effective against adherent bacterial cells. Unlike current research, they concluded that at a level of 200 mg/L peroxyacetic acid was effective at reducing planktonic bacterial cells by more than 5 logs. Trachoo and Frank (131) also found peroxyacetic acid to be quite effective against planktonic bacterial cells. They found that at 50 mg/L (27.5 % of hydrogen peroxide and 5.8 % of peracetic acid) the sanitizer effectively eliminated all planktonic cells of Campylobacterjejuni, also a gram negative bacteria, within 45 seconds. Even at this higher concentration, the sanitizer while effective against planktonic cells, was not able to inactivate the bacterial cells within a biofilm matrix after a 3 minute exposure.






69


Unlike the current research, Fatemi and Frank (44) found peroxyacetic acid to be an effective sanitizer against adherent bacterial cells. Although they utilized a higher concentration level, 40 mg/L, they found that in a mixed, adherent culture consisting of Listeria monocytogenes and Pseudomonas, peroxyacetic acid was able to reduce the bacterial population to less than 10 cfu/cm 2. Along this line, Farrell and others (43) found that after sanitizing treatment of stainless steel chips with adherent Escherichia coli 0157:H7 bacterial cells in a 0.2 % solution of peroxyacetic acid, viable bacteria was infrequently recovered. However, Farrell and others (43) also reported that even when inoculated at a level of only 102 cfu/ml of Escherichia coli 0157:H7, a more realistic scenario for a meat processing facility, 50 % of the stainless steel chips utilized in the study were positive for E. coli 0157:H7 after enrichment.

As shown by Table 17, significant differences existed between both strain and cell type in regards to log kills attained. While all log kill values were significantly different from each other, the iodine sanitizer was found to be effective against both the planktonic and adherent cells for both Escherichia coli 0157:H7 strains. Treatment of planktonic cells resulted in a greater than five-log reduction for both strains and treatment of adherent cells resulted in greater than a 3 log reduction (Table 17). Table 17. Mean log values (n=18) for planktonic and adherent bacterial isolates of
Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when
exposed to 0.5 mg/L of Zep-I-DineTM for 5 minutes at 22'C
Strain Cell type Mean log values (log cfu/ml or cfu/chip)
Before treatment After treatment Log kill
FSIS Planktonic 8.46 2.50 5.96b
FSIS Attached 5.85 2.69 3.16"
ATCC Planktonic 8.61 0.00 8.61a
ATCC Attached 5.94 1.23 4.71c
a,bc,d Different letters in the same column indicate a significant difference among means at the P <0.05 level.






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Mosteller and Bishop (93) also found iodine to be effective against planktonic bacterial cells. They showed a greater than five-log reduction in several bacterial populations within 30 seconds at a concentration of 25 mg/L. They found that iodine was not effective at reducing adherent bacterial populations by more than 3 logs in most cases. Adherent Listeria monocytogenes cells were not effectively reduced. Adherent Yersinia enterocolitica and Pseudomonasfluorescens cells were only reduced by more than 3 logs when attached to Teflon@ as determined by plate count method not the impedance method. Similarly, Joseph and others (64) found iodine to be effective at a level of only 1 mg/L on planktonic bacterial isolates of Salmonella species after a 5 minute treatment. However, in order to obtain a three-log or higher reduction on adherent cells the use of a higher concentration level and a longer exposure time was required.














CHAPTER 5
SUMMARY AND CONCLUSIONS

There is much evidence that Escherichia coli 0157:H7 is an adaptive organism capable of surviving hostile and harsh environments. This investigation demonstrated that the ability of various sanitizers to clearly provide a five-log or greater reduction in planktonic bacteria does not necessarily correspond to the sanitizers' ability to provide an adequate reduction (three-log) in attached bacteria. This study evaluated the ability of Escherichia coli 0157:H7 isolates to survive and adapt to four sanitizers common to the food industry.

Preliminary studies determined the minimum inhibitory concentration, sub-lethal and lethal concentration of sanitizers for Escherichia coli 0157:H7 strain FSIS 063-93 and strain ATCC 700599 which is known to be acid tolerant. The chemical sanitizers utilized included a sodium hypochlorite solution (bleach), a peroxyacetic acid compound (Zep-PerosanTM), a quaternary ammonium compound (Zepamine-ATM), and an iodine compound (Zep-I-DineTM). The concentration level for each sanitizer was adjusted depending upon the ability of the E. coli 0157:H7 isolates to survive treatment.

Data from the planktonic stage of testing showed that lack of recovery by plate count method after sanitizer treatment did not mean that an organism was no longer present. All of the sanitizers except Zep-PerosanTM demonstrated at least a five-log reduction. However, both strains demonstrated the ability to fully recover and grow in broth after treatment with all of the sanitizers. Zep-I-DineTM was found to be the most effective sanitizer for planktonic Escherichia coli 0157:H7 bacteria followed by


71






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Clorox@ and then Zepamine-ATM. At the concentration used in this study, ZepPerosanTM was not found to be effective.

Bacteria that survived the planktonic stage of testing were reassessed in regards to their minimum inhibitory concentration, sub-lethal and lethal concentration. No changes were found in the minimum inhibitory concentration for either bacterial strain when exposed to Clorox@ or Zep-I-DineTM. Substantial increases were found in the minimum inhibitory concentration levels for both bacterial strains for Zepamine-ATM and ZepPerosanTM. The repeated sub-lethal level of exposure with both sanitizers resulted in ability of the bacterial strains to withstand higher levels of the sanitizer(s), indicating that some kind of adaptation had occurred. With one exception, the increased minimum inhibitory concentrations for the two bacterial strains failed to provide any crossprotection. The FSIS 063-93 strain when pre-treated with sub-lethal levels of ZepamineATM seemed to invoke a response which allowed the bacteria to survive exposure to a higher level of Zep-I-DineTM.

Data from the adherent phase of testing supports previous research indicating that microorganisms become more resistant to sanitizers when they are attached to a surface. Zep-I-DineTM was the only sanitizer in this study that was effective against both planktonic and adherent bacterial isolates. Both Zepamine-ATM and Clorox@ which were effective against the planktonic bacteria were not effective against adherent bacteria. Zep-PerosanTM was not found to be effective against either planktonic or adherent bacteria.

The information obtained in this study suggests that disinfectants and sanitizers tested in suspension for use in the industry may not correlate with results obtained on






73


surfaces and perhaps even less in cases where a biofilm has developed. This study also indicates that methodology for recovery of bacteria is important. Plate count data may not be a reliable indication of the cleanliness of a surface. As chemical sanitizers are commonly utilized by the food industry as a tool for improving food quality and safety, it is essential to be able to accurately identify and quantify the bacterial organisms that may be present before and after cleaning and sanitation. While this study shows that organisms may still be present after sanitation, it is important to remember that the concentration levels utilized were minimal values not the recommended levels for sanitation or disinfection.

Currently, no data exists to suggest that the proper use of sanitizers in the food industry will lead to development of highly resistant microorganisms. However, the potential for an organism to adapt does exist. Therefore it is essential for the food industry to know as much as possible about the effectiveness of sanitizers and the microorganisms they are designed to eliminate or reduce to acceptable levels.

Further research opportunities presented by this study may include: 1) a study involving the use of additional bacterial strains; 2) the study of the effects of the sanitizing agents on both planktonic and adherent bacterial cells when using the recommended concentration instead of sub-lethal levels; 3) a comparative study of sublethal and recommended concentrations of the sanitizing agents; 4) the effect of sublethal exposure of the sanitizing agents on other food processing surfaces; 5) the combinations of manual or mechanical scrubbing and sub-lethal levels of the sanitizing agents 6) the investigation into the membrane of the Escherichia coli 0157:H7 bacterial isolates that exhibited increases in their minimum inhibitory concentration levels; and 7)






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the study of alternative enumeration methods to present a more accurate picture of the actual levels of bacteria remaining after treatment with various sanitizers.















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BIOGRAPHICAL SKETCH

Kristen Ann Goodfellow-Hunt was born on July 14, 1968, in Austin, Minnesota.

She attended Santa Fe Community College of Gainesville, Florida, where she received an Associate of Arts degree with high honors in the summer of 1989. She then transferred to the University of Florida and was awarded a Bachelor of Arts in Education with honors in the fall of 1992. She was admitted to graduate school in the spring of 1993 and received a Master of Education degree with a concentration in science in the spring of 1994.

After teaching for several years, Kristen decided in the spring of 1998 to return to the University of Florida part-time, while continuing to teach, in order to further her own education. In the fall of 1999 she was awarded an Animal Sciences Department assistantship and thus decided to return to the University of Florida in pursuit of a Doctor of Philosophy degree in the College of Agricultural and Life Sciences on a full-time basis. She was admitted to candidacy on March 26, 2001. Kristen earned her Doctor of Philosophy degree from the University of Florida in the summer of 2003.

Upon receiving her Doctor of Philosophy degree, Kristen plans to work in industry as a laboratory manager for a food testing company. This career choice should allow her to manage the lab, do consulting, and teach both microbiology and HACCP short courses.


86








I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.


Roge' West, Chair
Professor Emeritus of Animal Sciences

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philoso


Dwain D. Johns
Professor of Animal Sciences

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philoso y.


Sally K. ) iMams
Associate ofessor of Animal Sciences

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philoso


Gary E. Rodri
Professor o d Science and Human Nutrition

This dissertation was submitted to the Graduate Faculty of the College of
Agricultural and Life Sciences and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy.

August 2003
Dean, College of Agricultu 1 an ife Sciences


Dean, Graduate School




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THE FATE OF Escherichia coli 0157:H7 WHEN EXPOSED TO SUBLETHAL AND LETHAL CONCENTRATIONS OF COMMON INDUSTRIAL SANITIZERS By KRISTEN ANN HUNT A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2003

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Copyright 2003 by Kristen Ann Hunt

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I dedicate my dissertation to my husband, Philip, and to my two sons, Aaron and Jared. Their love, pride and guidance helped to keep me disciplined and motivated throughout my studies.

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ACKNOWLEDGMENTS I extend my sincere admiration and gratitude to my committee chairperson, professor and advisor, and friend. Dr. Roger L. West, for his continual guidance and support. I also wish to express my appreciation to the members of my supervisory committee. Dr. D. Dwain Johnson, Dr. Sally K. Williams, and Dr. Gary E. Rodrick, for their help in the completion of this project. Special thanks are given to Dr. West and the Department of Animal Sciences for financial support throughout my graduate studies at the University of Florida and this project. Special recognition is given to Deibel Laboratories, Incorporated for the use of their facilities, laboratory equipment, personnel and supplies. I wish to personally thank Dr. Robert H. Deibel for his support and understanding throughout the completion of this project. I also wish to thank LeaAnne B. Green as well, not only for her assistance but for the constant encouragement, non -judgmental listening, and above all else, her friendship. In addition, I would like to recognize Larry Eubanks, Byron Davis and Tommy and Brian Estevez for the friendship and humor they provided to me throughout this project. Regards go to fellow graduate students Gabriel Cosenza, Robin Hamm and Ben Warren for their camaraderie throughout our studies. In particular, I wish to thank Ben for his assistance with the statistical analysis of my project. My husband, Philip Hunt, and my parents, Steve and Colleen Goodfellow, deserve special credit for putting up with me throughout my studies, qualifying exams, and in iv

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particular, the completion of my dissertation. I sincerely thank them for their love, understanding and support. I thank them all for believing in me and encouraging me to better myself and above all else, I thank them for their patience. And, I thank God for helping me to keep the heart, faith and mind to make my dreams become realities. V

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TABLE OF CONTENTS page ACKNOWLEDGMENTS iv LIST OF TABLES viii LIST OF FIGURES x ABSTRACT xi CHAPTER 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 5 Characteristics of Escherichia coli 0157:H7 5 Clinical Aspects of Escherichia coli 0157:H7 7 Prevalence of Escherichia coli 0157:H7 9 Selected Outbreaks of Escherichia coli 0157:H7 10 Resistance and Adaptation Characteristics of Escherichia coli OI57:H7 13 Acid Resistance and Acid Tolerance 13 Antibiotic Resistance 19 Thermal Tolerance and Adaptation 23 Adaptation and Tolerance of Sanitizing Agents 25 Chemical Sanitizers in the Food Industry 27 Chlorine Sanitizers 28 Quaternary Ammonium Sanitizers 29 Peroxyacetic Acid Sanitizers 30 Iodine Sanitizers 31 The Ideal Sanitizer 32 Surfaces and Attachment 33 Recovery of Stressed Microorganisms 34 3 MATERIALS AND METHODS 36 Bacterial Cultures 36 Sanitizer Solutions 40 Determination of Minimum Inhibitory Concentration (MIC) 40 Zone Inhibition 42 Planktonic Bacteria 43 vi

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Bacterial Reassessment 44 Solid Substrate 45 Adhesion of Microbial Cells 45 Sanitizer Treatments for Adherent Cells 46 Microbiological Analysis 46 Statistical Analysis 47 4 RESULTS AND DISCUSSION 48 Determination of Minimum Inhibitory Concentration 48 Planktonic Bacteria 50 Chlorine Compound 50 Quaternary Ammonium Compound 53 Peroxyacetic Acid Compound 56 Iodine Compound 59 Bacterial Reassessment 60 Adherent Bacteria 63 Attachment Levels 63 Sanitizer Treatments for Adherent Cells 64 5 SUMMARY AND CONCLUSIONS 71 REFERENCES 75 BIOGRAPHICAL SKETCH 86 vii

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LIST OF TABLES Table page 1 The minimum inhibitory concentrations (MICs) of various sanitizers against Escherichia coll 0157:H7 strain ATCC 700599 and strain FSIS 063-93 after incubation at 37°C for 24 hours 48 2 Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 1.0 mg/L of Clorox® for 5 minutes at 22°C 51 3 Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 0.5 mg/L of Clorox® for 5 minutes at 22°C 52 4 Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 10.0 mg/L of Zepamine-A™ for 5 minutes at 22°C 53 5 Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to n.O mg/L of Zepamine-A™ for 5 minutes at 22°C 55 6 Mean log reduction values by trial (n=18) for Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 11.0 mg/L of ZepamineA™ for 5 minutes at 22°C 56 7 Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 1.0 mg/L per 4.4 mg/L of Zep-Perosan™ for 5 minutes at 22°C 57 8 Mean log reduction values by trial (n=18) for Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 1.0 mg/L of peroxyacetic acid per 4.4 mg/L of hydrogen peroxide of Zep-Perosan™ for 5 minutes at 22°C 57 9 Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 2.0 mg/L per 8.8 mg/L of Zep-Perosan™ for 5 minutes at 22°C 58 vui

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10 Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia coli 0157:H7 strain FSIS 063-93 when exposed to 0.25 mg/L of Zep-I-Dine™ and 0.50 mg/L of Zep-I-Dine for 5 minutes at 22°C 60 1 1 The minimum inhibitory concentrations for Escherichia coli 0157:H7 strain ATCC 700599 before and after pretreatment with sub-lethal levels of various sanitizers 61 12 The minimum inhibitory concentrations for Escherichia coli 0157:H7 strain FSIS 063-93 before and after pretreatment with sub-lethal levels of various sanitizers 62 13 The minimum inhibitory concentrations of various sanitizers for Escherichia coli 0157:H7 strain FSIS 063-93 planktonic bacterial isolates before and after pretreatment with sub-lethal levels of Zepamine-A™ 63 14 Mean log values (n=18) for planktonic and adherent bacterial isolates of Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 1.0 mg/L of Clorox® for 5 minutes at 22°C 65 15 Mean log values (n=18) for planktonic and adherent bacterial isolates of Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 1 1.0 mg/L of Zepamine-A™ for 5 minutes at 22°C 66 16 Mean log values (n=18) for planktonic and adherent bacterial isolates of Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 3.0 mg/L per 13.2mg/L of Zep-Perosan™ for 5 minutes at 22°C 68 17 Mean log values (n=18) for planktonic and adherent bacterial isolates of Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 0.5 mg/L of Zep-I-Dine™ for 5 minutes at 22°C 69 ix

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LIST OF FIGURES Figure page 1 Absorbance of Escherichia coli 0157:H7, strain ATCC 700599, in Lauryl Sulfate broth over 24 hours 38 2 Growth of Escherichia coli 0157:H7, strain ATCC 700599, on Violet Red Bile Agar over 24 hours 38 3 Absorbance of Escherichia coli 0157:H7, strain FSIS 063-93, in Lauryl Sulfate broth over 24 hours 39 4 Growth of Escherichia coli 0157:H7, strain FSIS 063-93, on Violet Red Bile Agar over 24 hours 39 5 Escherichia coli 0157:H7 strain ATCC 700599 Standard Curve 41 6 Escherichia coli 0157:H7 strain FSIS 063-93 Standard Curve 42 X

