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Phenotypic and Genotypic Properties of Vibrio Cholerae Non-O1, Non-O139 Isolates Recovered from Domestic Ducks in Germany

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Phenotypic and Genotypic Properties of Vibrio Cholerae Non-O1, Non-O139 Isolates Recovered from Domestic Ducks in Germany Characterization of Vibrio spp. isolates from Germany by reference to human clinical isolates using phenotypic and WGS-assisted genotypic methods Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry, Pharmacy of Freie Universität Berlin by Keike Schwartz Berlin 2020 This dissertation was prepared under supervision of Dr. Eckhard Strauch at the Vibrio consiliary laboratory, Department Biological Safety, German Federal Institute for Risk Assessment. May 2016 to December 2020 First examiner: Prof. Dr. Karsten Nöckler Second examiner: Prof. Dr. Jens Rolff Disputation: 05/05/2021 Danksagung An dieser Stelle möchte ich all denjenigen danken, die mich auf meinem Weg zur Promotion begleitet haben. Mein größter Dank gilt Dr. Eckhard Strauch für die Ermöglichung und engagierte Betreuung dieser Doktorarbeit am Konsiliarlabor für Vibrionen sowie seine immerwährende uneingeschränkte Unterstützung. Ganz herzlich bedanke ich mich bei Prof. Dr. Karsten Nöckler und Prof. Dr. Jens Rolff für die Begutachtung dieser Arbeit. Mein besonderer Dank gilt auch Dr. Martin Richter für die Möglichkeit, diese kumulative Dissertation in seiner Fachgruppe anfertigen zu können. Ich danke Dr. Claudia Metelmann und allen Mitarbeitern des IMD Greifswald für die Bereitstellung aktueller klinischer Vibrio-Isolate aus Humanproben sowie Dr. Nadja Bier und Dr. Jens André Hammerl für die bereitwillige Weitergabe wertvoller Erfahrungen zu sequenzbasierten bioinformatischen Analysen. Besonders herzlich möchte ich mich bei Dr. Claudia Jäckel, Cornelia Göllner, Nicole vom Ort und Jonas Nekat aus dem Vibrio-Team für die angenehme, kollegiale Atmosphäre sowie ihre Hilfsbereitschaft und stetige Unterstützung bedanken. In diesem Zusammenhang sei auch allen übrigen Kolleginnen und Kollegen der Fachgruppe 45 für die schöne gemeinsame Zeit gedankt. Mein weiterer Dank gilt Bruno Weser, der stets auf alle großen und kleinen IT-Fragen und -Probleme eine Antwort hatte. Abschließend möchte ich meinen Eltern und meiner Schwester Kerrin danken, die mir in allen Lebenslagen den Rücken gestärkt haben und ohne die ich wahrscheinlich nicht dort stehen würde, wo ich heute bin. III Table of Contents Table of Contents Danksagung............................................................................................................................ III List of Abbreviations .............................................................................................................. VI 1 Introduction ....................................................................................................................... 10 1.1 Vibrio cholerae and Vibrio navarrensis ........................................................................... 10 1.1.1 Taxonomy and Characteristics .............................................................................. 10 1.1.2 Isolation and Identification ................................................................................... 12 1.1.3 Habitats and Ecology ............................................................................................ 13 1.1.4 Virulence Factors .................................................................................................. 17 1.1.5 Human Infections and Infection Epidemiology .................................................... 25 1.2 Aims of the Study ............................................................................................................. 28 2 Publications ........................................................................................................................ 29 2.1 List of Publications and Own Contribution ...................................................................... 29 2.2 Publication 1 ..................................................................................................................... 30 2.3 Publication 2 ..................................................................................................................... 47 3 General Discussion ............................................................................................................ 61 3.1 Background ...................................................................................................................... 61 3.2 Characterization of Environmental Vibrio cholerae Isolates from Germany by Reference to Human Clinical Isolates .............................................................................. 62 3.3 Characterization of Veterinary Vibrio navarrensis Isolates From Germany by Reference to Environmental and Human Clinical Isolates .............................................. 