UNIVERSITY OF CINCINNATI Date:___________________ I, _________________________________________________________, hereby submit this work as part of the requirements for the degree of: in: It is entitled: This work and its defense approved by: Chair: _______________________________ _______________________________ _______________________________ _______________________________ _______________________________ Antibiotic Therapy in the Treatment of E. coli O157:H7. A dissertation submitted to the Division of Graduate Studies and Research of the University of Cincinnati In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY (Ph.D.) In the Department of Molecular Genetics, Biochemistry, & Microbiology of the College of Medicine 2008 Colleen M. McGannon B.S., Ashland University, 2002 Committee Chair: Alison A. Weiss, Ph.D. Abstract Escherichia coli O157:H7 causes an estimated 73,000 of food borne illness annually. Varying levels of disease severity exist and include diarrhea, bloody diarrhea, and hemolytic uremic syndrome which can result in kidney damage or death. E. coli O157:H7 produces Shiga toxins 1 and/or 2, although it is Shiga toxin 2 that is linked to severe disease. Currently, a Shiga toxin-producing isolate will yield a positive diagnostic result, regardless of which Shiga toxin variant is produced. We have developed an ELISA that can differentiate between Shiga toxin 1 and Shiga toxin 2 in the presence of fecal material, and if used in the clinical setting, can increase the accuracy of prognosis for a patient infected with E. coli O157:H7. Epidemiological studies have suggested that antibiotics may be linked to HUS development. As such, we have completed an extensive study to determine which, if any, antibiotics are safe for the treatment of E. coli O157:H7. The data suggest that antibiotic-mediated Shiga toxin induction or reduction is based on the mechanism. Antibiotics that target DNA increase Shiga toxin production while protein synthesis inhibitors decrease Shiga toxin production. Human intestinal E. coli have been shown to amplify Shiga toxin. Our data suggest that protein synthesis inhibitors may be effective, even if the patient harbors a Shiga toxin-amplifying isolate, provided that the commensal isolate is susceptible to the antibiotic. Conversely, antibiotics would likely be of no benefit if the Shiga toxin-amplifier is resistant to the antibiotic. Future studies should focus on characterizing the flora of HUS patients, and examining antibiotic treatments within the context of these strains. iii iv Follow your passion. ~ Alison A. Weiss v Acknowledgements Alison said, in a heart-warming fashion, Colleen, I encourage you to follow your passion. Kind in allowing me this latitude, there’s Not enough thanks and/or gratitude. Of course my committee supported me too, Willingly sharing their thoughts based on their point of view. Lyndsay, Mark, Shantini, Mike, Cindy, and Scott Eternally treasured, never forgot. Dad and Mom were supportive, and helped keep me sane. Goodness, I know it, bacteria must have a brain! Exuding such patience as the time it did crawl. Mr. O’Malley often took me to the Serpentine Wall. Emily Bradford, my friend, the dearest, the best. We’re Not really that different, who would have guessed? To RIVER and Skyline and St Monica St George. Sad to leave Cincinnati, but now to Albuquerque I’ll forge. vi Table of Contents Abstract……………………………………………………………………..………...iii Acknowledgements……………………………………………………………………v Table of Contents………………………………………………………………….....vi List of Figures and Tables……………………………………………………..…….ix List of Abbreviations…………………………………………………………..……..xi Chapter I. E. coli O157:H7 literature review………………………………..……..1 Introduction 2 E. coli O157:H7 is Associated with Disease 3 Food borne Transmission 4 Other Routes of Transmission 5 National Monitoring Systems 9 Inspection Protocols 11 Disease 13 Shiga toxins 15 Discovery 15 Toxin Nomenclature 16 Genetics and Expression 16 Structure 18 Toxin Receptor (Gb3) 20 Toxin-Gb3 Interaction 23 Toxin-Gb3 Internalization 27 Shiga toxin 2 Glycosidase Activity 28 Antibiotic Treatment of E. coli O157:H7 29 Ciprofloxacin 30 Trimethoprim/Sulfamethoxazole 34 Ampicillin 36 Ceftriaxone 37 Fosfomicin 38 Gentamicin 39 Doxycyline 39 Azithromycin 40 Rifampicin 41 vii Role of Normal Intestinal Flora in Disease 42 Lipopolysaccharide Fecal-Isolate 29 45 Laboratory Assays 48 Sorbitol MacConkey Agar 48 Latex Agglutination Assay 48 Polymerase Chain Assay 49 Western blot Assay 50 Rationale for Studies Performed 51 Chapter II. Glycoconjugates that specifically bind Stx1 or Stx2………………....53 Abstract 54 Introduction 55 Materials and Methods 59 Results 62 Discussion 67 Chapter III. Antibiotic treatment to prevent the systemic complications of Escherichia coli O157:H7 Infection………………………………………………...71 Abstract 72 Author Summary 74 Introduction 75 Materials and Methods 79 Results 85 Discussion 102 viii Chapter IV. Discussion………………………………………………………. 