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE FATE OF ESCHERICHIA C0LI0\51:W1 WHEN EXPOSED TO SUBLETHAL AND LETHAL CONCENTRATIONS OF COMMON INDUSTRIAL SANITIZERS By Kristen Ann Hunt August 2003 Chair: Roger L. West Major Department: Animal Sciences This study tested the hypothesis that pre-exposure of Escherichia coli 0157:H7 to sub-lethal levels of industrial sanitizers could affect the survival of cells to subsequent exposure at lethal levels. The susceptibility of planktonic and adherent cells to sanitizing compounds was compared. The ability for an acid tolerant Escherichia coli 0157:H7 strain to provide cross-protection to the cells when exposed to chemical sanitizers was also examined. E. coli 0157:H7 cells were exposed to a chlorine compound, an iodophor, a quaternary ammonium compound (quat) and a peroxyacetic acid compound (PAA). Results show that at the concentrations utilized in this study, the iodophor provided the greatest reduction in planktonic cells followed by chlorine, then quat and the PAA compound. All sanitizers, except the peroxyacetic acid, were effective (greater than 5 log reduction) against the planktonic Escherichia coli 0157:H7 bacterial isolates. Data from the planktonic stage of testing showed that lack of recovery by plate count method after xi

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sanitizer treatment did not mean that an organism was no longer present. Both strains demonstrated the abiUty to recover and grow in broth after treatment with all of the sanitizers All sanitizers tested were significantly less effective against the adherent cells than the planktonic cells. The iodophor compound was the only sanitizer found to be effective against both planktonic and adherent cells. Neither the chlorine compound nor the PAA compound was effective against the adherent cells. The quaternary ammonium compound was only effective against the acid tolerant strain for adherent cells. Throughout testing, the un-adapted Escherichia coli 0157:H7 strain typically showed higher survival rates than the known acid tolerant strain. Additionally, with one exception, the bacterial cells that exhibited an increased minimum inhibitory concentration did not demonstrate any increased resistance when exposed to other sanitizers. While the pretreatment of bacterial cells with a quaternary ammonium and a peroxyacetic acid compound resulted in survival at higher concentrations, the values were still far below the recommended usage level. This adaptation does however demonstrate the importance of proper cleaning and sanitation procedures to ensure a safe product. xii

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CHAPTER 1 INTRODUCTION Escherichia coli 0157:H7 is an important foodbome pathogen not only in the United States but globally (80, 95, 123, 129, 138). Increased knowledge and awareness of this human pathogenic bacterium by the scientific community and the general populace along with better surveillance systems and techniques have contributed to partial control of this organism. Despite the increased recognition and research devoted to E. coli 0157:H7, it remains a public health problem (129). Each year approximately 76 million people experience foodbome illness (84). The Centers for Disease Control (CDC) estimates that 73,000 persons become ill and 61 people die annually in the United States alone as a result of infection from E. coli 0157:H7 (24, 28). In addition to being a public health problem, Escherichia coli 0157:H7 is a familiar foe to the food industry. Even with governmental regulations and Hazard Analysis Critical Control Point (HACCP) in place, the industry continues to be plagued by this organism (1,2, 8). Millions of pounds of "contaminated" ground beef are recalled annually (8, 41, 46, 47). Although E. coli 0157:H7 was initially viewed as a threat associated primarily with ground beef, it now shows up in raw vegetables, unpasteurized juice, dairy products, and in the water in which we swim or drink (1, 2, 8, 25, 41, 95). Even foods which were once considered to be "safe" from enteric infections such as drycured salami and fermented sausage have now been associated with E. coli 0157:H7 infections and outbreaks (41, 129). In recent years, outbreaks and sporadic incidences of 1

PAGE 14

2 illness attributed to E. coli 0157:H7 have occurred in foods such as coleslaw, iceburg and romaine lettuce, fruit salad, cheese curds, yogurt, and even in cake (25, 41). Research has shown that Escherichia coli 0157:H7 is an adaptive organism that can survive hostile and harsh environments (8, 38, 39, 41). The organism is a hardy pathogen that has managed to survive and adapt to varied environmental stresses (39). Due to the hardy nature of the organism and its ability to adapt to new environments the need to be able to control or eliminate Escherichia coli 0157:H7 is of critical importance to the food industry (8, 103). Conditions designed to reduce bacterial counts such as refrigeration, freezing, and treatment of the carcass(es) with organic acids may in fact enhance the survival of the organism (132). When bacteria are exposed to certain food processing treatments designed to extend shelf life, sub-lethal injury can result. It is possible that bacteria which have been subjected to heating, refrigeration, freezing, acid, low pH, and even sanitizers may be present in foods. From a food safety standpoint this poses an unacceptable risk as inactivated or sub-lethally injured bacteria may be able to undergo repair and resume growth (120). As the majority of the responsibility for providing a "safe" product now falls on the food industry, it is essential to have and maintain an effective sanitation program (8, 81). Sanitation procedures are implemented in the food industry to aid in the production of a safe product with an acceptable level of quality (93). As defined in Principles of Food Sanitation, in regard to the food industry, sanitation is the "creation and maintenance of hygienic and healthful conditions." In regard to science, sanitation is "to provide wholesome food handled in a clean environment by healthy food handlers, to prevent contamination with microorganisms that cause foodbome illness and to minimize the

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3 proliferation of food spoilage microorganisms" (82, p.2). In simpler terms, sanitation is reducing the number of bacteria present on a surface. Sanitation does not mean sterile; in other words there may still be some bacteria present on a surface that has been sanitized. Three categories of sanitizers exist. They include physical sanitizing, radiation sanitizing, and chemical sanitizing. For this study, the focus will be on chemical sanitizing. The category of chemical sanitizers is extremely broad. It includes chlorine compounds, iodine compounds, bromine compounds, quaternary ammonium compounds, acid sanitizers, acid anionic sanitizers, acid-quaternary sanitizers, hydrogen peroxide compounds, peroxyacetic acids, ozone, glutaraldehydes, and microbicides (82). Chemical sanitizers are deemed to be effective on food contact surfaces if they demonstrate a five-log reduction in planktonic bacteria (43). Efficacy testing of sanitizers with non-adherent bacteria could be misleading as to the sanitizers' true effectiveness under processing conditions where the bacterial cells may be attached to a variety of surfaces. Research has found that various sanitizers failed to provide an adequate reduction (three-log) in attached bacteria where they had clearly demonstrated the ability to be effective against planktonic bacterial cells (71, 93). This study tested the hypothesis that pre-exposure of Escherichia coli 0157:H7 to sub-lethal levels of common industrial sanitizers could affect the survival of cells subsequently exposed to lethal levels of the sanitizers. The susceptibility of planktonic cells to sanitizing compounds was compared to the susceptibility of adherent cells when exposed to the same chemical treatment. The chemical sanitizers utilized included a sodium hypochlorite solution (bleach), a peroxyacetic acid compound (Zep-Perosan™), a quaternary ammonium compound (Zepamine A™), and an iodine compound (Zep-I-

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4 Dine™). Additionally, the survival of E. coli 0157:H7 cells exposed to sub-lethal levels of the sanitizers and the ability to recover and resuscitate said cells was investigated. Data were collected and compared for an un-adapted E. coli 0157:H7 strain as well as for an acid resistant strain.

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CHAPTER 2 REVIEW OF LITERATURE Characteristics of Escherichia coli 0157:H7 Escherichia coli 0157:H7 is a small, gram-negative, non-sporing, straight rod. This pathogenic organism is a facultative anaerobe and can therefore grow in the presence or absence of oxygen (1, 2, 7). It grows rapidly from 30°C to 42°C with generation times ranging from 0.49 hr at 37°C to 0.64 hr at 42°C (40). At temperatures of 44°C to 45°C, the organism grows poorly (104). Thermal inactivation studies of E. coli 0157:H7 in ground beef demonstrated that the pathogen has no unusual resistance to heat with D values of 270, 45, 24, and 9.6s at 57.2, 60.0, 67.8 and 64.3°C, respectively (40). However, it does appear to survive well in frozen storage at -20°C. Doyle and Schoeni (40) found that Escherichia coli 0157:H7 could survive in ground beef when frozen at -80°C and held at -20°C with no major changes in population for up to nine months. Escherichia coli 0157:H7 is one of hundreds of strains of the bacterium Escherichia coli. Most E. coli strains are harmless; however, some, such as Escherichia coli 0157:H7, are pathogenic and cause diarrheal illness (24, 41). The strains that cause disease are categorized into specific groups based on virulence properties, mechanisms of pathogenicity, clinical syndromes, and distinct 0:H serogroups (41, 95). Escherichia coli 0157:H7 is a member of the enterohemorrhagic E. coli (EHEC) group (1, 2, 41). Additionally, isolates of E. coli are serologically differentiated on the basis of three major surface antigens. The O (somatic) antigen, the H (flagella) antigen and the K (capsular) 5

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6 antigen enable serotyping of the organism. Currently, at least 174 O, 56 H, and 80 K antigens have been identified (41). Members of the EHEC group, such as Escherichia coli 0157:H7, are clonal in origin and phenotypically and genotypically very similar (73). The bacterium Escherichia coli 0157:H7 is distinct from other strains in that it is unable to ferment sorbitol in 24 hours and does not produce p-glucuronidase (41, 73, 98). It possesses a 60MDa plasmid, expresses an uncommon 5,000 to 8,000 molecular weight outer membrane protein, and has an attaching and effacing (eae) gene (41, 73, 95). The eae gene is responsible for the production of an attaching and effacing lesion (A/E) that causes the degeneration and effacement of intestinal epithelial cell microvilli, intimate adherence of bacteria to the epithelial cells, and assembly of highly organized cytoskeletal structures in the cells beneath intimately attached bacteria (69). The cytoskeletal structures are composed of components such as actin, talin, ezrin, and a-actinin (65). Once Escherichia coli 0157:H7 adheres to the bowel mucosa, it will grow and secrete an array of extracellular products including potent cytotoxins (73). These cytotoxins are referred to as Shiga toxins, Shiga-like toxins and verotoxins (95). The mechanism by which E. coli 0157:H7 causes disease is not yet fully understood, but an important factor in its virulence is the production of verotoxins (38). Escherichia coli 0157:H7 can produce Shiga toxin 1 (Stx 1), Shiga toxin 2 (Stx 2), or both (95). Most isolates of E. coli 0157 produce only Stx 2; occasionally isolates produce both Stx 1 and Stx 2, and rarely isolates are found to produce only Stx 1 (54). Shiga toxin 1 is homogeneous where Shiga toxin 2 is comprised of numerous variants. The toxins share about 60 percent of their DNA and amino acid homology but are

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7 immunologically distinct (73). Both are compound toxins consisting of an A subunit and a pentameric B subunit that are the active and binding portions of the toxins, respectively (38, 73). The hollow ring formed by the B subunit is where the C-terminus end of the A subunit is inserted. The B subunits mediate binding to specific glycosphingolipids, termed globotriosyl ceramides (Gb3 and Gb4), which are receptors on the surface of cells of specific body tissues in eukaryotes (38, 73). The globotriosyl ceramide, Gb3, is abundant in the cortex of the human kidney (38). Both toxins have similar modes of action, which involve blocking protein synthesis by inhibiting elongation factor1dependent aminoacyl binding of t-RNA to ribosomal subunits (38, 73). The toxicity of the two toxins however is dissimilar. Shiga toxin 2 has been found to be 1,000 times more cytotoxic than Shiga toxin 1 towards human renal microvascular endothelial cells (73). Although Stx 1 and Stx 2 have similar structures and similar mechanisms of action, the genes for Shiga toxin 1 and Shiga toxin 2 are carried on two separate bacteriophages. Bacteriophages are viruses capable of infecting bacterial cells (1,2). Clinical Aspects of Escherichia coli 0157:H7 Illness associated with Escherichia coli 0157:H7 is primarily the result of a foodbome infection. The disease may also be transmitted via person to person, especially in institutional settings or child care facilities. Escherichia coli 0157:H7 can also be transmitted by consumption of inadequately chlorinated drinking water or by swirmning in contaminated lakes and pools (24, 25, 41, 95). Once the bacterium has been ingested, the onset of illness averages from 18 to 36 hours (7). Under the right conditions, anyone can become infected with Escherichia coli 0157:H7, but many will be asymptomatic (41). As with many foodbome infections, the susceptibility of those exposed varies greatly with the very young, the elderiy and the immunocompromised being more likely

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8 to develop severe illness and serious complications that may lead to chronic sequelae or death (1, 2, 25, 41). Other factors such as the number of bacteria ingested, the amount of toxin produced in the intestine, the degree to which the toxin is absorbed and the sensitivity of an individual's cells to the toxin also contribute to the severity of the disease (1, 2). An Escherichia coli 0157:H7 infection may involve a variety of symptoms including mild diarrhea, hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS), and thrombotic thrombocytopenic purpura (TTP) (24, 38). Hemorrhagic colitis consists of sudden and severe abdominal cramping followed by watery diarrhea that progresses to grossly bloody diarrhea. Vomiting may also occur but there is little to no fever with the illness and the condition can last an average of four to ten days (38, 41, 98). Approximately 5 to 7 percent of persons infected will suffer from HUS. Hemolytic uremic syndrome generally affects children and has a 3 to 5 percent mortality rate (24). Hemolytic uremic syndrome causes red blood cells within the capillaries of the kidneys and other organs to clot, resulting in the accumulation of waste products in the blood, kidney failure, heart failure, blindness, seizures, strokes, coma and death (24, 38). One third of those with hemolytic uremic syndrome will have abnormal kidney function for many years, some will require long term dialysis, and 8 percent of HUS patients will have lifelong complications such as high blood pressure, seizures, blindness, paralysis or the effects of having part of their bowel removed (24). Rarely, a person will develop thrombotic thrombocytopenic purpura. This largely affects adults and histologically resembles HUS except that the central nervous system is principally involved and there is

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9 a high mortality rate. In such cases, blood clots develop in the brain causing neurological abnormalities and death (38, 41, 98). The infectious dose of the microorganism is unknown (41). It is believed to be very low with the Centers for Disease Control estimating that as little as 10 organisms would be able to cause illness (24). Analysis of frozen ground beef patties involved in an outbreak revealed that as little as 0.3 cells per gram to 15 cells per gram caused illness. Similarly, analysis of salami associated with a foodbome outbreak showed that 0.3 to 0.4 cells of Escherichia coli 0157:H7 per gram of salami were able to cause illness (41). Additional evidence of a low infectious dose is the capability of the disease to be transmitted from person to person as well as in water (38, 41). Prevalence of Escherichia coli 0157:H7 Studies performed by the Centers for Disease Control have established a yearly baseline of Escherichia coli 0157:H7 of an average of 2.4 cases per 100,000 persons (28). Cases in Canada reported over a five year period spanning from 1990 to 1994 ranged from 3.0 to 5.3 per 100,000 (138). In the United Kingdom there were considerable regional variations in the isolation rates within individual countries. In Northern Ireland, there have been fewer than 3 isolates per year since 1989. In contrast, the mean annual isolation rate in England and Wales in 1994 was 0.80 per 100,000 with Scotland experiencing substantially higher incidence. The mean annual isolation rate in Scotland was found to be 2.4 per 100,000 inhabitants. The reason for this variation is unknown (123). Epidemiologic data from Argentina show that this country has the highest rate in the worid. With data regarding only children from 6 to 48 months in age, the incidence of infection annually is approximately 22 per 100,000 in Buenos Aires alone. The incidence of disease related to Escherichia coli 0157:H7 in this country

