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Mueller Online.Pdf Aus dem Institut für Mikrobiologie und Tierseuchen des Fachbereichs Veterinärmedizin der Freien Universität Berlin Molecular and functional typing of isolates of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex with emphasis on multi-drug resistant Acinetobacter baumannii Inaugural-Dissertation zur Erlangung des Grades eines Doktors der Veterinärmedizin an der Freien Universität Berlin vorgelegt von Stefanie Müller Tierärztin aus Jena Berlin 2017 Journal-Nr.: 3964 Gedruckt mit Genehmigung des Fachbereichs Veterinärmedizin der Freien Universität Berlin Dekan: Univ.-Prof. Dr. Jürgen Zentek Erster Gutachter: Univ.-Prof. Dr. Lothar H. Wieler Zweiter Gutachter: PD Dr. Gottfried Wilharm Dritter Gutachter: Univ.-Prof. Dr. Thomas Alter Deskriptoren (nach CAB-Thesaurus): Acinetobacter calcoaceticus; Acinetobacter baumannii; antibiotics; multiple drug resistance; Carbapenems; zoonoses; restriction fragment length polymorphism; genome analysis Tag der Promotion: 19.09.2017 Bibliografische Information der Deutschen Nationalbibliothek Die Deutsche Nationalbibliothek verzeichnet diese Publikation in der Deutschen Nationalbibliografie; detaillierte bibliografische Daten sind im Internet über <http://dnb.ddb.de> abrufbar. ISBN: 978-3-86387-758-3 Zugl.: Berlin, Freie Univ., Diss., 2017 Dissertation, Freie Universität Berlin D 188 Dieses Werk ist urheberrechtlich geschützt. Alle Rechte, auch die der Übersetzung, des Nachdruckes und der Vervielfältigung des Buches, oder Teilen daraus, vorbehalten. Kein Teil des Werkes darf ohne schriftliche Genehmigung des Verlages in irgendeiner Form reproduziert oder unter Verwendung elektronischer Systeme verarbeitet, vervielfältigt oder verbreitet werden. Die Wiedergabe von Gebrauchsnamen, Warenbezeichnungen, usw. in diesem Werk berechtigt auch ohne besondere Kennzeichnung nicht zu der Annahme, dass solche Namen im Sinne der Warenzeichen- und Markenschutz-Gesetzgebung als frei zu betrachten wären und daher von jedermann benutzt werden dürfen. This document is protected by copyright law. No part of this document may be reproduced in any form by any means without prior written authorization of the publisher. Alle Rechte vorbehalten | all rights reserved Mensch und Buch Verlag 2018 Choriner Str. 85 - 10119 Berlin [email protected] – www.menschundbuch.de This work has been funded by the H. Wilhelm Schaumann Stiftung, Hamburg Table of Contents TABLE OF CONTENTS TABLE OF CONTENTS ......................................................................................................... I LIST OF FIGURES .............................................................................................................. VII LIST OF TABLES ................................................................................................................ IX ABBREVIATIONS ............................................................................................................... XII INTRODUCTION ................................................................................................................... 1 LITERATURE REVIEW ......................................................................................................... 3 I General characteristics and taxonomy of Acinetobacter spp. ................................... 3 II A. baumannii as a nosocomial pathogen in human medicine ................................... 6 1 Clinical relevance and treatment ................................................................................. 6 2 Epidemiology ............................................................................................................... 9 III A. baumannii in veterinary medicine .........................................................................11 1 Livestock ....................................................................................................................11 2 Companion animals ....................................................................................................12 IV Possible transmission of A. baumannii between humans and animals .................14 V Quinolones – mechanisms of action and resistance ................................................17 1 Mechanisms of action .................................................................................................17 2 Resistances against quinolones .................................................................................19 VI Mechanisms of antimicrobial resistance in A. baumannii .......................................20 1 Intrinsic resistances and efflux pumps ........................................................................20 2 Resistances against beta-lactam antibiotics ...............................................................22 3 Resistances against other antimicrobial classes .........................................................23 VII Immune defense during A. baumannii infection .....................................................26 - I - Table of Contents MATERIALS AND METHODS .............................................................................................28 I Materials ........................................................................................................................28 1 Origin of Acinetobacter isolates ..................................................................................28 2 Consumables and media for bacterial cultivation ........................................................29 3 Chemicals, enzymes and devices ..............................................................................29 4 Buffers and solutions ..................................................................................................29 II Methods ........................................................................................................................30 1 General methods ........................................................................................................30 1.1 Cultivation and conservation of bacteria ...............................................................30 1.2 Isolation of chromosomal DNA .............................................................................30 2 Identification of species of the Acb-complex ...............................................................31 2.1 Molecular methods ...............................................................................................31 2.1.1 Restriction fragment length polymorphism of the16S-23S intergenic spacer region ................................................................................................31 2.1.2 16S-23S intergenic spacer sequencing .........................................................32 2.1.3 Partial RNA polymerase beta subunit (rpoB) sequencing ..............................33 2.2 Phenotypic methods ............................................................................................34 2.2.1 Omnilog® Phenotypic MicroArray ..................................................................34 2.2.1.1 Experimental procedure ..........................................................................34 2.2.1.2 Evaluation of data ...................................................................................35 2.2.2 Species identification based on selected carbon sources using the Acinetobacter test medium ............................................................................36 3 Analysis of human and animal clinical Acb-complex isolates ......................................37 3.1 Species identification ...........................................................................................37 3.2 Antimicrobial susceptibility testing ........................................................................37 - II - Table of Contents 3.3. Whole genome sequencing .................................................................................39 3.3.1 Selection of isolates ......................................................................................39 3.3.2 Evaluation of data ..........................................................................................40 4 Investigation of fluoroquinolone resistance in A. baumannii ........................................40 4.1 Induction of fluoroquinolone resistance ................................................................40 4.1.1 Preparation of gradient plates ........................................................................40 4.1.2 Cultivation in subinhibitory fluoroquinolone concentrations ............................41 4.1.3 Species confirmation of fluoroquinolone resistant colonies ............................42 4.2 Whole genome sequencing ..................................................................................42 4.2.1 Selection of isolates ......................................................................................43 4.2.2 Evaluation of data ..........................................................................................43 4.3 Phenotypic analysis .............................................................................................44 4.3.1 Macroscopic and microscopic investigation ...................................................44 4.3.2 Antimicrobial susceptibility testing .................................................................44 4.3.3 Conjugation experiments ...............................................................................45 4.3.3.1 Identification of a conjugative plasmid .....................................................45 4.3.3.1.1
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