106 Summary 107 Future Directions 108 Concluding Remarks 113 References……………………………………………………………………… 114 ix Figures and Tables Chapter I. Figure 1. The structure of Stx. (Page 19) Figure 2. The structure of Gb3. (Page 21) Figure 3. Sequence alignment of the Stx1 and Stx2 B subunits. (Page 26) Figure 4. The structure of lipopolysaccharide (LPS). (Page 47) Chapter II. Figure 1. Structure of Gb3 and the O117 LPS. (Page 57) Figure 2. Representation of the tailored biotinylated glyconjugate and the structures of the five molecules. (Page 58) Figure 3. Antibodies specific to O107 and O117 block Stx2 binding to LPS. (Page 64) Figure 4. Differential binding of Stx1 or Stx2 to synthetic glycoconjugates. (Page 66) Chapter III. Table 1. Plasmids and strains used in this study. (Page 84) Figure 1. The Luc2p-MigR1 retroviral vector. (Page 87) Figure 2. Quantifying Shiga toxin production. (Page 88) Table 2. The Minimum Inhibitory Concentrations (MIC) of antibiotics. (Page 90) Figure 3. The effect of ciprofloxacin and trimethoprim/sulfamethoxazole on the production of Stx. (Page 92) Figure 4. Antibiotics that induce production of Stx. (Page 93) Figure 5. Determining if antibiotics induce Stx1, Stx2, or both in E. coli O157:H7 strain PT-32 (stx1, stx2). (Page 95) Figure 6. Determining the effect of antibiotics on the production of Stx. (Page 96) Figure 7. The efficacy of antibiotics in the presence of a susceptible amplifer strain. (Page 99) x Figure 8. The efficacy of antibiotics in the presence of a resistant amplifier strain. (Page 101) xi Abbreviations CAT scan computer axial tomography scan CIP ciprofloxacin CDC Centers for Disease Control E. coli Escherichia coli ED50 effective dose 50% ELISA enzyme-linked immunosorbent assay EHEC enterohemorhagic Escherichia coli EPEC enteropathogenic Escherichia coli FDA Food and Drug Administration FI fecal isolate Gb3 globotriaosylceramide GFP green fluorescent protein HUS hemolytic uremic syndrome HuSAP human serum amyloid P IL interleukin kDa kilodalton KDEL lysine-glutamic acid-aspartic acid-lysine KDO 3-deoxy-D-manno octulosonic acid LB Luria Bertani LEE Locus of Enterocyte Effacement LPS lipopolysaccharide MH Mueller-Hinton MIC minimum inhibitory concentration PEST proline-glutamic acid-serine-threonine PBS phosphate buffered saline RPLA reversed passive latex agglutination RT-PCR reverse transcriptase-polymerase chain reaction SDS-PAGE sodium dodecyl sulfate poly acrylamide gel electrophoresis SAP serum amyloid P SOS save-our-ship Stx Shiga toxin Tir translocated intimin receptor TMP-SMX trimethoprim-sulfamethoxazole USDA United States Department of Agriculture xii Chapter I. Literature Review 1 INTRODUCTION Escherichia coli is a Gram-negative bacterium present within the human microbiota. E. coli is a component of animal gut flora as well. For example, cattle are natural carriers of E. coli serotype O157:H7 [1-3]. While adult cattle harbor this strain asymptomatically, [4], E. coli O157:H7 is responsible for a likely underestimated 73,000 cases of human disease per year, and is the primary cause for the development of hemorrhagic colitis and hemolytic uremic syndrome (HUS) in children [5]. First associated with disease in 1982 [6] [7], E. coli O157:H7 is classified as a newly emerging food borne pathogen, with ground beef being the primary source for human infection. After ingestion of an infectious dose as low as 50 organisms, the bacteria colonize the large intestine, causing a disruption of the microvilli. Subsequently, the bacteria produce its major virulence factors called Shiga toxins, which inhibit protein synthesis within target cells. Unfortunately, antibiotic treatment has been linked to the development of HUS in children infected with E. coli O157:H7 [8]. Therefore, the current treatments are limited to supportive therapies such as rehydration. This review will first provide background information regarding E. coli O157:H7 and its Shiga toxins, then introduce topics related to the two major courses of experimental study reported in this document. Those topics include the development of an assay that can distinguish between the two types of Shiga toxin, and in addition determining which, if any, antibiotics are safe for use in the treatment of E. coli O157:H7. 2 E. COLI O157:H7 IS ASSOCIATED WITH DISEASE E. coli O157:H7 was initially isolated in 1975 from a sporadic case of hemorrhagic colitis. This strain was not seen again until 1983 when it was first linked to disease as a food borne pathogen [7]. Two groups of patients, one in Oregon and one in Michigan, displayed