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10 appears to be 7 to 10 times greater than data reported from "high-risk" areas in the world (80). Data reported do not seem to indicate any pattern. The incidence of infection, even in the United States, is sporadic. In the United States the numbers of incidence recorded annually have roughly remained the same with slight increases or decreases occurring each year. The reported cases were 2.7, 2.3, 2.8, 2.1, 2.9, and 2.1 per 100,000 persons from 1996 to 2001, respectively (28). Selected Outbreaks of Escherichia coli 0157:H7 Escherichia coli 0157:H7 has been isolated from samples of foods linked to human illness (54). While E. coli 0157:H7 is typically associated with foods of bovine origin, it has been found in a variety of food vehicles including ground beef, raw milk, yogurt, roast beef, salad dressing, cantaloupe, coleslaw, cake, mayonnaise, orange juice, apple cider, and water, both in un-chlorinated municipal water and swimming water (28, 41, 137). The first documented outbreak of Escherichia coli 0157:H7 occurred in Oregon in 1982, with 26 cases and 19 persons requiring hospitalization (41, 137). All patients had bloody diarrhea and severe abdominal pain with the age of those infected ranging from 8 to 76 years. This outbreak was associated with eating undercooked hamburgers from fast food restaurants of a particular chain. Only 3 months later, a second outbreak occurred with the same fast food chain being implicated again. This time the incidence occurred in Michigan with 21 cases and 14 persons being hospitalized. The range in age spanned from 4 years to 58 years. Escherichia coli 0157:H7 was isolated from the patients and frozen ground beef patties (109). A large outbreak of E. coli 0157:H7 associated with contaminated municipal drinking water occurred in Missouri between December of 1989 and January of 1990. Of

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11 the 243 persons affected, 86 had bloody diarrhea, 32 were hospitalized and 4 died. The four persons where the infection resulted in death were women 79 years of age or older. The outbreak occurred after two large water mains broke as a result of cold weather and before chlorination of the water supply (128). In the fall of 1991 an outbreak of Escherichia coli 0157:H7 occurred in Massachusetts involving 23 cases of which 6 required hospitalization. The Centers for Disease Control and the State of Massachusetts public health officials found that freshpressed apple cider made at one mill was significantly associated with the illness (16, 137). Escherichia coli 0157:H7 was not isolated from the apple cider made by the implicated processor, but it was revealed in inoculated studies that the pathogen could survive in apple cider for twenty days at 8°C (141). The largest outbreak documented in the United States occurred in 1993 and involved four Western states, Washington, Idaho, California and Nevada. The incident resulted from eating undercooked hamburgers from a single fast-food chain. In all, 731 cases were identified: 629 in Washington, 13 in Idaho, 57 in Nevada, and 34 in California. Of the 629 cases in Washington, 48 were found to be the result of person-toperson transmission. The age of patients ranged from 4 months to 88 years and before the outbreak was over, a total of 178 persons were hospitalized, 56 developed hemolytic uremic syndrome (HUS), and 4 children died (41, 133, 137). In 1994, an unusual outbreak of E. coli 0157:H7 occurred in Washington and California that involved 19 cases. The food associated with the incident was dry fermented salami. Among the 15 confirmed cases in Washington, 3 patients developed HUS and of the 4 cases in California, 2 patients developed HUS. The age of the persons

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12 affected ranged from 6 to 77 years of age (23). Dry cured salami is not cooked but is fermented and dried. Inoculated studies have revealed that Escherichia coli 0157:H7 can survive the fermentation, drying and storage processes involved in the production of fermented sausage (52). More recently, the first reported outbreak of E. coli 0157:H7 in the United States involving the direct transmission of the pathogen from farm animals to humans occurred during the spring and fall of 2000 in Pennsylvania and Washington. The outbreak resulted in 56 cases of illness of which 19 required hospitalization. The persons affected were preschool to school age children. The farm implicated had no separate area designated for the interaction between visitors and farm animals and the wash areas were not supplied with soap (26). One of the largest product recalls in the history of the United States resulted following a multi-state outbreak of Escherichia coli 0157:H7 in 2002. The Colorado Department of Public Health and Environment identified the outbreak among Colorado residents. To date, six other states have also been linked to the outbreak involving 28 cases of which 7 required hospitalization. The outbreak has been linked to the consumption of contaminated ground beef and ground beef products recalled by the Con Agra Beef Company. The recall, which originally involved 354,200 pounds of ground beef, preceded the outbreak. The recall was initiated due to detection oiE. coli 0157:H7 during routine microbiological testing by the United States Department of Agriculture (USDA). Following the detection of this multi-state outbreak and the initiation of an inplant inspection of the Con Agra Beef Company by USDA, the nationwide recall of

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13 354,200 pounds was expanded to a recall of 18.6 million pounds of fresh and frozen ground beef and ground beef trimmings (27). As a direct result of these outbreaks and the many others that have occurred, changes in food processing procedures and regulatory actions have been implemented in an attempt to prevent further outbreaks from occurring. Ultimately, we are still "searching for solutions" (8). Resistance and Adaptation Characteristics of Escherichia coli 0157:H7 Escherichia coli 0157:H7 is an organism capable of adapting to new and hostile environments in order to survive (8). Some have suggested that Escherichia coli 0157:H7 cells can enter into a viable but non-culturable (VBNC) state and survive for long periods of time. Regardless of the existence of a VBNC state, the organism has proven to be a hardy pathogen that has managed to survive and adapt to varied environmental stresses (39). The hurdle approach to food processing may not be sufficient to eliminate or reduce to an acceptable level this pathogenic organism. In fact, such treatments may in fact enhance the survival of the organism (120, 132). Additionally, concern has recently been raised that pathogenic microorganisms like Escherichia coli 0157:H7 can develop resistance to the antimicrobials and sanitizing agents used in food processing and manufacturing (33). Acid Resistance and Acid Tolerance Unlike many foodbome pathogens, E. coli 0157:H7 has proven to be uniquely tolerant to acidic environments (31, 38, 41, 1 17, 132). Although it will not flourish at pH values below 5.5, it is able to survive at pH values as low as 2.0 (36). Escherichia coli 0157:H7 can also survive extended storage at low pH values and at low temperatures. Glass and others (52) showed that E coli 0157:H7 can survive the fermentation, drying

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14 and storage process in fermented sausage with a pH of 4.5 at 4°C for up to two months with only a sUght reduction in cell populations. In inoculated studies, Escherichia coli 0157:H7 survived in ketchup with a pH of 3.6 to 3.9 at 5°C for up to seven weeks (140). Zhao and others (141) reported that E. coli 0157:H7 survived for up to thirty-one days in unpasteurized apple cider with a pH of 3.6 to 4.0 at 8°C. Resistance of Escherichia coli 0157:H7 to acidic conditions may be the result of a genetically induced acid response system (51). Acid resistance, also termed acid tolerance response (ATR), and acid tolerance, also termed acid shock response (ASR), are considered to be important determinants in Escherichia coli 0157:H7's virulence, and contribute to its ability to survive and cause infection (21, 51). As defined by Foster (49), acid resistance (ATR) occurs when a microorganism is exposed for an extended period of time to moderately acidic conditions, for example, a pH of 5.0, triggering a response which enables it to be able to withstand pH values of < 2.5. The ATR requires the induction of protein synthesis to provide protection against acid stress. In ATR, certain outer membrane components of E. coli are genetically modified to protect against internal cell acidification by preventing the passage of hydrogen ions into the cell (112). Conversely, acid tolerance (ASR) occurs when a microorganism exhibits enhanced survival when exposed to pH values between 2.5 to 4.0 after no exposure or only a brief exposure to moderately acidic conditions (49). Both responses appear to result in the production of proteins responsible for the prevention of or the recovery from damage to cells (105, 106). Heyde and Portalier (59) found that a shift from pH of 6.9 to 4.3 induced at least sixteen polypeptides, seven of which were identified as acid shock proteins.

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15 Garren and others (51) investigated the survival of Escherichia coli 0157:H7 and non-0157:H7 isolates due to induced acid tolerance (ATR) or acid shock responses (ASR) when exposed to lactic acid. Both treatment groups were incubated at 25°C and 32°C for twenty one days to determine if E. coli isolates could demonstrate a sustained ATR or ASR. Temperature, pH, strain of E. coli, and phase of growth were all important variables in the survival of both acid tolerance and acid shock treated isolates of Escherichia coli. Highest survival rates for all isolates occurred at a pH of 4.0 at 25°C while no detectable survivors were found at a pH of 3.5 at 32°C. Pathogenic isolates (0157:H7) outperformed non-pathogenic strains. In cases where a difference occurred, acid shocked cells had approximately a twolog higher survival rate than acid tolerance (acid adapted) cells, likely due to the growth phase of the cells. Law (73) stated that stationary-phase bacteria are 1,000 times more resistant to acid than exponentially growing organisms and do not need prior exposure to low pH to exhibit resistance. Benjamin and Datta (14) stated that there is an altered gene expression during stationary-phase. The rpo-regulated proteins which may provide resistance to chemical and physical stresses are associated with the stationary-phase. In addition, many physiological changes that are regulated by the rpoS gene product of this phase have been linked to increased acid resistance in enteric bacteria (29,48, 53, 59, 72, 76). Garren and others (51) reported that acid shocked bacteria in foods could survive over a sustained period of time at lower temperatures provided that the contaminating bacteria are in stationary-phase. These results are important to the meat-processing industry as acidic dips and washes are utilized in an attempt to control microbial growth

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16 on carcasses, and equally important to the general food industry as acidulants are used to extend shelf life and improve flavor (35, 121). Such treatments may induce ASR and cause the proteins needed for protection against this stress to be produced (51). Berry and Cutter (15) found that acid adapted Escherichia coli 0157:H7 negatively influenced the effectiveness of acetic acid washes in reducing the numbers of this organism on carcasses. Additionally, foods which rely on acidic pH to inactivate pathogens, such as fermented sausage, mayonnaise, yogurt, and apple juice, are known vehicles of infection with Escherichia coli 0157:H7 (85). Buchanan and Edelson (21) studied the effect of acidulant identity on the pHdependent stationary-phase acid resistance response of enterohemorrhagic Escherichia coli cells grown in BHI (Brain Heart Infusion) broth at 37°C. The study utilized four acids, 0.5% citric, malic, lactic, and acetic, which were adjusted to a pH of 3.0 using hydrochloric acid. The results were compared to data utilizing only hydrochloric acid (HCl). Hydrochloric acid was found to be the least damaging to cells while lactic acid was the most detrimental. In general, acetic acid had a greater effect than citric or malic acid. Buchanan and Edelson (21) found, as did Garren and others (51), that exponentially growing cells were more sensitive than those in stationary-phase. The exponential-phase cells were sensitive to all four acids with a four-log reduction in two hours whereas the stationary-phase cells decreased by less then one-log with all acids except for lactic acid. Additionally, the acid-adapted stationary-phase showed even further increased survival with only a two-log reduction when exposed to lactic acid. It again appears that maximum survival of Escherichia coli in strongly acidic environments is associated with

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17 the stationary-phase acid habituation. Researchers (13, 20, 29, 76) suggested that both the constitutive rpoS gene-regulated and the inducible pH-dependent acid resistance systems are active in this acid-adapted stationary-phase. The mechanism of acid tolerance has been speculated but not yet fully elucidated. Doyle and others (41) suggested that it is associated with protein(s) that may appear as a result of pre-exposure to acid conditions. Stress proteins have been found to enhance the ability of an organism to withstand a number of challenges such as hydrogen peroxide, acid and alkaline pH, heat and osmolarity (79). Lin and others (78) proposed that there are several acid resistance systems that are involved in the acid tolerance of pathogenic Escherichia coli and each system is needed to survive the different acid stress environments of the stomach (pH 1 to 3) and the intestine (pH 4.5 to 7 with high concentrations of volatile fatty acids). Lin and others (77) identified three distinct low-pH induced acid survival systems for Escherichia coli. They are an acid-induced oxidative system that requires rpoS, an acid-induced argininedependent system and a glutamate-dependent system (77). RpoS, an alternative sigma factor (ct^) that is involved in regulating the expression of a variety of stress response genes, is only partially involved in the later two systems (58, 72, 78, 96). The acid induced system is expressed in oxidatively metabolizing bacteria grown in complex media but will protect cells in minimal medium to pH 2.5. This system is not apparent in fermentatively metabolizing cells (77, 78). The arginine-dependent system involves an arginine decarboxylase system that utilizes adi, an arginine decarboxylase gene, and its regulators, cysB and adiY to provide acid resistance. This system will only function if

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18 arginine is present (78). Similarly, the glutamate-dependent system will only function in the presence of glutamate (78). When testing the three acid resistance systems against extreme acid exposure at a pH < 2.0, mild acid exposure at a pH of 4.0 utilizing benzoic acid, and mild acid exposure at a pH of 4.4 utilizing a volatile fatty acid (VPA) cocktail comprised of acetic, propionic and butyric acid at levels approximate to those in the intestine, Lin and others (78) found that the involvement and effectiveness of each system varied. At extreme acid shock the oxidative system was ineffective with < 1 percent survival of enterohemorrhagic Escherichia coli strains. The arginine-dependent system had 10 to 50 percent survival at pH 2.5 and limited survival at pH 2.0. The glutamate-dependent system was effective at both a pH of 2.0 and 2.5 with 80 to 100 percent survival of EHEC isolates. In the weakly acidic benzoic acid solution, the glutamate system again was most effective with the oxidative system being modestly effective and the arginine-dependent system being ineffective. When exposed to the VFA cocktail, both the arginine and glutamate-dependent systems were very effective for at least 7 hours; however, the oxidative system was only mildly effective for 3 hours and ineffective at 7 hours. Lin and others (78) suggested that the arginine and glutamate-dependent systems functioned to maintain a less acidic intracellular pH in extremely acidic environments, while the oxidative system minimizes the actual damage to macromolecules. Generally, it has been considered that all stages and components involved in stress tolerance induction are intracellular (94, 126). Rowbury and Goodson (113) proposed that stress responses may in fact be associated with the appearance in medium of extracellular agents that are essential for habituation. They termed such agents

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19 "extracellular induction components" (ETC). Rowbury and Goodson (113) suggested that a heat-stable protein (EIC) is present in the media and is converted by mildly acidic pH (4.5 to 6.0) to an EIC, also a protein, that induces acid tolerance in Escherichia coli. While there is little information of the conversion of EIC to EIC, Rowbury and Goodson (113) found that it doesn't involve proteolytic removal of the fragment because a mixture of protease inhibitors did not stop the conversion. EIC is not significantly effected by chloramphenicol, not destroyed at 75°C or by exposure to pH 2.0 or pH 11.5. It is however reversibly activated to EIC at mildly acidic pH values (4.5 to 6.0). The benefit of an extracellular induction component would be that it might provide earlier warning of impending lethal stress. Antibiotic Resistance Escherichia coli 0157:H7 has further shown its ability to adapt to environmental conditions and stress with a new trend towards antibiotic resistance (41). Early research by Ratnam and others (107) revealed that E. coli 0157:H7 isolates were sensitive to most antibiotics. Ratnam and others (107) found that only 2.9 percent of 174 Escherichia coli 0157:H7 isolates were resistant to an antibiotic. Similarly, Kim and others (67) found that in 1987 all of the isolates that they tested were susceptible to the antibiotics tested. However, when they performed the study again in 1991, it was discovered that several of the strains had adapted and developed a resistance to streptomycin, sulfisoxazole, and tetracycline (67). Meng and others (86) found antibiotic resistance in E. coli 0157:H7 and E. coli 0157:NM strains isolated from animals, humans, and food. They found twenty-four percent of the isolates tested to be resistant to at least one antibiotic, nineteen percent of the isolated strains to be resistant to three or more antibiotics and two of the

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20 isolated strains were resistant to six antibiotics including ampicillin, kanamycin, sulfisoxazole, streptomycin, tetracycline, and ticamillan. Researchers today feel they have a relatively good insight into the mechanism(s) by which bacteria, such as Escherichia coli 0157:H7, have become resistant to antibiotics. As bacteria have evolved, they have developed diverse mechanisms to transmit resistance traits not only to members within their species but also to other species (66). The genetic traits that code for antibiotic resistance are located either in the chromosomes of the bacteria, in plasmids or transposons (86). Plasmids and transposons are extrachromosomal elements that a bacterium may possess and if a bacterium possesses either of these they may code for antibiotic resistance or may serve other functions (66, 86). Both plasmids and transposons consist of tiny circular DNA that is about 1 percent the size of a chromosome (66, 74). There are three primary mechanisms of antibiotic resistance. First, resistance can be a natural attribute of an organism, such as an impermeable outer membrane that resists penetration (18, 97, 136). Gram-negative bacteria have a thick lipopolysaccharide layer that can act as a barrier to limit the diffusion of antibiotic molecules into the cell (18). Gram-negative bacteria are more resistant to lipophilic and amphiphilic inhibitors such as dyes, detergents, and antibiotics due to their outer membrane (97, 136). Additionally, gram-positive bacteria have lipophilic substances in their cell walls that retard penetration of hydrophilic, cationic and antimicrobial compounds (18). Second, resistance can be spontaneously acquired (18). Single point mutations to drugs can occur. In the lab, resistance to Naladixic Acid and Rifampin occurred spontaneously (66). Finally, resistance can be acquired through genetic exchange (4, 5, 66, 89).