71 3.4 Vibrio spp. Surveillance in Germany and Future Perspectives ........................................ 74 4 Summary ............................................................................................................................ 77 5 Zusammenfassung ............................................................................................................. 79 6 References .......................................................................................................................... 81 IV Table of Contents 7 Appendix .......................................................................................................................... 109 7.1 Supplementary Material ................................................................................................. 109 7.1.1 Publication 1 ....................................................................................................... 109 7.1.2 Publication 2 ....................................................................................................... 131 7.2 Publication 3 ................................................................................................................... 137 7.3 Publications and Congress Presentations ....................................................................... 155 7.4 Curriculum Vitae ............................................................................................................ 157 7.5 Eidesstattliche Erklärung ................................................................................................ 159 V List of Abbreviations List of Abbreviations ACD actin cross-linking domain Acf accessory colonization factor ADP adenosine diphosphate AI-2 autoinducer-2 BON domain bacterial OsmY and nodulation domain bp base pair(s) BPI bactericidal/permeability-increasing protein BWM Ballast Water Management Caco-2 human epithelial colon adenocarcinoma cell line CAI-1 cholera autoinducer-1 cAMP cyclic adenosine monophosphate CDC Centers for Disease Control and Prevention CFU colony forming unit(s) Changliver HeLa derivative ChxA cholix toxin cps gene encoding a putative capsule biosynthesis protein CqsA CAI-1 autoinducer synthase CqsS CAI-1 autoinducer receptor CTX cholera toxin ctxA gene encoding the A subunit of the cholera toxin CTXΦ lysogenic filamentous bacteriophage whose genome encodes the cholera toxin dctM, dctP, dctQ genes encoding proteins involved in sialic acid transport δ-VPH delta V. parahaemolyticus hemolysin DLV double-locus variant DNA deoxyribonucleic acid DncV V. cholerae dinucleotide cyclase DT diphtheria toxin DTH delta thermostable hemolysin ECDC European Centre for Disease Prevention and Control eEF eukaryotic elongation factor VI List of Abbreviations HA/P; HapA hemagglutinin/protease Hcp hemolysin-coregulated protein HeLa human epithelial cervix adenocarcinoma cell line HHM Hafen Hamburg Marketing e.V. HisF protein of the baseplate structure of the V. cholerae T6SS HlyA hemolysin A HlyAET El Tor variant of hemolysin A HlyD hemolysin D protein HlyIII hemolysin III protein IfSG Infektionsschutzgesetz IMO International Maritime Organization ISO International Organization for Standardization kb kilobase(s) kbp kilobase pair(s) KLIWAS Auswirkungen des Klimawandels auf Wasserstraßen und Schifffahrt in Deutschland (research program) LDH lecithin-dependent hemolysin LuxPQ AI-2 autoinducer receptor LuxS AI-2 autoinducer synthase MARTX multifunctional-autoprocessing repeats-in-toxin toxin Mb megabase(s) MDa megadalton μm micrometer MLSA multilocus sequence analysis MLST multilocus sequence typing MPN most probable number MSHA mannose-sensitive hemagglutinin NaCl sodium chloride nagA, nanA, nanE, nanK genes necessary for sialic acid catabolism nanH gene necessary for sialic acid scavenging Neu5Ac N-acetylneuraminic acid O-antigen surface antigen O1 O1-antigen O139 O139-antigen VII List of Abbreviations OmpT outer membrane protein T OmpU outer membrane protein U ORF open reading frame OsmY putative BON domain-containing hemolysin in V. navarrensis PAAR-motif proteins proline-alanine-alanine-arginine motif proteins PCR polymerase chain reaction pilV, pilW genes encoding proteins for type IV pilus biosynthesis ppt parts per thousand pVH plasmid VH QMRA quantitative microbial risk assessment RadC DNA repair protein RAW 264.7 murine macrophage cell line rfb O1 O1-antigen-specific genomic region rfb O139 O139-antigen-specific genomic region RNA ribonucleic acid RNase H1 ribonuclease H1 rpoB gene encoding the β subunit of RNA polymerase RstA protein required for replication of CTXΦ RstB protein required for site-specific integration of CTXΦ RstR repressor of rstA and rstB transcription rtx repeats-in-toxin gene SA sialic acid SLV single-locus variant SNP single nucleotide polymorphism SST sea surface temperature ST sequence type
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