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Detection of Shigella and Enteroinvasive Escherichia Coli, the Causative Agents of Bacillary Dysentery from Environmental and Clinical Samples

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Detection of Shigella and Enteroinvasive Escherichia Coli, the Causative Agents of Bacillary Dysentery from Environmental and Clinical Samples EVELYN DÓRA SZAKÁL Detection of Shigella and enteroinvasive Escherichia coli, the causative agents of bacillary dysentery from environmental and clinical samples Ph.D. DISSERTATION PTE AOK Faculty of Medicine, University of Pécs Department of Clinical Microbiology and Immunology Hungary Pécs 2003 EVELYN DÓRA SZAKÁL Detection of Shigella and enteroinvasive Escherichia coli, the causative agents of bacillary dysentery from environmental and clinical samples Ph.D. DISSERTATION Title of PhD Programme: Molecular pathogenesis of infectious diseases Programme leader: Professor Levente Emődy, M.D., Ph.D., D.Sc. Supervisor: Professor Tibor Pál, M.D., Ph.D., D.Sc. PTE AOK Faculty of Medicine, University of Pécs Department of Clinical Microbiology and Immunology Hungary Pécs 2003 To my brother, my parents and my grandparents CONTENTS ABBREVIATIONS........................................................................................................8 1 INTRODUCTION ......................................................................................................9 2 REVIEW OF THE LITERATURE ........................................................................11 2.1. THE SIGNIFICANCE OF BACILLARY DYSENTERY IN THE PAST AND TODAY.............11 2.2. THE CAUSATIVE AGENTS OF BACILLARY DYSENTERY, BIOCHEMICAL AND SEROLOGICAL CHARACTERISTICS ........................................................................13 2.3. CLINICAL FEATURES OF BACILLARY DYSENTERY .................................................16 2.4. IMMUNITY............................................................................................................18 2.5. VIRULENCE AND PATHOGENESIS OF SHIGELLA AND EIEC STRAINS......................18 2.6. EPIDEMIOLOGY OF BACILLARY DYSENTERY.........................................................28 2.6.1. Epidemiology of Shigella and EIEC ............................................................28 2.6.2. Waterborne diseases, pathogen microbes in water .....................................30 2.6.3. Shigella and EIEC as waterborne pathogens..............................................31 2.6.4. Food poisonings, pathogen microbes in food..............................................33 2.6.5. Shigella and EIEC as food borne pathogens...............................................36 2.7. THERAPY, PREVENTION AND CONTROL OF BACILLARY DYSENTERY.....................37 2.7.1. Treatment with antibiotics, antibiotic resistance ........................................37 2.7.2. Vaccine development ...................................................................................38 2.7.3. Personal hygiene, water and food safety.....................................................40 2.8. LABORATORY DIAGNOSIS IN SHIGELLA AND EIEC INFECTIONS............................43 2.8.1. Traditional methods.....................................................................................43 2.8.2. Molecular methods ......................................................................................48 2.8.3. Immunological methods...............................................................................50 3 OBJECTIVES OF THE STUDY.............................................................................53 4 MATERIALS AND METHODS .............................................................................54 4.1. BACTERIAL STRAINS (PAPERS I-III)......................................................................54 4.2. MEDIA (PAPERS I-III)...........................................................................................54 4.3. MAIC-1 ANTIBODY (PAPERS I-III).......................................................................55 4.4. VIRULENCE TESTS VERIFYING ENTEROINVASIVE STRAINS (PAPERS I-III) .............55 4.4.1. IpaC ELISA..................................................................................................55 4.4.2. Plasmid electrophoresis...............................................................................56 4.4.3. Cell invasion test..........................................................................................56 4.4.4. Guinea pig keratoconjunctivitis test (Serény test).......................................57 4.5. PREPARATION OF THE BACTERIAL CULTURES FOR THE ARTIFICIAL CONTAMINATION OF SAMPLES (PAPER I-III)....................................................................................57 4.6. THE COLONY IMMUNOBLOT METHOD (PAPERS I-III) ............................................57 4.7. TESTING WATER SAMPLES (PAPER I) ....................................................................58 4.8. OPTIMISING