Genotyping and Genomotyping of Tropheryma Whipplei – the Causative Agent of Whipple's Disease»

Genotyping and Genomotyping of Tropheryma Whipplei – the Causative Agent of Whipple's Disease»

Aus der Klinik für Innere Medizin der Medizinischen Fakultät Charité – Universitätsmedizin Berlin und der Unité de recherche sur les Maladies infectieuses et tropicales émergentes Marseille DISSERTATION «Genotyping and Genomotyping of Tropheryma whipplei – The Causative Agent of Whipple's Disease» zur Erlangung des akademischen Grades Doctor medicinae (Dr. med.) vorgelegt der Medizinischen Fakultät Charité – Universitätsmedizin Berlin von Nils Wetzstein aus Hanau Datum der Promotion: 08.12.2017 1 Table of Contents 1. Abstracts.................................................................................................................................................7 1.1. Abstract...........................................................................................................................................7 1.2. Zusammenfassung..........................................................................................................................7 2. Introduction...........................................................................................................................................9 2.1. Historical introduction....................................................................................................................9 2.2. Epidemiology and transmission......................................................................................................9 2.3. Clinical manifestations.................................................................................................................10 2.4. Antibiotic treatment......................................................................................................................12 2.5. Diagnostics...................................................................................................................................12 2.6. Bacteriology and phylogeny.........................................................................................................13 2.7. Bacterial typing systems...............................................................................................................14 2.8. Geographical distribution and disease outcome............................................................................14 2.9. Bacterial genomics and application in TW...................................................................................15 2.10. Bacterial core and pan-genomes.................................................................................................15 2.11. Problems addressed in this study................................................................................................16 3. Materials & Methods...........................................................................................................................17 3.1. Utilized reagents and devices........................................................................................................17 3.2. Strains of TW................................................................................................................................19 3.3. Axenic culture of TW...................................................................................................................19 3.4. Harvesting....................................................................................................................................19 3.5. Verification of purity on cellular level..........................................................................................20 3.5.1. Classical staining methods...................................................................................................21 3.5.2. Immunofluorescence...........................................................................................................21 3.6. DNA-extraction............................................................................................................................21 3.7. Verification of purity on the DNA-level.......................................................................................22 3.8. Genotyping based on HVGS-regions............................................................................................23 2 3.9. Epidemiological, phylogenetic and geographical analysis of typing data.....................................25 3.10. Basic genomic data and sequencing............................................................................................26 3.11. Genomic analysis........................................................................................................................26 3.12. Geographic distribution of sequenced genomes..........................................................................27 3.13. Calculation of core and pan-genome...........................................................................................28 4. Results...................................................................................................................................................29 4.1. Patients' characteristics and clinics...............................................................................................29 4.2. Genotyping of remaining strains...................................................................................................29 4.3. Analysis of genotyping data in patient group including prior genotyping results..........................30 4.4. Sequenced genomes, genome length and GC-content...................................................................32 4.5. Geographic distribution of sequenced genomes and ANI distance matrix....................................32 4.6. Phylogenetic analysis of sequenced strains using genotyping data...............................................35 4.7. Phylogenetic analysis of sequenced strains using whole-genomes...............................................35 4.8. ANI-comparison of most important genotypes and different clinical entities...............................36 4.10. Comparison of TW with other intracellular pathogenic bacteria and other species in the group of Actinobacteria......................................................................................................................................37 4.11. Comparison of RAST annotation in TW strains..........................................................................39 4.12. Prediction of antibiotic resistance genes and phage DNA...........................................................39 4.13. Core-genome and pan-genome...................................................................................................40 4.14. WiSPs in the core and pan-genome.............................................................................................41 5. Discussion.............................................................................................................................................43 5.1. Patients' characteristics and clinics...............................................................................................43 5.2. Analysis of genotyping data..........................................................................................................43 5.3. Sequenced genomes, genome length and GC-content...................................................................44 5.4. Geographic distribution of sequenced genomes............................................................................45 5.5. Phylogenetic analysis of sequenced strains...................................................................................46 5.6. Core and pan-genome...................................................................................................................46 5.7. Host and pathogen factors in WD.................................................................................................47 3 5.8. Prediction of antibiotic resistance genes: fluoroquinolone resistance gene...................................48 5.9. Limitations of this study...............................................................................................................48 5.10. Conclusion..................................................................................................................................48 6. Bibliography.........................................................................................................................................50 7. Eidesstattliche Erklärung....................................................................................................................55 8. Anteilserklärung an erfolgten Publikationen.....................................................................................56 9. Curriculum Vitae.................................................................................................................................57 10. Acknowledgments..............................................................................................................................58 4 Abbreviations AC asymptomatic carriers / asymptomatic carriage ANI average nucleotide identity APHM Assistance Publique – Hôpitaux de Marseille BCYE-Agar buffered charcoal yeast extract agar BDBH bidirectional best hit algorithm BLAST basic local alignment search tool CDC Centers for Disease Control and Prevention COG cluster of orthologous groups COS-Agar Columbia agar with 5% sheep blood CSF cerebrospinal fluid CWD classical Whipple’s disease DMEM F12 Dulbecco's modified Eagle medium F12 DNA deoxyribonucleic acid NW neurological manifestations due to Whipple's

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