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21 Although all three modes of resistance are important, resistance through genetic exchange is the clinically relevant form of antibiotic resistance (114). As antibiotics utilized for therapeutic purposes generally have specific target sites in microbial cells, they have greater potential to result in mutations and in the development of acquired resistance (18, 1 16). Some of the methods of resistance that occur genetically include the inactivation of the antibiotic, an alteration of the target, synthesis of an alternate pathway, and efflux of the antibiotic (75). Genetic exchange of drug resistance has been documented through both epidemiological observations (11, 130) and experimental models (37). Additionally, Mizan and others (89) found that the transfer of genetic elements such as plasmids between microflora and enterohemorrhagic Escherichia coli appeared to occur readily in rumen fluid. Genetic exchange has been found to occur in three ways, transformation, transduction, and conjugation (4, 5, 18, 135). Transformation is used to move DNA between bacteria, plants, and animals. In transformation, DNA is removed from donor cells and added to recipient cells that are in a competent state (one that is capable of binding the DNA). The recipient cells either take the donor DNA into their cytoplasm where it may exchange into the recipient DNA or if it is a plasmid, it will replicate (135). Transformation involves many techniques such as the use of calcium chloride solutions, salt solutions, coated beads, and electricity depending upon if the organism is bacteria, a prokaryotic cell or a eukaryotic cell (4, 5) Transduction involves the mediation of viruses called bacteriophages. The bacteriophages actually transport the DNA. Scientists who were studying conjugation discovered transduction accidentally in 1952. Gene transfer occurred even when the two

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22 bacterial membranes were separated and DNase did not inhibit the transfer. This process came to be known as transduction (5). Transfer involving bacteriophages can be advantageous and efficient. In contrast to other methods, intimate contact between bacteria is not required. Additionally, bacteriophages can carry large blocks of deoxyribonucleic acid (DNA) and can survive harsh conditions that eliminate bacterial populations. Thus, DNA important to a population can be preserved until a host is re-introduced into an environmental niche (87). Conjugation is basically the ability of the bacterial cells to transfer DNA between cells that are in physical contact (135). It occurs between members of the same species, members of closely related species, and even from bacteria to prokaryotes and from bacteria to some eukaryotic cells (4, 5). In order for conjugation to occur, the donor cells must carry a unique plasmid that contains a set of genes that makes the transfer possible (4, 5, 89). A novel system, believed to play a role in the acquisition and dissemination of antibiotic resistant genes in bacteria that exhibit multiple resistances, has also been identified (56). The system is referred to as bacterial integrons (139). Hall and Stokes (57) defined integrons as mobile DNA elements with a specific structure consisting of two conserved segments flanking a central region containing gene cassettes that usually code for resistance to specific antimicrobials. Several classes of integrons have been identified to date with the majority of those identified belonging to class 1 type (63). Hall and Stokes (57) found that class 1 type integrons consist of a 5' conserved region that encodes a site-specific recombinase (integrase) and strong promoter(s) that ensures

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23 the expression of the integrated cassettes. Additionally, the 3' conserved region in class type 1 integrons carries the genes ^acAE and sul1 , and an open reading frame of unknown function. Hall and Stokes (57) determined that qacAE specifies resistance to antiseptics and disinfectants and that sul-l confers sulfonamide resistance. Zhao and others (139) conducted a study to characterize antimicrobial susceptibility patterns among Shiga toxin-producing Escherichia coli (STEC), including Escherichia coli 0157:H7, isolated from cattle, ground beef, and humans and to determine if resistant phenotypes observed could be attributed to integron-mediated resistant gene cassettes. They found that of the 50 isolates tested, seventy-eight percent exhibited resistance to two or more antimicrobials. Multiple resistances to streptomycin, sulfamethoxazole, and tetracycline were observed most often. Integrons were found in STEC isolates, including Escherichia coli 0157:H7 and were demonstrated to be transferable via conjugation to other strains. However, Zhao and others (139) also identified isolates that displayed multiple antibiotic resistances that did not contain any gene cassettes. Therefore, while integrons may play an important and active role in multiple resistances, other mechanisms also contribute to the antibiotic resistance phenotypes. Thermal Tolerance and Adaptation Characteristically, the optimum growth temperature for Escherichia coli 0157:H7 is 37°C. Although the organism has been associated with milk and cooked ground beef patties, it does not appear to possess any unusual resistance to heat (41, 98). Mongold and others (91) found that thermotolerant mutants were more likely to occur in isolates that had previously been adapted to 41-42C but that such mutants could also result out of isolates previously adapted to only 32°C. Additionally, it was found that such mutants afforded little advantage over other strains at lethal temperatures. Research into the

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24 upper thermal limits of E. coli 0157:H7 suggested that some strains can survive at temperatures between 49-52°C (99, 120). Another study found that certain strains could survive at temperatures as high as 55°C relevant to prior storage and holding conditions (62). Corry (32) suggested that the composition of the food may in fact provide protection for bacteria at elevated temperatures. A study by Splittstoesser and others (124) found that high concentrations of solutes in apple juice did afford protection to Escherichia coli 0157:H7 in regard to thermotolerance. The organism was still found to be relatively heat sensitive with D-values at 52, 55, and 58°C of 12, 5.0 and 1.0 minutes respectively. Arsene and others (9) investigated the heat shock response of Escherichia coli that allows cells to adapt to environmental and metabolic changes and to survive the stress conditions. It was found that an upshift from 30 to 42°C resulted in the rapid induction of synthesis of more than 20 heat shock proteins (HSPs). Major heat shock proteins are molecular chaperones and proteases. The two major chaperone systems of E. coli, determined by their abundance (15-20 percent of total protein at 46°C), are the DnaK and GroE systems. These systems play a vital role in preventing aggregation and refolding proteins. Studies investigating the minimal temperature for E. coli 0157:H7 have shown that the organism is capable of growth at temperatures as low as 8°C and can produce verotoxins at 10°C (99, 100). Additionally, Jackson and others (62) demonstrated the organism's ability to remain viable when stored at -18°C for up to fifteen days and Semanchek and Golden (120) found viable cultures of E. coli 0157:H7 after seven months of storage at -20°C. Knudsen and others (68) found that Escherichia coli

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25 0157:H7 survived without significant decline on cut strawberries held at 5°C and that frozen inoculated strawberries evaluated 19 months after the initial sampling date still had populations of Escherichia coli 0157:H7 present. Populations were highest (>log 4.6 cfu/sample) in strawberries with added sucrose. Populations without added sucrose could only be detected upon enrichment of the sample. Barkocy-Gallagher and others (12) evaluated isolates of Escherichia coli 0157:H7 at -20, 1, 4, and 7°C in ground beef samples to see if genomic differences could account for differing abilities to survive at the various temperatures. They found that no one strain or genomic cluster was more successful at survival in persisting low temperatures. All strains evaluated showed limited growth at temperatures of 4°C and 7°C and while small losses in cell numbers occurred at both TC and -20°C, it is evident from this study that freezing cannot be expected to eliminate Escherichia coli 0157:H7 at least in ground beef. When assessing the ability of Escherichia coli 0157:H7 strains to resist heating or freezing, care must be taken by the food industry as variations in the temperature ranges capable of supporting or sustaining the bacteria may occur. These variations may be due to storage conditions, the growth phase of the organism, product formulations, sampling techniques, and the particular serotype of the strain (62, 120). Adaptation and Tolerance of Sanitizing Agents As stated previously, chemical sanitizers are considered to be effective on food contact surfaces if they demonstrate a five-log reduction in planktonic bacteria (43). In order to obtain maximum benefits sanitizing should be performed after cleaning as the use of the sanitizer(s) leads to the inactivation of cells which may result in biofilm formation (61, 82). The continued adherence of the inactivated cells and cell fragments

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26 may potentially create a suitable environment for future bacteria. The creation of a biofilm can lead to the enhancement and survival of future cells (22, 43, 61, 82). It has been documented that microorganisms of a biofilm matrix on food contact surfaces display more resistance to toxic compounds than their single-celled counterparts in suspension (64, 71). Unlike antibiotics, biocides do not have a specific target site. Biocides usually exert their cytotoxic effects through multiple non-specific targets (18). Therefore, while a single mutation or chemical transformation of a cellular target can provide antibiotic resistances in bacteria, biocidal action is rarely affected by such an event. Resistance to such compounds can occur when a cell develops reduced permeability, like a mucopolysaccharide outer layer within a biofilm (18). Additionally, there is some evidence that a plasmid could be responsible for changes in the cell envelope which increases the resistance to biocides (1 14, 1 15). Attachment to surfaces can also have an impact on bacterial resistance to disinfection as a planktonic organism is susceptible to a disinfectant from all sides and angles but an attached organism is only susceptible from one side (18). Research conducted by Farrell and others (43) illustrated that enumeration by plate count techniques alone is insufficient for indicators of sanitizer efficacy. They found that low numbers of typical and/or injured E. coli 0157:H7 cells could remain on contact surfaces after sanitation with chlorine and peroxyacetic acid. Ronner and Wong (110) found similar results when studying the effects of hypochlorite sanitizers on Listeria monocytogenes and Salmonella typhimurium. Additionally, Mosteller and Bishop (93) found that various sanitizers failed to provide an adequate reduction (three-log) in

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27 attached bacteria where they had clearly demonstrated the ability to be effective against the planktonic bacteria. Krysinski and others (71) found similar results. Chemical Sanitizers in the Food Industry Sanitizing agents have been used to reduce microorganisms on processing equipment for nearly 100 years. The selection of a sanitizer is usually based on economics, equipment type and its ability to be cleaned, and clean-up scheduling (33). Chemical sanitizers available for use in the food industry vary in chemical composition and activity. In general, the more concentrated a sanitizer, the more rapid and effective its action (7). The individual characteristics of a chemical sanitizer must be known and fully understood so that the most appropriate sanitizer can be selected. For a sanitizer to be effective when combined with cleaning compounds the temperature of the cleaning solution should be < 55°C and the soil should be light. The efficacy of chemical sanitizers is affected by the following physical-chemical factors: exposure time, temperature, concentration, pH, equipment cleanliness, water hardness, and bacterial attachment (82). As defined by the Environmental Protection Agency, sanitizers are "pesticide products that are intended to disinfect or sanitize, reducing or mitigating growth or development of microbiological organisms including bacteria, fungi or viruses on inanimate surfaces in the household, institutional, and/or commercial environment" (40 Code of Federal Regulations [CFR] 455.10) (30). Sanitizers utilized by the food industry include chlorine and chlorine derivatives, iodine derivatives, quaternary ammonium compounds, acid-anionic sanitizers, hydrogen peroxide, peroxyacetic acid, and acidified sodium chlorite (21 CFR 178.1010) (30). Generally, sanitizers are utilized to inactivate target microorganisms on the food contact surfaces of cleaned food processing and food

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28 service equipment. More recently, these compounds have been used for the inactivation of bacteria on raw, unprocessed food products such as meat and poultry carcasses as well as fruits and vegetables (33). The usage level of each sanitizer on food products, food contact surfaces and plant equipment is regulated by the United States Department of Agriculture (USD A). As per the Food and Drug Administration (FDA), (21 CFR §178.1010) (30), the maximum permitted use levels on food-contact surfaces without rinsing for chlorine is 200 parts per million (ppm), 25 ppm for iodophors and 200 ppm for quaternary ammonium compounds. The allowable level for peroxyacetic acid was not found. Chlorine Sanitizers Chlorine sanitizers are commonly used in the food industry. They are likely the most commonly utilized surface sanitizer as they have proven to be effective on a broad spectrum of microorganisms and are relatively inexpensive (82). As little as 0.6 to 13 parts per million (ppm) of free available chlorine is able to inactivate 90 percent of most planktonic bacteria within 10 seconds (82). Further illustration of chlorines ability to act rapidly was demonstrated by Kotula and others (70) when they showed that cultures of Clostridium perfringens, Escherichia coli, Proteus vulgaris, Kocuria varians, Salmonella spp., and Pseudomonas spp. were destroyed by 3 ppm of free available chlorine in 15 seconds. When added to water, chlorine immediately oxidizes all inorganic and organic compounds in the solution. Chlorine sanitizers damage proteins essential for enzymatic activity and diminish bacterial cell function until the bacterial cells ultimately die. Although these compounds are generally said to be lethal to the widest variety of microorganisms, their ability to destroy microorganisms, required concentration, and residual properties vary with the specific sanitizer used. Additionally, the effectiveness

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29 is affected to varying degrees by temperature, pH, organic residues, and water hardness. Chlorine sanitizers include calcium and sodium hypochlorite (household bleach), chloramines, and chlorine dioxides. These sanitizers are most effective at a slightly acidic pH and at temperatures below 120 degrees Fahrenheit. They have limited residual activity and can be very corrosive to metals. Also, they deteriorate when stored at temperatures above 60°C or in the presence of light. While they are relatively inexpensive, they dissipate rapidly during storage and can produce a poisonous gas if mixed with a more acidic compound. Organic residues neutralize the hypochlorites whereas the chloramines are less sensitive to the residues, are more shelf stable and are active over a broader pH range (6.0 9.5). Finally, the chlorine dioxides, unlike the two previously mentioned, are not corrosive, not effected by organic residues, and can be used at very low concentrations (1-5 ppm). The chlorine dioxides however, are fairly expensive in comparison (7, 61, 82). Quatemarv Ammonium Sanitizers Quaternary Ammonium sanitizers are often referred to as "quats". Quats are ammonium compounds in which four organic groups are linked to a nitrogen atom that produces a positively charged ion (cation) (82). They form a bacteriostatic film that inhibits bacterial growth. They also are very effective on porous surfaces because of their penetration ability. For these reasons, they are widely used on floors, walls, equipment and furnishings of meat and poultry plants (82). Additionally, quaternary ammonium sanitizers are non-corrosive, have definite residual activity, and are more resistant to inactivation by organic material (7, 42, 82). In general, quats are coloriess, odorless, stable against temperature fluctuation, non-toxic and non-irritating to skin (82). Quaternary ammonium compounds also work well against gram-positive bacteria.

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30 especially Listeria monocytogenes, but are less effective against gram-negative bacteria (7, 82). The mechanism of germicidal activity of quaternary ammonium compounds is not fully understood. It is believed that the surface-active nature of the compounds surrounds and covers the cells outer membrane, causing a failure of the wall, resulting in the leakage of internal organs and enzyme inhibition (82). McDonnell and Russell (83) suggest that after the quaternary ammonium compound compromises the bacterial cell walls it reacts with the cytoplasmic membrane to produce membrane disorganization, leakage of the intracellular material, and degradation of proteins and nucleic acids. Some disadvantages to using quaternaries are that they must be used in higher concentrations (generally 200 400 ppm), are slower acting and require a contact time of several minutes for total effectiveness. Also, anionic surfactants like soaps and synthetic detergents neutralize the sanitizer. Calcium, iron, and aluminum ions also react with quaternaries to lower their efficacy. Finally, hard water may reduce the activity of the quaternary ammonium compounds but this can be overcome with use of higher concentrations (7, 42, 50, 61, 82). Peroxvacetic Acid Sanitizers Peroxyacetic acid sanitizers are antimicrobial compounds that act as both an oxidizer and an acid. They consist of hydrogen peroxide and peroxyacetic acid and are relatively new to the food industry (81). Peroxyacetic acids are considered to be effective against bacteria, yeasts, molds, and fungal and bacterial spores (10). They are fast acting and very effective against biofilm formation (81,111). It is believed that peroxyacetic acids denature proteins and enzymes and increase cell wall permeability by disrupting sulfhydryl (-SH) and disulfide (S-S) bonds. Additionally, they are less affected by organic matter than other sanitizers (83). They are environmentally safe, as the

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31 compounds break down into oxygen and water. Peroxyacetic acids do not react with proteins to produce toxic or carcinogenic compounds (7). Peroxyacetic acid compounds are effective over a broad pH range as well as a broad temperature range, as low as 40 degrees Fahrenheit. Additionally, they are non-foaming and lethal to a broad spectrum of microorganisms (6, 7, 81). Some disadvantages to using peroxyacetic acids are that they are corrosive to non-stainless steel metals and copper alloys and lose their effectiveness in the presence of some metals and organic materials (81). Additionally, the odor released by such compounds can be irritating to the nose and throat (7). Iodine Sanitizers lodophors are antimicrobial compounds containing iodine and a surfactant (92). They are deemed to be effective at killing 99.999% of planktonic bacterial cells at a concentration as low as 6.25 mg/L in only 30 seconds (82). The mode of antibacterial action of iodine has not been studied in detail. It is believed that diatomic iodine is the major antimicrobial agent, which disrupts bonds that hold cell protein together and inhibits protein synthesis (82). They are generally inexpensive although they can be more expensive than the chlorine compounds. They are very rapid acting; however, they are ineffective at alkaline pH values. Iodine compounds are most effective at a pH of 2.5 to 3.5. They are also rapidly inactivated by contact with organic matter (6, 7, 82). Additionally, iodine loss may occur during the storage of the iodophor compounds (42). Iodine sanitizers maintain more residual activity than the chlorine compounds and are not as corrosive. Unlike chlorine compounds, iodophors are usually not used as surface sanitizers and instead are primarily used as employee skin sanitizers in food-processing plants. lodophors, but not iodine itself, are generally mild and non-irritating to the skin. They have residual bactericidal activity on the skin that can last for up to an hour, making

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32 them ideal for sanitizing workers' hands (7, 42, 82). A potential disadvantage is that they do tend to cause discoloration of some materials (7). Iodine is vaporized at temperatures of > 50°C and is inefficient at low temperatures. Iodine sanitizers may also cause off flavors in foods. Consequently, the use of iodophors on food contact surfaces is limited (42). Additionally, iodine sanitizers are incompatible with wastewater treatment systems (7). The Ideal Sanitizer According to Marriott (82), for a sanitizer to be desirable and useful it should possess several properties. First, an ideal sanitizer should have uniform microbial destruction properties and broad spectrum activity against vegetative bacteria, yeasts and molds in order to kill rapidly. Water hardness and pH values should not alter its effectiveness. Additionally, it should maintain its effectiveness in the presence of organic matter, detergents, and soap residues. It should offer good cleaning properties, be nontoxic and non-irritating, and have an acceptable odor or no odor. The ideal sanitizer should be easy to use, for example it should be water soluble in all proportions and be easy to measure in use solution. It should also be stable in both concentrated and use dilution forms, inexpensive and readily available. Obviously, no one sanitizer can possess all of the ideal properties. The chemical selected as a sanitizer depends on the type of processing plant and the product being produced (42). Sanitizers selected should produce a 99.999% kill of 75 to 125 million Escherichia coli and Staphylococcus aureus within 30 seconds after application at 20°C to be deemed effective (82).