THE COLONY IMMUNOBLOT METHOD FOR FAECAL SAMPLES (PAPER II) ............................................................................................................................58 4.9. ELIMINATION OF THE ENZYME ACTIVITY OF FAECAL SAMPLES (PAPER II)............59 4.10. ARTIFICIAL CONTAMINATION OF FAECAL SAMPLES (PAPER II)...........................60 4.11. BACTERIOLOGICAL EXAMINATION OF CLINICAL FAECAL SPECIMENS (PAPER II) 60 4.12. TESTING MILK SAMPLES.....................................................................................61 4 4.13. IPAH PCR (PAPERS I-III)...................................................................................61 4.14. DNA HYBRIDISATION (PAPER I).........................................................................62 4.15. GROWTH OF ISOLATED STRAINS ON SOLID MEDIA (PAPER III) ............................62 4.16. GROWTH OF ISOLATED STRAINS IN LIQUID MEDIA..............................................63 4.17. ARTIFICIAL CONTAMINATION AND SELECTIVE CULTURE OF FAECAL SAMPLES (PAPER III) ..........................................................................................................63 4.18. ASSESSMENT OF SELECTIVE MEDIA IN THE RECOVERY OF EIEC FROM CLINICAL FAECAL SAMPLES (PAPER III) ..............................................................................64 4.19. STATISTICAL ANALYSIS (PAPERS I-III)...............................................................65 5 RESULTS ..................................................................................................................66 5.1. DEVELOPING THE SHIGELLA /EIEC SPECIFIC COLONY IMMUNOBLOT METHOD (PAPER I).............................................................................................................66 5.2. APPLYING THE COLONY IMMUNOBLOT METHOD FOR DIFFERENT CLINICAL AND ENVIRONMENTAL SAMPLES .................................................................................68 5.2.1. Applying the colony immunoblot method for water samples (paper I) .......68 5.2.2. Applying the colony immunoblot method for faecal samples (paper II) .....71 5.2.3. Applying the colony immunoblot method for milk samples.........................76 5.3. SELECTIVE AND ENRICHMENT MEDIA FOR CULTURING EIEC (PAPER III).............78 5.3.1. Growth of EIEC and other isolated strains as pure culture........................78 5.3.2. Recovery of EIEC from artificially contaminated faecal samples...............80 6 DISCUSSION............................................................................................................84 6.1. DIAGNOSTIC METHODS PRESENTLY AVAILABLE FOR THE DETECTION OF SHIGELLA AND EIEC STRAINS (PAPERS I-III) ......................................................................84 6.2. DEVELOPING THE COLONY IMMUNOBLOT METHOD AND ITS APPLICATION TO ENVIRONMENTAL AND CLINICAL SAMPLES (PAPERS I, II) ....................................86 6.3. APPLICATION OF THE COLONY IMMUNOBLOT METHOD FOR WATER SAMPLES (PAPER I).............................................................................................................86 6.4. APPLICATION OF THE COLONY IMMUNOBLOT METHOD FOR FAECAL SAMPLES (PAPER II)............................................................................................................87 6.5. APPLICATION OF THE COLONY IMMUNOBLOT METHOD FOR MILK SAMPLES..........90 6.6. SUMMARY OF THE APPLICATION OF THE COLONY BLOT METHOD FOR ENVIRONMENTAL AND CLINICAL SAMPLES (PAPERS I, II) ....................................90 6.7. SELECTIVE AND ENRICHMENT MEDIA IN THE DIAGNOSIS OF BACILLARY DYSENTERY (PAPER III) ..........................................................................................................91 7 CONCLUSIONS .......................................................................................................96 8 SUMMARY ...............................................................................................................97 9 ÖSSZEFOGLALÁS..................................................................................................99 ACKNOWLEDGEMENTS ......................................................................................103 REFERENCES...........................................................................................................105 ORIGINAL PUBLICATIONS .................................................................................124 5 LIST OF ORIGINAL COMMUNICATIONS This PhD dissertation is based on the following original communication (I-III.) The papers are referred to in the text by their Roman numerals. In addition, some previously unpublished data are also presented. A. Published articles I. Szakál D., I. Gadó, T. Pál (2001): A colony blot immunoassay to detect enteroinvasive Escherichia coli and Shigella in water samples. Journal of Applied Microbiology 90 (2): 229-236 II. Szakál D., Gy. Schneider, T. Pál (2003): A colony blot immune assay to identify enteroinvasive Escherichia
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