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Rabold Denise Dissertation.Pdf Aus dem Institut für Mikrobiologie und Tierseuchen des Fachbereichs Veterinärmedizin der Freien Universität Berlin Occurrence and characterization of Clostridioides difficile in small companion animals and their owners Inaugural-Dissertation zur Erlangung des Grades eines Doktors der Veterinärmedizin an der Freien Universität Berlin vorgelegt von Denise Rabold, geb. Hildebrand Tierärztin aus Berlin Berlin, 2019 Journal-Nr. 4122 Gedruckt mit Genehmigung des Fachbereichs Veterinärmedizin der Freien Universität Berlin Dekan: Univ.-Prof. Dr. Jürgen Zentek Erster Gutachter: Prof. Dr. Lothar H. Wieler Zweiter Gutachter: Univ.-Prof. Dr. Barbara Kohn Dritter Gutachter: Univ.-Prof. Dr. Thomas Alter Deskriptoren (nach CAB Thesaurus): dogs, cats, man, Clostridium difficile, faeces, polymerase chain reaction, ribotyping, prevalence, epidemiology, Germany Tag der Promotion: 17.09.2019 A wise man proportions his belief to the evidence. David Hume TABLE OF CONTENTS Table of Contents List of Figures ............................................................................................................................ ii List of Tables ............................................................................................................................. iii List of Abbreviations ................................................................................................................. iv 1. Introduction ......................................................................................................................... 1 2. Literature Review ................................................................................................................ 3 2.1. Taxonomy .................................................................................................................... 3 2.2. Historical background .................................................................................................. 3 2.3. Characteristics of C. difficile and CDI ......................................................................... 5 2.4. C. difficile in veterinary medicine .............................................................................. 11 2.5. Food as a possible source of infection ....................................................................... 19 3. Material ............................................................................................................................. 23 3.1. Project pseudonym and symbol ................................................................................. 23 3.2. Data collection ........................................................................................................... 23 3.4. Sample collection ....................................................................................................... 31 4. Methods ............................................................................................................................. 34 4.1. Partners involved in the interdisciplinary project ...................................................... 34 4.2. Ethics approval ........................................................................................................... 34 4.3. Data acquisition .......................................................................................................... 35 4.4. Microbiological examination ..................................................................................... 41 5. Results ............................................................................................................................... 43 5.1. Study participant recruitment ..................................................................................... 43 5.2. Microbiological examination ..................................................................................... 44 5.3. Risk assessment .......................................................................................................... 52 6. Discussion ......................................................................................................................... 64 6.1. Microbiological examination vs. diagnostical detection of C. difficile ..................... 64 6.2. Isolation rates and molecular characteristics of C. difficile isolates .......................... 64 6.3. Epidemiological factors associated with C. difficile isolation in dogs, cats and their owners ........................................................................................................................ 69 7. Summary ........................................................................................................................... 73 8. Zusammenfassung ............................................................................................................. 74 9. Reference List ................................................................................................................... 76 10. Appendix .............................................................................................................................. I 11. Publication List ...............................................................................................................XIV 12. Danksagungen .................................................................................................................XVI 13. Selbstständigkeitserklärung ............................................................................................XVII i LIST OF FIGURES List of Figures Figure 1: Ribosomal RNA operon (rrn) from C. difficile .......................................................... 8 Figure 2: Project pseudonym and symbol ................................................................................ 23 Figure 3: Part 1 of the questionnaire requesting general information about animal contacts .. 25 Figure 4: Field for the animal ID belonging to part 2 of the questionnaire ............................. 25 Figure 5: Part 2 of the questionnaire requesting information about the animal participant ..... 26 Figure 6: Field for the animal owner ID belonging to part 3 of the questionnaire .................. 28 Figure 7: Part 3 of the questionnaire requesting information about the animal owner ............ 29 Figure 8: Part 4 of the questionnaire requesting contact allowance from the animal owner ... 31 Figure 9: Distribution of participating households in the federal states of Germany .............. 44 Figure 10: Isolation rates of C. difficile in different participant groups ................................... 45 Figure 11: Minimum spanning tree of 44 C. difficile isolates originating from animal and human participants typed by multiple-locus variable number tandem-repeat analysis (MLVA). .................................................................................................... 51 ii LIST OF TABLES List of Tables Table 1: Variables created for animal participants ................................................................... 39 Table 2: Variables created for human participants ................................................................... 40 Table 3: Return rates on various participant recruitment routes .............................................. 43 Table 4: C. difficile isolation and characterization results ....................................................... 46 Table 5: Ribotypes and detection of toxin genes in C. difficile isolates .................................. 47 Table 6: MLVA results for animal C. difficile isolates ............................................................ 48 Table 7: MLVA results for human C. difficile isolates ............................................................ 49 Table 8: Characteristics of two C. difficile pairs isolated from four cats from two independent households ............................................................................................................... 50 Table 9: Univariate analysis for faecal shedding of C. difficile in dogs and cats .................... 53 Table 10: Distinctive characteristics from the univariate analysis for faecal shedding of C. difficile in dogs ........................................................................................................ 56 Table 11: Distinctive characteristics from the univariate analysis for faecal shedding of C. difficile in cats ......................................................................................................... 57 Table 12: Univariate analysis for faecal shedding of C. difficile in animal owners................. 58 Table 13: Multivariate analysis for faecal shedding of C. difficile in dogs and cats................ 62 Table 14: Multivariate analysis for faecal shedding of C. difficile in dogs.............................. 62 Table 15: Multivariate analysis for faecal shedding of C. difficile in cats ............................... 62 Table 16: Multivariate analysis for faecal shedding of C. difficile in animal owners .............. 63 iii LIST OF ABBREVIATIONS List of Abbreviations AAC antibiotic-associated colitis AAD antibiotic-associated diarrhoea ADR European Agreement concerning the International Carriage of Dangerous Goods by Road BMBF Bundesministerium für Bildung und Forschung (German Federal Ministry of Education and Research) bp basepairs CA-CDI community-acquired Clostridioides difficile infection C. difficile Clostridioides difficile CDAD Clostridioides difficile-associated diarrhoea CDI Clostridioides difficile infection CDT CD ADP-ribosyltransferase, meaning the binary toxin
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