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33 Surfaces and Attachment It has been shown that even when cleaning and sanitation procedures are followed and are consistent with good manufacturing practices that microorganisms can still remain on all food processing surfaces (34, 1 1 1). Even with the inactivation of bacterial cells, a desired result of sanitation, the cells or fragments of the cells may remain attached. This would create a conditioning layer that could enhance future attachment of other bacteria (22). It is therefore important to examine the surfaces associated with bacterial attachment in the food processing industry as bacterial attachment is the first step in biofilm formation (34). The ability for microorganisms to attach seems to be affected by nutrient conditions and environmental factors such as temperature and atmosphere (34). Additionally, it has been suggested that low-nutrient systems may enhance that ability for an organism to attach (19). Sasahara and Zottola (119) also found that pure cultures may show reduced adherence capabilities in comparison to mixed populations. Researchers have also shown that bacterial cells in early or late log phase attached at twice the rate of those in stationary phase. Similarly, bacterial cells in the log phase were found to have greater adherence when compared to the stationary or death phase (45, 125). Stainless steel is one of the most common surfaces found in food processing facilities (60). To the unaided eye the surface appears to be smooth, but when viewed under a microscope it is in fact found to be very rough. The flaws could potentially harbor spoilage or pathogenic organisms as bacteria tend to accumulate in scratches and other irregularities on metal surfaces (127). Buna-N rubber and Teflon® are other materials that may be found in a food processing plant, especially in a dairy processing facility. Such materials are commonly

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34 used for gaskets (93, 110). These surfaces represent those that are most difficult to sanitize. As with stainless steel, these gasket materials appear to be smooth. They are in fact covered with minute holes and cracks which may provide adherent cells protection from antimicrobial agents and surfactants (93). Other surfaces that have been investigated as potential food contact surfaces include a high density polyethylene plastic, polyvinyl chloride plastic and cement (64, 131). Joseph and others (64) found that organisms had a greater propensity to attach to plastic (107) followed by cement (106) and steel (105). Such cells in a food processing facility may not be removed by routine cleaning and sanitation procedures and therefore could be a source of contamination of foods coming into contact with such surfaces (34, 64, ill). Recoverv of Stressed Microorganisms Both viable and injured microorganisms that have been exposed to stress such as chemical shock may still remain on the surface or food that has been treated (34, 111). Therefore, it is essential for recovery methodology to be accurate. The use of a selective media may prohibit the recovery of sub-lethally injured bacteria (122). Silk and Donnelly (122) found that an incubation step prior to the plating of Escherichia coli 0157:H7 with a selective media increased the level of recovery. However, these levels were still below recovery levels obtained when using a non-selective media. Additionally, researchers have suggested that plate count methodology does not accurately represent the bacterial population present of a surface (3, 93). Andrade and others (3) stated that plate count methodology underestimates the number of cells on a surface since some cells may not be removed. They found the impedance method to recover 25 times more cells than the plate count method. Similarly, Mosteller and Bishop

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35 (93) found recovery of bacteria with the plate count method to be less representative of the actual numbers present than the impedance method. The impedance method, which measures metabolic growth of individual cells, detects both reversibly and irreversibly attached cells (3, 93). Mosteller and Bishop (93) found direct epifluorescent filter technology (DEFT) to be superior in recovery of bacteria to both the impedance and plate count methodology. The DEFT method enumerates both reversibly and irreversibly attached bacteria as well as single cells and clumps of bacteria (93). Rossoni and Gaylarde (111) have suggested that one way to resolve the issue of recovery counts is to utilize non-parametric statistics.

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CHAPTER 3 MATERIALS AND METHODS This study was conducted in two phases. Phase one of the study involved the use of four commonly used food-processing sanitizers, a chlorine compound, a quaternary ammonium compound, an iodine compound and a peroxyacetic acid compound in conjunction with planktonic cells from two distinct Escherichia coli 0157:H7 strains, one unadapted and one known to be acid tolerant, in three trials, each of which lasted for three days. Phase two consisted of the use of the same four sanitizers in conjunction with adherent bacterial cells from the two Escherichia coli 0157:H7 strains utilized in phase one. Phase two was also performed in three trials, with each trial lasting one day. All trials were conducted at Deibel Laboratory in Gainesville, Florida. The protocols for the three trials for phase one and the protocol for the three trials of phase two were identical. Bacterial Cultures One of the Escherichia coli 0157:H7 strains utilized in this study was obtained from the American Type Culture Collection (ATCC). The strain is designated as ATCC 700599. It was isolated from a salami product in 1994 and is known to be acid tolerant. This strain is identified as a biohazard type 1 as it is not known to have caused human illness. Upon receiving the freeze-dried isolate, the bacterium was resuscitated in Nutrient Broth (Difco Laboratories, Detroit, Michigan) at 37°C under aerobic conditions. The stock culture was then maintained through monthly transfers on slants of Trypticase soy agar (TSA) (Difco) supplemented with 0.5 percent yeast extract (TSAYE) (Difco) 36

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37 and stored at 2.5°C. The other Escherichia coli 0157:H7 strain used throughout this study was obtained from Dr. Robert Deibel of Deibel Laboratories, Incorporated. The strain is designated as FSIS 063-93. It was isolated from a meat product in 1993. This culture was also maintained through monthly transfers on slants of Trypticase soy agar supplemented with 0.5 percent yeast extract (TSAYE) (Difco). Before use, the cultures were grown in Trypticase soy broth (TSB) (Difco) supplemented with 0.5 percent yeast extract (TSBYE) (Difco) overnight (at least 12 hours) at 37°C. Ten-fold dilutions were performed using 0.1 percent peptone solution from Bacto Peptone (Difco). Growth characteristics were established for both strains of Escherichia coli 0157:H7 by performing 24 hour growth curves in Lauryl Sulfate broth at 37°C. Aliquots of the incubating cultures were removed after 1, 3, 5, 8, 12, 15, 18, 21, and 24 hours respectively. The Optical Density (OD) of each aliquot was determined on a Spectrophotometer 20 (Milton Roy Company, Item 333172, USA) and portions of each aliquot were plated in triplicate on Violet Red Bile Agar (VRBA) (Difco). Both the absorbance curve and the growth curve for E. coli 0157:H7, strain ATCC 700599, showed a rapid increase during the first five hours and then tend to plateau (Figure 1 and Figure 2). The absorbance curve and growth curve for E. coli 0157:H7, strain FSIS 06393, also exhibited a rapid increase during the first five hours but fluctuated more than the other strain over the next nineteen hours (Figure 3 and Figure 4).

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38 0.8 0.7 0.6 E 0.5 c o o S 0.3 < 0.2 0.1 0 0 5 10 15 20 25 30 Time (hours) Figure 1. Absorbance of Escherichia coli 0157:H7, strain ATCC 700599, in Lauryl Sulfate broth over 24 hours 9 5.5 6 -I -r— , , , 0 5 10 15 20 25 30 Time (hours) Figure 2. Growth of Escherichia coli 0157:H7, strain ATCC 700599, on Violet Red Bile Agar over 24 hours

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39 0.6 0 5 10 15 20 25 30 Time (hours) Figure 3. Absorbance of Escherichia coli 0157:H7, strain FSIS 063-93, in Lauryl Sulfate broth over 24 hours 9 8.5 8 7.5 1 t5 7 65 6 5.5 5 15 20 25 Time (flours) Figure 4. Growth of Escherichia coli 0157:H7, strain FSIS 063-93, on Violet Red Bile Agar over 24 hours

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40 Sanitizer Solutions Escherichia coli 0157:H7 cells were exposed to four commonly utilized industrial plant sanitizers which included a Chlorine compound (Clorox® regular bleach), a Peroxyacetic Acid compound (Zep Perosan^'^), a Quaternary Anmionium compound (Zepamine-A™), and an Iodine compound (Zep-I-Dine™). The active ingredient for Clorox is 6.0% Sodium Hypochlorite. The active ingredients of Zep Perosan are 5.1% Peroxyacetic Acid and 21.7% Hydrogen Peroxide. The active ingredients of Zepamine A are 5.0% n-Alkyl Dimethyl benzyl ammonium chloride and 5.0% n-Alkyl Dimethyl ethylbenzyl ammonium chloride. The active ingredient for Zep-I-Dine is 1.75% iodinej from the alpha (p-nonylphenyl)-omega-hydroxypoly (oxythylene)-iodine complex. All dilutions of the sanitizing agents were prepared in 99 ml quantities in 250 ml sterile dilution bottles on each day of testing. The sanitizers were diluted with autoclaved distilled water to obtain the necessary concentration. Determination of Minimum Inhibitory Concentration (MIC) The minimum inhibitory concentration for each sanitizer was determined by a method similar to that utilized by Pickett and Murano (102). The MIC (defined as the lowest concentration of a sanitizer that will prohibit growth of Escherichia coli 0157:H7 cultures) was determined by inoculating lO'^ E. coli 0157:H7 cells per ml into sterile test tubes containing serial dilutions of the sanitizing solutions and then incubating at 37° C for 48 hours. Once an initial MIC was determined, further dilutions were made between that dilution and the next lower dilution to more precisely approach the actual MIC. Growth was measured at 580 nm on a Spectrophotometer 20 after 24 and 48 hours. All tests were performed in triplicate. The average of the lowest concentration of each sanitizer showing a bacterial population with an OD value of ^0.01 was designated as

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41 the minimum inhibitory concentration. A standard curve relating bacterial counts obtained by standard plate count on Violet Red Bile agar with the optical densities was determined and utilized throughout the study to provide an estimate of the bacterial population present in suspension (Figure 5 and Figure 6). The actual number of bacteria present in the cell suspension was determined more precisely by plate count values. Once the minimum inhibitory concentration was determined, the sub-lethal and lethal concentrations were determined. The sub-lethal level is the concentration of sanitizer corresponding to one dilution lower than the minimum inhibitory concentration. The lethal level is the concentration of sanitizer corresponding to one dilution greater than the minimum inhibitory concentration. 0.6 0.5 g 0.4 C o 00 in O 0.3 c n .o k_ o (0 ja < 0.2 >^ y = 0.1641x0.963 >^ = 0.8033 • ,_* -y'^^ 5 5.5 S 6.5 7 75 8 8.5 9 0.1 Log Count (CFU/ml) Figure 5. Escherichia coli 0157:H7 strain ATCC 700599 Standard Curve

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42 E * * / — y = 0.1585x0.9492 = 0.9422 ^ 5.5 6 6.5 7 75 8 8.5 9 Log Count (CFU/ml) Figure 6. Escherichia coli 0157:H7 strain FSIS 063-93 Standard Curve Zone Inhibition Zone inhibition tests were performed to determine the bacteriostatic activity of the sanitizer solutions. Tests were performed utilizing the Kirby-Bauer technique (88). In this method, seeded agar plates are utilized. The seeded agar plates were created by dipping sterile swabs into each of the Escherichia coli 0157:H7 cultures, which were grown overnight at 37°C to a level of 10^ cfu/ml, and streaking the bacteria onto MeullerHinton (Difco) agar plates. Plates were cross-streaked in at least three different directions to cover the agar surface. Using aseptic technique, blank test discs (Difco, Detroit, Michigan, B31039) were dipped for 30 seconds into various concentrations of the sanitizing agents. The impregnated filter discs were then applied immediately to the seeded agar surface of the Meuller-Hinton agar plates. The plates were incubated upright

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43 at 37°C for 48 hours. After incubation, the plates were examined and clear zones were measured in millimeters from the edge of the discs. In this method, as the substance diffuses from the filter discs into the agar, the concentration decreases as a function of the square of the distance of diffusion, until at some point it is no longer effective at inhibiting microbial growth. The effectiveness of a particular antimicrobial agent is determined by the diameter of the clear zones produced. The clear areas are growth-inhibited zones that appear where the sanitizers or other antimicrobial agents were able to prevent bacterial growth (88). It was determined that this method was not suitable for the purposes of this study. The diffusion discs were only able to hold approximately 10 microliters of the sanitizing agents and such small amounts were rapidly oxidized by the media itself. Only concentrations much greater than the minimum inhibitory concentration were able to produce a clear zone. Therefore, the results for this method are not meaningful and are not included. Planktonic Bacteria Planktonic cells from growth flasks were harvested by centrifugation at 12,400 rpm in a Micro Centrifuge (Fisher Scientific, Model 235C, Pittsburgh, Pennsylvania) at room temperature, approximately 22°C, for 15 minutes. Cell pellets were then resuspended in the same volume of 0.5% saline solution, as a wash, and re-centrifuged for an additional 15 minutes also at 12,400 rpm. Cell pellets were then resuspended in 0.5% saline solution to a final concentration of approximately 10^ colony forming units (cfu)/milliliter (ml). One milliliter of the cell suspension was then added to 9 milliliters of a sanitizing solution at a concentration lower than the previously determined minimum inhibitory concentration. The bacterial cells were exposed to the sanitizer solutions at room temperature for 5 minutes. One milliliter of this cell-sanitizer mixture was then

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44 immediately removed and added to 9 milliliters of neutralizer (D/E Neutralizing broth, Difco). The mixture was allowed to set in the neutralizer solution for 30 seconds. Then an aliquot of the mixture was removed, diluted as necessary, and plated on Violet Red Bile agar in triplicate. The spread plate method was utilized for all samples and will be explained in detail in the microbiological analysis section. Concentration levels for all stages were verified by direct plating. All plates were incubated upright at 37°C overnight and then results were recorded. A milliliter of the cell-sanitizer-neutralizer solution was also removed and placed into a tube of Lauryl Sulfate broth and incubated at 37°C for 24 hours. This allowed for growth of uninjured cells and also enabled potentially injured cells to recover and grow. The cells of this tube were then utilized for day 2 and day 3 of this project. This procedure was repeated for each of the four sanitizers in three trials with each trial lasting for three consecutive days of sanitizer exposure at a previously determined sub-lethal level for the planktonic cells. Numbers of removed cells were calculated as cfu/ml and the results for all sanitizer experiments were the average of the three trials. Cultures that exhibited survival at the initial concentration of a samtizer(s) underwent subsequent experimentation following the above procedure at a higher concentration. Cultures that were unable to survive at the initial concentration of a sanitizer(s) underwent subsequent experimentation at a lower concentration also following the same protocol as outlined above. Bacterial Reassessment The bacteria isolates that survived the planktonic stage of the study were harvested and maintained on a culture slant (TSAYE) with monthly transfers as previously described. Upon completion of the planktonic testing phase, these surviving bacterial

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45 isolates were reassessed for their minimum inhibitory concentration, sub-lethal and lethal concentration levels. For any culture that showed an increase for any sanitizer treatment, this culture was then tested against other sanitizers to determine if the resistance (i.e. the increase in the minimum inhibitory concentration, sub-lethal and lethal concentration) could then be increased against other sanitizers by cross-protection. Solid Substrate Stainless steel plates (1 mm thick), type 304 with a mill finish (Thompson Sheet Metal, Gainesville, Florida), were utilized for the sanitizer challenge experiments. They were cut into coupons, size 2.54 x 7.62 centimeters. All coupons were degreased and cleaned with Dawn® dishwashing detergent, washed with 70% Isopropyl Alcohol (LabChem Inc., product no. LCI 5760-2, Pittsburgh, Pennsylvania) and then rinsed thoroughly with distilled water. All coupons were then air dried before autoclaving in sealed pouches (Fisher Scientific, product no. 01-812-54, Pittsburgh, Pennsylvania). Adhesion of Microbial Cells Escherichia coli 0157:H7, strain ATCC 700599 and strain FSIS 063-93, were cultured overnight in TSB at 37°C. These cultures were then centrifuged, washed in the same volume of 0.5% saline solution, re-centrifuged, and then resuspended in 0.5% saline solution to give a final concentration of 10^ cfu/ml. These concentrations were estimated by spectrophotometry (Spectrophotometer 20), using a standard curve of optical density at 580 nm against colony forming units. Concentrations were also verified through direct plating. Sterile stainless steel coupons were then suspended in the bacterial solutions and incubated at room temperature, approximately 22°C, without shaking, for 4 hours. Static incubation was used to better mimic the conditions in a processing facility and also because Blanchard and others (17) stated that attachment formed in this manner may be

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46 more resistant to sanitation. Coupons were then removed with sterile forceps, rinsed with distilled water for roughly 1 minute to remove poorly adhering cells and allowed to dry under a hood for about 20 minutes. Several of the dry coupons were swabbed (a 6.45 centimeter square area) to get an approximation of the level of bacteria that was able to attach. The remaining dry coupons were then aseptically placed into a sterile wire staining rack and the rack was then aseptically placed into a sterile staining dish and treated with the sanitizing agents or control distilled water as described below. Sanitizer Treatments for Adherent Cells The stainless steel coupons with adherent cells were submerged at room temperature, approximately 22°C, for 5 minutes in the sanitizer solution or in distilled water for a control. The coupons were then removed and rinsed in sterile distilled water for the removal of loosely attached cells. A 6.45 centimeter square area of the coupons, which was previously marked with a permanent pen, was then swabbed for 30 seconds. The swabs were then put into the appropriate neutralizing solution, vortexed, and diluted in 0.5% peptone and plated on VRBA (Difco) for enumeration of cells. Each of the four sanitizers was utilized and each experiment was performed in triplicate. Microbiological Analvsis The inoculum level of the Escherichia coli 0157:H7 bacterial cultures were verified by direct plating. Spread plating was utilized for both phase one and phase two of this study. For each initial dilution plated, a volume of 0.5 ml was dispensed onto prepoured Violet Red Bile agar plates. Subsequent dilutions, which utilized a volume of 0.1 ml, were also dispensed onto pre-poured VRBA plates. A 60 mm sterile plastic spreader (Fisher Scientific, product no. 05-541-11, United Kingdom) was then used to evenly distribute the sample over the plate as the plate was spun (134). All samples were plated

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47 in triplicate. The VRBA plates were incubated upright at 37°C for 48 hours. Plates with 30 to 300 colonies were counted and recorded. Statistical Analysis Plate count data were converted to log values for analysis. Data from the planktonic stage of the experiment were analyzed with the mixed model program (PROC MIXED) of SAS (SAS Institute, Gary, North Carolina) (118). Comparisons between the strains of Escherichia coli 0157:H7, day of treatment, sanitizer, concentration, nested within each sanitizer, and trial were made using the Ismeans statement of SAS. Treatment effects and differences were considered to be significant when P < 0.05. Data from the adherent stage of testing were also analyzed by SAS. The general linear model (PROC GLM) was utilized; however, the data was again analyzed using a mixed model with the random component being trials nested within the interaction of the other three variables (strain, sanitizer, attached). The /test option statement was used at the end of the statement to provide the appropriate f-tests. Comparisons were again made using the Ismeans statement. Treatment effects and differences were considered to be significant when P < 0.05.

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CHAPTER 4 RESULTS AND DISCUSSION Determination of Minimum Inhibitory Concentration Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 exhibited numerically different minimum inhibitory concentration (MIC) levels except with the Zep-Perosan™ treatment (Table I). Escherichia coli 0157:H7 strain FSIS 063-93 demonstrated a higher minimum inhibitory concentration level when exposed to Clorox®, Zep-I-Dine™, and Zepamine-A™ than the ATCC 700599 strain (Table 1). Table 1. The minimum inhibitory concentrations (MICs) of various sanitizers against Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 after incubation at 37°C for 24 hours E. coli 0151 -.W E. coli 0157:W FSIS 063-93 ATCC 700599 Sanitizer Minimum Inhibitory Concentration Clorox® 3.25 mg/L 1.0 mg/L Zep-I-Dine™ 1.5 mg/L -* Zepamine-A™ 13.0 mg/L 12.5 mg/L Zep-PerosanTM 3.5 mgyl. 3.5 mg/L *No minimum inhibitory concentration was found The two bacterial strains did not perform as expected. It was hypothesized that the ATCC 700599 strain, which is known to be acid tolerant, would outperform the FSIS 063-93 strain. Literature reviewed suggested that acid tolerant bacteria often exhibit increased resistance to other stresses such as heat, irradiation, and other antimicrobial agents (21). In this study, strain ATCC 700599, did not exhibit any increased resistance over strain FSIS 063-93 and did not appear to offer any cross-protection to sanitation treatment. Perhaps the pH of the sanitizing solutions was not sufficient to induce the stress response system(s) which would afford strain ATCC 700599 greater protection 48

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49 than the FSIS 063-93 strain (78, 1 13). With the exception of the peroxyacetic acid, the pH of the sanitizing solutions at the concentrations utilized in this study was essentially neutral. Marriott (82) stated that as little as 0.6 mg/L to 13 mg/L of chlorine is able to inactivate 90% of most planktonic bacteria. The minimum inhibitory concentration values obtained for both strains for the Clorox® treatment were within this range. Marriott (82) also stated that 6.25 mg/L of an iodine solution is sufficient to reduce a planktonic bacterial population by 99.999%. The minimum inhibitory concentration for strain FSIS 063-93 fell well below this value and no MIC could be found for the ATCC strain. At a value of only 0.25 mg/L of the iodine sanitizing solution the ATCC 700599 strain was unable to survive. Pickett and Murano (102) found that the minimum inhibitory concentration for quaternary ammonium against Listeria monocytogenes was 5 mg/L. In this study the ATCC 700599 strain and the FSIS 063-93 strain produced a MIC of 12.5 mg/L and 13.0 mg/L, respectively. As quaternary ammonium compounds have been found to be very effective against gram positive bacteria but less effective against gram negative bacteria, it is not surprising that the MIC for the two Escherichia coli 0157:H7 strains was higher than that reported by Pickett and Murano (102). GuerinMechin and others (55) found the MIC for Pseudomonas aeruginosa, a gram negative bacterium, to fall within the range of 10 to 20 mg/L. This is comparable to the data found in this study for both Escherichia coli 0157:H7 strains. No comparable data was available for the MIC of the peroxyacetic acid solution.

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50 Planktonic Bacteria Chlorine Compound A chlorine concentration of 1.0 mg/L killed approximately 10^ cfu/ml of Escherichia coli 0157:H7 strain FSIS 063-93 after a 5 minute exposure and more than 10^ cfu/ml of Escherichia coli 0157:H7 strain ATCC 700599 (Table 2). After the chemical shock of 1 mg/L for 5 minutes, a milliliter of the cell-sanitizer-neutralizer solution was removed and placed into a 9 ml tube of Lauryl Sulfate broth and incubated at 37°C for 24 hours to allow for potential recovery of the exposed cells. This procedure was repeated for three consecutive days. On day 2 and day 3, the FSIS 063-93 strain demonstrated a greater than 10^ log reduction while the ATCC 700599 strain showed a mean log reduction of 6.62 and 2.14 for day 2 and day 3 respectively (Table 2). The lower mean log reduction values for the ATCC strain 700599 are a result of a failure of the bacterial cells to fully recover to a 10^ cfu/ml level in the Lauryl Sulfate broth after the initial chemical exposure on day 1 (Table 2). Additionally, there were significant differences between the ATCC 700599 strain and the FSIS 063-93 strain for day 1, day 2 and day 3 respectively. This is also attributed to the fact that after the initial chemical shock on day 1, the ATCC 700599 strain was not able to recover to a 10^ cfu/ml level in the Lauryl Sulfate broth while the FSIS 063-93 strain underwent full recovery in the broth solution (Table 2). Both Escherichia coli 0157:H7 strains exhibited slightly greater than a 1 log survival after treatment on day 1 and both demonstrated less than a 1 log survival after repeated treatment on day 2 and day 3 (Table 2).

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51 Table 2. Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 1.0 mg/L of Clorox® for 5 minutes at 22°C Day E. coli 0157:W E. coli 0151 Ml FSIS 063-93 (cfu/ml) ATCC 700599 (cfu/ml) Before After Log kill Before After Log kill treatment treatment treatment treatment 1 8.46 1.54 6.92'" 8.94 1.14 7.80"'^ 2 8.75 0.27 8.48"'" 7.26 0.64 6.62"'^ 3 8.50 0.00 8.50''' 2.40 0.26 2.14'-' '' Different letters in the same column indicate a significant difference among means at the P < 0.05 level. "'^ Different letters in the same row indicate a significant difference between FSIS and ATCC means at the P < 0.05 level. A similar pattern was observed for both E. coli 0157:H7 strains when the concentration of chlorine was reduced to 0.5 mg/L. The FSIS 063-93 strain showed a greater than 10^ log reduction on day 1 with a greater than 10^ log reduction on day 2 and day 3 (Table 3). The ATCC 700599 strain showed a greater than 10^ log reduction at this concentration of chlorine for day 1 and had a 3.87 and 1.99 mean log reduction value on day 2 and day 3, respectively (Table 3). The lower mean log reduction values on day 2 and day 3 for the ATCC 700599 strain can again be attributed to the lack of ability to recover to a level of 10^ cfu/ml in the Lauryl Sulfate broth after the chemical shock on day 1 (Table 3). There was no significant difference between the ATCC 700599 strain and the FSIS 063-93 strain for day 1, P=0.37, however, significant differences were observed between the two strains for day 2 and day 3. This is also attributed to the fact that after the initial chemical shock on day 1, the ATCC 700599 strain was not able to recover to a 10^ cfu/ml level in the Lauryl Sulfate broth while the FSIS 063-93 strain underwent full recovery in the broth solution (Table 3).

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52 Table 3. Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 0.5 mg/L of Clorox® for 5 minutes at 22°C Day £. co//0157:H7 E. coli OISIMI FSIS 063-93 (cfu/ml) ATCC 700599 (cfu/ml) Before After Log kill Before After Log kill treatment treatment treatment treatment 1 8.79 2.03 6.76"'" 8.61 1.55 7.06"" 2 8.70 0.65 8.05"" 4.23 0.36 3.87"'^ 3 n h 8.32 0.00 8.32'''' 2.22 0.23 1.99'-^ Different letters in the same column indicate a significant difference among means at the P < 0.05 level. "'^Different letters in the same row indicate a significant difference between FSIS and ATCC means at the P < 0.05 level. Statistical analysis revealed that there were no trial differences for either strain at either concentration level of chlorine. Additionally, neither Escherichia coli 0157:H7 strain was able to be recovered on the Violet Red Bile agar after repeated exposure of chlorine at a concentration of 1.0 mg/L or 0.5 mg/L. The FSIS 063-93 was able to grow in the Lauryl Sulfate broth at 37°C after repeated exposure at 1.0 mg/L (Table 2) and at 0.5 mg/L (Table 3). This would indicate the presence of sub-lethally injured bacteria (21, 122). The results obtained in this study are similar to those of Joseph and others (64). They found that planktonic Salmonella cells were unable to survive a five minute exposure to chlorine at a concentration of 10 mg/L. In general, Zhao and others (142) found similar results to this study. They tested seven strains of Escherichia coli 0157:H7 and found that six of the seven were susceptible to chemical shock of chlorine at a concentration of only 0.25 mg/L after 1 minute of exposure. They did find one unusual strain that they labeled as E coli 0157:H7 G which was able to withstand chemical exposure of chlorine at a level of 2 mg/L. It appears that there may be an innate

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53 difference among E. coli 0157:H7 isolates in regard to their ability to tolerate chlorine. A similar finding was cited by Mokgatla and others (90) when testing Salmonella strains isolated from a poultry abattoir. They reported that one of the isolates was able to survive chemical exposure to hypochlorous acid at a level of 72 mg/L and could grow in the presence of a chlorine concentration considered to be antibacterial. Quaternary Ammonium Compound An initial chemical shock of Zepamine-A™ at a concentration of 10 mg/L resulted in a greater than 10^ log reduction for both Escherichia coli 0157:H7 strains utilized in this study (Table 4). Subsequent exposure resulted in a mean log reduction of 7.39 and 8.49 on day 2 and day 3 respectively for the FSIS 063-93 strain. In comparison, the repeated exposure to the quaternary ammonium compound resulted in a mean log reduction value of 5.94 and 7.44 on day 2 and day 3 respectively for the ATCC 700599 strain (Table 4). While both strains were able to recover to a level of 10^ cfu/ml in the Lauryl Sulfate broth after the chemical exposure, it appears that the ATCC 700599 strain had the ability to adapt and survive as determined by plate count data (cfu/ml) on Violet Red Bile agar (Table 4). Table 4. Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 10.0 mg/L of Zepamine-A™ for 5 minutes at 22°C Day E coli 0157:H7 E. coli 0157:H7 FSIS 063-93 (cfu/ml) ATCC 700599 (cfu/ml) Before After Log kill Before After Log kill 1 treatment treatment treatment treatment 8.46 3.29 5.17'^-'' 8.61 2.95 5.66''" 2 8.78 1.39 7.39"'" 8.46 2.52 5.94"'^ 3 a,b, 8.52 0.03 8.49'" 8.52 1.08 7.44''y Different letters in the same column indicate a significant difference among means at the P < 0.05 level. "•^Different letters in the same row indicate a significant difference between FSIS and ATCC means at the P < 0.05 level.

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54 In addition to exhibiting significant differences by day within each strain (Table 4), there were also significant differences by day between the two strains. Analysis of the data revealed no significant difference between the two strains at day 1 (P=0.23) however, there was significant differences between the two strains for day 2 and day 3. This is likely due to the fact that the ATCC 700599 strain had less injured cells and therefore exhibited higher colony counts on the VRBA plates for both day 2 and day 3 (Table 4). Perhaps, the E. coli 0157:H7 FSIS 063-93 strain had a greater number of injured cells and therefore was not able to grow as well on the selective media (21, 122). Additionally, there were no significant differences between trials for this concentration of Zepamine-A™. Although Escherichia coli 0157:H7 strain FSIS 063-93 exhibited minimal to no survival after three days of repeated exposure to 10 mg/L of Zepamine-ATM (Table 4) on the VRBA, it was decided that both strains, not just the ATCC 700599 strain, would be subjected to 1 1 mg/L, a higher concentration, of the sanitizer. The results of the exposure of the two strains to the higher level of the sanitizer did not conform to expectations. For reasons that remain unclear, the two strains demonstrated a role reversal at the higher level of sanitizer. At a concentration of 1 1 mg/L of the quaternary ammonium compound, Zepamine-A, the E. coli 0157:H7 FSIS 063-93 strain showed a higher level of survival than it did at 10 m^. The E. coli 0157:H7 ATCC 700599 strain showed a decrease in its survival rate (Table 5). Perhaps at this increased level of exposure, strain FSIS 063-93 underwent habituation. This phenomenon occurs when enterobacteria, especially Escherichia coli spp., respond to low doses of chemical or physical stresses by inducing responses which allow mildly stressed organisms to subsequently resist higher.

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55 potentially lethal doses of the same stress (113). There were significant differences within each strain for day of exposure (Table 5). Additionally, there were significant differences between strains in reference to day of exposure. Table 5. Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 1 1.0 mg/L of Zepamine-A™ for 5 minutes at 22°C Day E.coliOl51:W E. coli 0151 Bl FSIS 063-93 (cfu/ml) ATCC 700599 (cfu/ml) Before After Log kill Before After Log kill treatment treatment treatment treatment 1 8.87 3.38 5.49^" 8.91 2.11 2 8.63 2.86 5.77"''' 8.57 1.28 7.29"'^ 3 8.52 0.71 7.81'''' 8.49 0.07 8.42'''y Different letters in the same column indicate a significant difference among means at the P < 0.05 level. "'^Different letters in the same row indicate a significant difference between FSIS and ATCC means at the P < 0.05 level. Trial was also a source of variation for mean log reduction values for both of the Escherichia coli 0157:H7 strains when exposed to Zepamine-A™ at a concentration of 1 1 mg/L (Table 6). Trial 2 was significantly different from both trial 1 and trial 3 for strain ATCC 700599. Strain FSIS 063-93 in trial 2 was also significantly different from trial 1 and trial 3. The difference in trial 2 for both strains is due to higher survival rates on the VRBA plates (Table 5). Possible reasons for this variation may be fluctuation changes in room temperature during trials or fluctuations in incubation temperatures which could lead to variations in bacterial growth. Another explanation is experimental error.

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56 Table 6. Mean log reduction values by trial (n=18) for Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 11.0 mg/L of Zepamine-A™ for 5 minutes at 22°C Trial E.coliOl51:W ATCC 700599 (cfu/ml) E. CO// 0157:H7 FSIS 063-93 (cfu/ml) 1 7.72' 6.70' 2 7.10" 5.92" 3 7.68' a.bT^w-,TT—. — -. ; : — r. : — — 6.45" '• Different letters in the same column indicate a significant difference among means at the P< 0.05 level. Review of the plate count data reveal a downward trend in reference to survival on Violet Red Bile agar after repeated exposure at the sub-lethal sanitizer concentration of 10 mg/L (Table 4) and 1 1 mg/L (Table 5) for both bacterial strains. These results are similar to that reported by Pickett and Murano (102). They found that sub-lethal exposure of Listeria monocytogenes to quaternary ammonia failed to result in any acquired resistance to subsequent exposure of the same sanitizer. In contrast, GuerinMechin and others (55) found that repeated exposure of sub-lethal levels of quaternary ammonium compounds to Pseudomonas aeruginosa resulted in increased survival of the bacteria upon exposure to subsequently higher levels of the sanitizer. Peroxyacetic Acid Compound A concentration of 1.0 mg/L of peroxyacetic acid per 4.4 mg/L of hydrogen peroxide of Zep-Perosan™ only killed approximately 10^ cfu/ml of both E. coli 0157:H7 strains after initial exposure to the sanitizer for 5 minutes at 22°C. Further exposure to the sanitizer resulted in roughly a three log reduction for day 2 and day 3 for each strain (Table 7). Significant differences exist within both strains for day of exposure (Table 7) however, no significant differences between strains were found for day of exposure.

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57 Table 7. Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 1.0 mg/L per 4.4 mg/L of Zep-Perosan™ for 5 minutes at 22°C Day E. coli 0157:H7 E. coli 0157:H7 FSIS 063-93 (cfu/ml) Before After Log kill Before After Log kill treatment treatment treatment treatment 1 8.46 6.47 1.99" 8.61 6.45 2.16" 2 8.69 5.51 3.18' 8.43 5.52 2.91'" 3 8.52 4.84 3.68' 8.51 4.97 3.54' the P < 0.05 level. Trial was also a source of variation for mean log reduction values for both of the Escherichia coli 0157:H7 strains when exposed to Zep-Perosan™ at a concentration of 1.0 mg/L of peroxyacetic acid per 4.4 mg/L of hydrogen peroxide (Table 8). Trial 1 was significantly different from both trial 2 and trial 3 for both Escherichia coli 0157:H7 strains. The difference in trial 1 for both strains is due to lower survival rates on the VRBA plates (Table 7). Possible explanations for this decrease in survival rate of trial 1 could be an error in the initial sanitizer concentration, an error during dilutions or perhaps a temperature fluctuation of the incubator. Table 8. Mean log reduction values by trial (n=18) for Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 1.0 mg/L of peroxyacetic acid per 4.4 mg/L of hydrogen peroxide of Zep-Perosan™ for 5 minutes at 22°C ^"riai E. coli 0157:H7 E. coli 0157 Ml . ATCC 700599 (cfu/ml) FSIS 063-93 (cfu/ml) 1 4.00' 4.17' 2 2.24" 2.16" -Ip— 2.36" 2,53^ Different letters in the same column indicate a significant difference among means at the P < 0.05 level. As only a small log reduction was found when the two strains of Escherichia coli 0157:H7 were exposed to 1 .0 mg/L of peroxyacetic acid per 4.4 mg/L of hydrogen

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58 peroxide of Zep-Perosan^^, the concentration was increased to 2.0 mg/L of peroxyacetic acid per 8.8 mg/L of hydrogen peroxide of Zep-Perosan^'^ and the experiment was repeated. At this higher concentration, very Httle difference was observed in the mean log reduction values of either strain of Escherichia coli 0157:H7 (Table 9). The FSIS 063-93 strain averaged a 10^ cfu/ml reduction for all three days of exposure and the ATCC 700599 strain exhibited a 10^ cfu/ml reduction upon initial exposure to the sanitizer and then had approximately a 10^ cfu/ml log reduction for day 2 and day 3 of exposure (Table 9). E. coli 0157:H7 strain FSIS 06-93 showed a slightly higher level of growth based on plate count data from VRBA for day 2 and day 3 (Table 9). Significant differences for day of exposure were found for the ATCC 700599 bacterial strain but not for the FSIS 063-93 strain (Table 9). No significant difference was demonstrated between the strains for the initial chemical shock however, there were significant differences between the two Escherichia coli 0157:H7 strains for treatment and log kill on day 2 and day 3. There were no differences observed for trials at a concentration of 2.0 mg/L of peroxyacetic acid per 8.8 mg/L of hydrogen peroxide of Zep-Perosan™. Table 9. Mean log values by day (n=18) for planktonic bacterial isolates Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 2.0 mg/L per 8.8 mg/L of Zep-Perosan™ for 5 minutes at 22°C Day E. coli 0151 :H1 E. co//0157:H7 FSIS 063-93 (cfu/ml) ATCC 700599 (cfu/ml) Before After Log kill Before After Log kill treatment treatment treatment treatment 1 8.87 6.59 2.28'-'' 8.91 6.58 2.33'''^ 2 8.81 6.50 2.31''''' 8.76 5.52 3.24'-y 3 a.b. 8.68 6.30 2.38^''' 8.69 5.42 3.27"'^ Different letters in the same column indicate a significant difference among means at the P < 0.05 level. "'^Different letters in the same row indicate a significant difference between FSIS and ATCC means at the P < 0.05 level.

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59 The data suggested that Zep-Perosan, a peroxyacetic acid sanitizer, was not effective against either of the planktonic E. coli 0157:H7 strains when utiHzed at such low concentrations. In contrast, Mosteller and Bishop (93) found peroxyacetic acid to be capable of producing a greater than 9 log reduction in planktonic bacterial isolates from Pseudomonas fluorescens, Yersinia enterocolitica, and Listeria monocytogenes after only a thirty second exposure. However, the study conducted by Mosteller and Bishop (93) treated the bacterial cells at the recommended in use concentration of 200 mg/L (4% peroxyacetic acid and 25% hydrogen peroxide) and this study utilized values just below the minimum inhibitory concentration. The data suggested that Zep-Perosan, a peroxyacetic acid sanitizer, was not effective against either of the planktonic E. coli 0157:H7 strains when utilized at such low concentrations. In contrast, Mosteller and Bishop (93) found peroxyacetic acid to be capable of producing a greater than 9 log reduction in planktonic bacterial isolates from Pseudomonas fluorescens. Yersinia enterocolitica, and Listeria monocytogenes after only a thirty second exposure. However, the study conducted by Mosteller and Bishop (93) treated the bacterial cells at the recommended in use concentration of 200 mg/L (4% peroxyacetic acid and 25% hydrogen peroxide) and this study utilized values just below the minimum inhibitory concentration. Iodine Compound As no minimum inhibitory concentration for the Escherichia coli 0157:H7 ATCC 700599 strain was determined, only the FSIS 063-93 strain was treated with the iodine sanitizer during the planktonic phase of this study. The FSIS 063-93 strain was not able to grow on the Violet Red Bile agar plates after repeated exposure at either concentration. Additionally, the bacterial isolates were unable to recover to a detectable level in the

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60 Lauryl Sulfate broth after day 2 (Table 10). Significant differences were found between each day of treatment at both concentration levels but when comparing the log kills for each concentration there was only a significant difference at day 2 (Table 10). No trial differences occurred at either concentration of the iodine sanitizer. Table 10. Mean log values by day (n=18) for planktonic bacterial isolates of Escherichia coli 0157:H7 strain FSIS 063-93 when exposed to 0.25 mg/L of Zep-I-Dine™ and 0.50 mg/L of Zep-I-Dine for 5 minutes at 22°C Day Concentration of 0.25 mg/L (cfu/ml) Concentration of 0.50 mg/L (cfu/ml) Before After Log kill Before After Log kill treatment treatment treatment treatment 1 8.79 2.57 6.12" 8.46 2.51 5.95" 2 8.37 0.77 7.60' 8.74 0.08 8.66' 3 a.b.c T-^0.00 0.00 0.00' 0.00 0.00 0.00' '' '' Different letters in the same column indicate a significant difference among means at the P < 0.05 level. The results found in this study are similar to those of Ronner and Wong (1 10). These researchers found that planktonic cells of Listeria monocytogenes and Salmonella typhimurium were reduced by 7 to 8 logs when treated with 25 mg/L of iodine for 10 minutes. Joseph and others (64) also showed that planktonic bacteria were unable to survive treatment with an iodine sanitizer. They found that Salmonella weltevreden and Salmonella FCM 40 were completely killed, a 6 log reduction, when exposed to 10 mg/L of iodine for 5 minutes. Finally, Marriott (82) stated that iodine sanitizers could reduce planktonic bacterial cell populations by 99.999% within 30 seconds when treated with a concentration level of 6.25 mg/L. Bacterial Reassessment No Escherichia coli 0157:H7 bacterial isolates for strain ATCC 700599 survived the planktonic stage of testing when treated with Clorox® at 0.5 mg/L or at 1.0 mg/L. Additionally, no survival occurred when the ATCC 700599 strain was treated with Zep-I-

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61 Dine™ at 0.25 mg/L or at 0.50 mg/L. Repeated sub-lethal exposure to Zep-Perosan™ and Zepamine-A^M did result in an increase in the minimum inhibitory concentration for this strain of Escherichia coli 0157:H7. When repeatedly exposed to sub-lethal levels of Zep-Perosan™, a peroxyacetic acid sanitizer, the minimum inhibitory concentration of E. coli 0157:H7 strain ATCC 700599 increased by 142.8%. Similarly, pretreatment of the ATCC 700599 bacterial isolates with sub-lethal levels of Zepamine-A™ resulted in a 68% increase of the minimum inhibitory concentration (Table 11). Table 11. The minimum inhibitory concentrations for Escherichia coli 0157:H7 strain ATCC 700599 before and after pretreatment with sub-lethal levels of various sanitizers Sanitizer Minimum Inhibitory Concentration Untreated Treated Clorox® 1.0 mg/L 1.0 mg/L Zep-I-Dine™ .* -* Zepamine-A^'^ 12.5 mg/L 21.0 mg/L Zep-Perosan^M 3.5 mg/L 8.5 mg/L *No minimum inhibitory concentration was found The bacterial isolates for E. coli 0157:H7 strain ATCC 700599 that demonstrated an increase in their minimum inhibitory concentrations when treated with sub-lethal levels of Zep-PerosanTM and Zepamine-A^M were then further tested against the other sanitizers utilized in this study. The purpose for this was to determine if this increased resistance would provide cross protection against the other sanitizers and result in a similar increase in the minimum inhibitory concentrations for them. No increase was found in any isolates for Clorox® or Zep-I-Dine™. The isolates did maintain their increased minimum inhibitory concentration for Zepamine-ATM and Zep-PerosanTw. Guerin-Mechin and others (55) observed similar results when treating Pseudomonas aeruginosa, also a gram-negative bacteria, with quaternary ammonium compounds. They found that the minimum bactericidal concentrations for five different subcultures of

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62 Pseudomonas aeruginosa could be substantially increased upon pre -exposure to sublethal levels with two quaternary ammonium sanitizers. Additionally, it was determined that the increased levels of resistance to these sanitizers did not offer any cross protection when the subcultures were subsequently treated with other sanitizing compounds. In contrast, Pickett and Murano (102) found that exposure of Listeria monocytogenes to sub-lethal levels of sanitizers, including a chlorine based compound, an iodine base compound and a quaternary ammonium compound, did not affect the minimum inhibitory concentrations. The results for Escherichia coli 0157:H7 strain FSIS 063-93 were similar to those of strain ATCC 700599. The pretreatment of the bacterial cells with sub-lethal levels of Clorox® and Zep-I-Dine^M did not result in an increase in the minimum inhibitory concentration for either sanitizer. When previously exposed to sub-lethal levels of Zepamine-A™ and Zep-Perosan™ a 61.5% increase and a 185.7% increase in the minimum inhibitory concentration occurred respectively (Table 12). Results obtained for the FSIS 063-93 strain are similar to that of Guerin-Mechin and others (55). As previously stated, they determined that the minimum inhibitory concentration for a sanitizer, specifically quaternary ammonium compounds, could be increased by the pretreatment of the bacterial cultures with sub-lethal levels of the sanitizer. Table 12. The minimum inhibitory concentrations for Escherichia coli 0157:H7 strain FSIS 063-93 before and after pretreatment with sub-lethal levels of various sanitizers Sanitizer Minimum Inhibitory Concentration Untreated Treated Clorox® Zep-I-Dine™ Zepamine-A™ Zep-PerosanTM 3.25 mg/L 1.5 mg/L 13.0 mg/L 3.5 mg/L 3.25 mg/L 1.5 mg/L 21.0 mg/L 10.0 mg/L

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63 Additionally, when the bacterial isolates that were exposed to sub-lethal levels of Zep-Perosan^^ were again tested against Clorox®, Zep-I-Dine™ and Zepamine-A™, no cross protection occurred as no increase in the minimum inhibitory concentrations resulted. The cells did maintain their previous increase in minimum inhibitory concentration for the quaternary ammonium sanitizer and the peroxyacetic acid sanitizer. In contrast to the ATCC 700599 strain, the FSIS 063-93 strain did exhibit crossprotection when pretreated with sub-lethal levels of Zepamine-A™, the quaternary ammonium sanitizer. The repeated sub-lethal chemical shock with Zepamine-A™ on E. coli 0157:H7 strain FSIS 063-93 resulted in a decrease in the minimum inhibitory concentration for the chlorine compound, the maintenance of the increased minimum inhibitory concentrations for the quaternary ammonium sanitizer and the peroxyacetic acid sanitizer, and an increase of 186.7% for the minimum inhibitory concentration for the iodophor compound (Table 13). Table 13. The minimum inhibitory concentrations of various sanitizers for Escherichia coli 0157:H7 strain FSIS 063-93 planktonic bacterial isolates before and after pretreatment with sub-lethal levels of Zepamine-A™ Minimum Inhibitory Concentration Sanitizer Untreated Treated Clorox® Zep-I-DineTM Zepamine-A™ Zep-Perosan™ 3.25 mg/L 1.5 mg/L 13.0 mg/L 3.5 mg/L 2.2 mg/L 4.3 mg/L 21.0 mg/L 10.7 mg/L Adherent Bacteria Attachment Levels Microbial attachment and the development of biofilms are known to occur on many surfaces and in many different environments (34, 64, 93). In this study it was determined that both Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 attached

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64 to the stainless steel chips after a 4 hour static suspension at a level of approximately lO'* cfu/cm . Dewanti and Wong (34) found similar results. When stainless steel chips were inoculated with Escherichia coli 0157:H7 at a level of lO^cfu/ml, they found the bacteria were able to adhere to the surface at a level from 10^ to 10^ cfu/cm^ after a 1 hour incubation at room temperature. Similar results were obtained by Hood and Zottola (60) who found that Salmonella typhimurium, Listeria monocytogenes, Pseudomonas fragi, Pseudomonas fluorescens, and Escherichia coli 0157:H7 bacterial cells were all able to attach to stainless steel within 1 hour in various test growth media. In particular, they determined that depending upon the test media, Escherichia coli 0157:H7 cells were able to attach at levels of 10^ to 10^ cfu/cml Farrell and others (43) also observed a similar attachment level for Escherichia coli 0157:H7 bacteria on stainless steel. Their research showed that after only a 5 minute incubation period, E. coli 0157:H7 cells were able to attach to the surface at a level of 10^ to 10'^ cfu/cm^. Sanitizer Treatments for Adherent Cells As previously stated, chemical sanitizers are considered to be effective on food contact surfaces if they demonstrate a five-log reduction in planktonic bacteria (43) and a greater than three-log reduction for adherent cells (93). In this study the treatment of Escherichia coli 0157:H7 bacterial isolates with 1.0 mg/L of Clorox® was effective against planktonic cells but failed to provide an adequate reduction in adherent E. coli 0157:H7 cells (Table 14). There were no significant differences between the strains for log kill for adherent cells however significant differences existed between the adherent cells and the planktonic cells (Table 14). The chlorine treatment of the adherent bacterial cells provided slightly less than a two-log reduction for the FSIS 063-93 strain and slightly

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65 greater than a two-log reduction for the ATCC 700599 strain. In this case, the use of the chlorine sanitizer was no more effective than the use of water alone. Treatment of the stainless steel coupons with water also provided a 1 to 2 log decrease in the bacterial counts for both E. coli 0157:H7 strains. Table 14. Mean log values (n=18) for planktonic and adherent bacterial isolates of Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 1.0 mg/L of Clorox® for 5 minutes at 22°C Strain Cell type Mean log values (log cfu/ml or cfu/chip) Before treatment After treatment Log kill FSIS Planktonic 8.46 1.54 6.92" FSIS Attached 5.47 3.77 1.70^= ATCC Planktonic 8.94 1.14 7.80' ATCC a.b.C -r^-rr Attached 5.56 3.48 2.08' ' '^ Different letters in the same column indicate a significant difference among means at the P < 0.05 level. Researchers have found similar results when treating adherent pathogenic bacteria with a chlorine sanitizer. Restaino and others (108) found that chlorine at 100 mg/L did not provide a significant difference from water in reducing the bacterial population of adherent Staphylococcus aureus cells on Formica surfaces. Both the chlorine sanitizer and water provided only a two-log decrease in the bacterial isolates. In another study, Joseph and others (64) found that at a concentration of 10 mg/L, CI2 was able to reduce greater than 10^ log of planktonic Salmonella spp. after a 5 minute contact time. The same sanitizer was only able to reduce the adherent bacterial cells by 1 log after a 25 minute exposure. Additionally, when Joseph and others (64) increased the CI2 concentration up to 50 mg/L, adherent bacteria were still only reduced by 2 logs after 25 minutes. Andrade and others (3) also found that adherent bacterial cells were more resistant than non-adherent cells when treated with chlorine. In contrast, Farrell and others (43) found a three-log reduction or higher when adherent Escherichia coli

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66 0157:H7 cells on stainless steel were treated with chlorine. However, they treated the cells at a level of 200 mg/L and when the stainless steel coupons were enriched after sanitizer treatment 63 to 88 % of the chips were found to be positive for Escherichia coli 0157:H7. This indicates that injured organisms remained on the surface after treatment and were able to recover upon enrichment. This phenomenon also occurred in the current study when planktonic E. coli 0157:H7 cells were exposed to chemical shock with Clorox® (Table 2 and Table 3). As shown by Table 15, significant differences existed between both strain and cell type in regards to log kills attained. The quaternary ammonium compound was found to be effective for both planktonic and adherent cells for the ATCC 700599 strain. In contrast, the sanitizer was effective against only the planktonic cells of the FSIS 063-93 strain. Treatment of adherent Escherichia coli 0157:H7 FSIS 063-93 cells resulted in less than a 1 log reduction. Table 15. Mean log values (n=18) for planktonic and adherent bacterial isolates of Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 1 1.0 mg/L of Zepamine-A™ for 5 minutes at 22°C Strain Cell type Mean log values (log cfu/ml or cfu/chip) FSIS Planktonic Before treatment 8.87 After treatment 3.38 Log kill 5.49" FSIS Attached 5.58 4.88 0.70" ATCC Planktonic 8.91 2.11 6.80' ATCC a.B.C T^-rr Attached 5.67 1.62 4.05' Different letters in the same column indicate a significant difference among means at the P < 0.05 level. Results found by Mosteller and Bishop (93) supports the current research. They found that quaternary ammonia at 200 mg/L killed more than 5 logs of planktonic bacterial cells of Pseudomonasfluorescens, Yersinia enterocolitica, and Listeria monocytogenes in just 30 seconds. Nonetheless, the same sanitizer was unable to provide

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67 a three log reduction in the same bacterial isolates when the cells were attached to rubber or Teflon®. Additionally, Restaino and others (108) showed quaternary ammonium at 150 mg/L to be ineffective against adherent Staphylococcus aureus cells both in the presence and absence of organic material. After a 60 minute exposure time, they found a 2 and 2.5 log reduction, respectively. Similarly, Trachoo and Frank (131) found that a quaternary ammonium compound at a level of 50 mg/L was able to inactivate Campylobacter jejuni cells with no biofilms present in 45 seconds. With biofilms, the same sanitizer even at an increased level of 200 mg/L did not inactivate the bacterial cells. Peng and others (101) also found planktonic cells to be the most susceptible to sanitizers, followed by attached, single cells and then cells present in a biofilm. They determined that 100 mg/L of quaternary ammonia resulted in a greater than five-log reduction in planktonic Bacillus cereus cells within 15 seconds. However, neither 100 mg/L or 200 mg/L of the same sanitizer was effective against the cells in a biofilm. Results of this study found Zep-Perosan™, a peroxyacetic acid, to be somewhat ineffective against both planktonic and adherent Escherichia coli 0157:H7 cells at the concentrations utilized. Both strains showed a three-log reduction for planktonic cells with no significant differences between the two strains. The strains did show a significant difference for the adherent cells (Table 16). The FSIS 063-93 strain showed less than a one-log decrease for the adherent cells while the ATCC 700599 strain showed a two-log reduction. As in this study, Mosteller and Bishop (93) found that peroxyacetic acid was not effective against adherent bacterial cells. Unlike current research, they concluded that at a level of 200 mg/L peroxyacetic acid was effective at reducing planktonic bacterial cells

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68 by more than 5 logs. Trachoo and Frank (131) also found peroxyacetic acid to be quite effective against planktonic bacterial cells. They found that at 50 mg/L (27.5 % of hydrogen peroxide and 5.8 % of peracetic acid) the sanitizer effectively eliminated all planktonic cells of Campylobacter jejuni, also a gram negative bacteria, within 45 seconds. Even at this higher concentration, the sanitizer while effective against planktonic cells, was not able to inactivate the bacterial cells within a biofilm matrix after a 3 minute exposure. Table 16. Mean log values (n=18) for planktonic and adherent bacterial isolates of Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 3.0 mg/L per 13.2mg/L of Zep-Perosan™ for 5 minutes at 22°C Strain Cell type Mean log values (log cfu/ml or cfu/chip) Before treatment After treatment Log kill FSIS Planktonic 8.49 5.27 3.22' FSIS Attached 5.49 5.36 0.13' ATCC Planktonic 8.59 5.04 3.55' ATCC a.b.c r-N/•
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69 Unlike the current research, Fatemi and Frank (44) found peroxyacetic acid to be an effective sanitizer against adherent bacterial cells. Although they utilized a higher concentration level, 40 mg/L, they found that in a mixed, adherent culture consisting of Listeria monocytogenes and Pseudomonas, peroxyacetic acid was able to reduce the bacterial population to less than 10 cfu/cm^. Along this line, Farrell and others (43) found that after sanitizing treatment of stainless steel chips with adherent Escherichia coli 0157:H7 bacterial cells in a 0.2 % solution of peroxyacetic acid, viable bacteria was infrequently recovered. However, Farrell and others (43) also reported that even when inoculated at a level of only 10^ cfu/ml of Escherichia coli 0157:H7, a more reaUstic scenario for a meat processing facility, 50 % of the stainless steel chips utilized in the study were positive for E. coli 0157:H7 after enrichment. As shown by Table 17, significant differences existed between both strain and cell type in regards to log kills attained. While all log kill values were significantly different from each other, the iodine sanitizer was found to be effective against both the planktonic and adherent cells for both Escherichia coli 0157:H7 strains. Treatment of planktonic cells resulted in a greater than five-log reduction for both strains and treatment of adherent cells resulted in greater than a 3 log reduction (Table 17). Table 17. Mean log values (n=18) for planktonic and adherent bacterial isolates of Escherichia coli 0157:H7 strain ATCC 700599 and strain FSIS 063-93 when exposed to 0.5 mg/L of Zep-I-Dine™ for 5 minutes at 22°C Strain Cell type Mean log values (log cfu/ml or cfu/chip) Before treatment After treatment Log kill FSIS Planktonic 8.46 2.50 5.96*' FSIS Attached 5.85 2.69 3.16'' ATCC Planktonic 8.61 0.00 8.61* ATCC Attached 5^94 L23 4.71" Different letters in the same column indicate a significant difference among means at the P < 0.05 level.

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70 Mosteller and Bishop (93) also found iodine to be effective against planktonic bacterial cells. They showed a greater than five-log reduction in several bacterial populations within 30 seconds at a concentration of 25 mg/L. They found that iodine was not effective at reducing adherent bacterial populations by more than 3 logs in most cases. Adherent Listeria monocytogenes cells were not effectively reduced. Adherent Yersinia enterocolitica and Pseudomonas fluorescens cells were only reduced by more than 3 logs when attached to Teflon® as determined by plate count method not the impedance method. Similarly, Joseph and others (64) found iodine to be effective at a level of only 1 mg/L on planktonic bacterial isolates of Salmonella species after a 5 minute treatment. However, in order to obtain a three-log or higher reduction on adherent cells the use of a higher concentration level and a longer exposure time was required.

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CHAPTER 5 SUMMARY AND CONCLUSIONS There is much evidence that Escherichia coli 0157:H7 is an adaptive organism capable of surviving hostile and harsh environments. This investigation demonstrated that the ability of various sanitizers to clearly provide a five-log or greater reduction in planktonic bacteria does not necessarily correspond to the sanitizers' ability to provide an adequate reduction (three-log) in attached bacteria. This study evaluated the ability of Escherichia coli 0157:H7 isolates to survive and adapt to four sanitizers common to the food industry. Preliminary studies determined the minimum inhibitory concentration, sub-lethal and lethal concentration of sanitizers for Escherichia coli 0157:H7 strain FSIS 063-93 and strain ATCC 700599 which is known to be acid tolerant. The chemical sanitizers utilized included a sodium hypochlorite solution (bleach), a peroxyacetic acid compound (Zep-PerosanTM), a quaternary ammonium compound (Zepamine-A™), and an iodine compound (Zep-I-Dine™). The concentration level for each sanitizer was adjusted depending upon the ability of the E. coli 0157:H7 isolates to survive treatment. Data from the planktonic stage of testing showed that lack of recovery by plate count method after sanitizer treatment did not mean that an organism was no longer present. All of the sanitizers except Zep-Perosan™ demonstrated at least a five-log reduction. However, both strains demonstrated the ability to fully recover and grow in broth after treatment with all of the sanitizers. Zep-I-Dine™ was found to be the most effective sanitizer for planktonic Escherichia coli 0157:H7 bacteria followed by 71

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72 Clorox® and then Zepamine-A^^'^. At the concentration used in this study, ZepPerosan™ was not found to be effective. Bacteria that survived the planktonic stage of testing were reassessed in regards to their minimum inhibitory concentration, sub-lethal and lethal concentration. No changes were found in the minimum inhibitory concentration for either bacterial strain when exposed to Clorox® or Zep-I-Dine™. Substantial increases were found in the minimum inhibitory concentration levels for both bacterial strains for Zepamine-A™ and ZepPerosan™. The repeated sub-lethal level of exposure with both sanitizers resulted in ability of the bacterial strains to withstand higher levels of the sanitizer(s), indicating that some kind of adaptation had occurred. With one exception, the increased minimum inhibitory concentrations for the two bacterial strains failed to provide any crossprotection. The FSIS 063-93 strain when pre-treated with sub-lethal levels of ZepamineA™ seemed to invoke a response which allowed the bacteria to survive exposure to a higher level of Zep-I-Dine™. Data from the adherent phase of testing supports previous research indicating that microorganisms become more resistant to sanitizers when they are attached to a surface. Zep-I-Dine™ was the only sanitizer in this study that was effective against both planktonic and adherent bacterial isolates. Both Zepamine-A™ and Clorox® which were effective against the planktonic bacteria were not effective against adherent bacteria. Zep-Perosan™ was not found to be effective against either planktonic or adherent bacteria. The information obtained in this study suggests that disinfectants and sanitizers tested in suspension for use in the industry may not correlate with results obtained on

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73 surfaces and perhaps even less in cases where a biofilm has developed. This study also indicates that methodology for recovery of bacteria is important. Plate count data may not be a reliable indication of the cleanliness of a surface. As chemical sanitizers are commonly utilized by the food industry as a tool for improving food quality and safety, it is essential to be able to accurately identify and quantify the bacterial organisms that may be present before and after cleaning and sanitation. While this study shows that organisms may still be present after sanitation, it is important to remember that the concentration levels utilized were minimal values not the recommended levels for sanitation or disinfection. Currently, no data exists to suggest that the proper use of sanitizers in the food industry will lead to development of highly resistant microorganisms. However, the potential for an organism to adapt does exist. Therefore it is essential for the food industry to know as much as possible about the effectiveness of sanitizers and the microorganisms they are designed to eliminate or reduce to acceptable levels. Further research opportunities presented by this study may include: 1) a study involving the use of additional bacterial strains; 2) the study of the effects of the sanitizing agents on both planktonic and adherent bacterial cells when using the recommended concentration instead of sub-lethal levels; 3) a comparative study of sublethal and recommended concentrations of the sanitizing agents; 4) the effect of sublethal exposure of the sanitizing agents on other food processing surfaces; 5) the combinations of manual or mechanical scrubbing and sub-lethal levels of the sanitizing agents 6) the investigation into the membrane of the Escherichia coli 0157:H7 bacterial isolates that exhibited increases in their minimum inhibitory concentration levels; and 7)

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74 the study of alternative enumeration methods to present a more accurate picture of the actual levels of bacteria remaining after treatment with various sanitizers.

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BIOGRAPHICAL SKETCH Kristen Ann Goodfellow-Hunt was bom on July 14, 1968, in Austin, Minnesota. She attended Santa Fe Community College of Gainesville, Florida, where she received an Associate of Arts degree with high honors in the summer of 1989. She then transferred to the University of Florida and was awarded a Bachelor of Arts in Education with honors in the fall of 1992. She was admitted to graduate school in the spring of 1993 and received a Master of Education degree with a concentration in science in the spring of 1994. After teaching for several years, Kristen decided in the spring of 1998 to return to the University of Florida part-time, while continuing to teach, in order to further her own education. In the fall of 1999 she was awarded an Animal Sciences Department assistantship and thus decided to return to the University of Florida in pursuit of a Doctor of Philosophy degree in the College of Agricultural and Life Sciences on a full-time basis. She was admitted to candidacy on March 26, 2001. Kristen earned her Doctor of Philosophy degree from the University of Florida in the summer of 2003. Upon receiving her Doctor of Philosophy degree, Kristen plans to work in industry as a laboratory manager for a food testing company. This career choice should allow her to manage the lab, do consulting, and teach both microbiology and HACCP short courses. 86

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fially adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Roge(X)west, Chair Professor Emeritus of Animal Sciences I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosoj Dwain D. Johnsc Professor of Animal Sciences I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosopfhy. Sally K. WiUiams Associate Professor of Animal Sciences I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosof Gary E. Rodric Professor of?4ibd Science and Human Nutrition This dissertation was submitted to the Graduate Faculty of the College of Agricultural and Life Sciences and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doct or of Philosoph y. , August 2003 <^~. ^Q ^^ ^^-T"^-^ Dean, College of Agricultural anosLife Sciences Dean, Graduate School