Identifiication and Characterization of Novel Carbapenemases

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Identifiication and Characterization of Novel Carbapenemases DISSERTATION Identification and characterization of novel carbapenemases Dissertation to obtain the degree Doctor Rerum Naturalium (Dr. rer. nat.) at the Faculty of Biology and Biotechnology International Graduate School of Biosciences Ruhr-University Bochum Department of Medical Microbiology submitted by Niels Ernst Pfennigwerth from Essen Advisor: Prof. Dr. Sören G. Gatermann Second advisor: Prof. Dr. Franz Narberhaus Bochum, April 2015 DISSERTATION Identifizierung und Charakterisierung neuer Carbapenemasen Dissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat) an der Fakultät für Biologie und Biotechnologie Internationale Graduiertenschule Biowissenschaften Ruhr-Universität Bochum Abteilung für Medizinische Mikrobiologie eingereicht von Niels Ernst Pfennigwerth Essen Referent: Prof. Dr. Sören G. Gatermann Korreferent: Prof. Dr. Franz Narberhaus Bochum, April 2015 Danksagung Viele haben zu einem erfolgreichen Gelingen dieser Dissertation beigetragen. Einigen möchte ich besonders danken. Meinem Doktorvater Herrn Prof. Dr. Sören G. Gatermann danke ich sehr für seine fortwährende Unterstützung, sein großes Vertrauen in meine Arbeit und die Möglichkeit, in diesem interessanten Fachbereich zu promovieren. Herrn Prof. Dr. Franz Narberhaus danke ich sehr für die freundliche Übernahme des Korreferats. Herrn Dr. Alexander Stang und Herrn Prof. Klaus Überla danke ich für die Möglichkeit, das in dieser Arbeit gefundene Plasmid in der Abteilung für Virologie zu sequenzieren. Allen Mitarbeitern der Abteilung für medizinische Mikrobiologie danke ich für das tolle, nette und freundschaftliche Arbeitsklima und für eine Hilfsbereitschaft, die nie zu enden scheint. Besonders danke ich hierbei Frau Anja Kaminski für die Hilfe bei den isoelektrischen Fokussierungen, Frau Anke Albrecht für ihre unverzichtbare Unterstützung bei den Lokalisationsstudien und Frau Susanne Friedrich für ein immer offenes Ohr bei experimentellen Problemen. Danke auch an meine Masterstudent(in)en Lisei Meining, Alexander Hoffmann und Felix Lange und meine S-Moduler für ihr Mitwirken an Teilen dieser Arbeit. Ein besonders großer Dank geht an meine KoMaNePf-Mitinsassen Dr. Sandra Neumann, Dr. Lennart Marlinghaus und Dr. Miriam Korte-Berwanger, ohne euch wären die letzten vier Jahre um mindestens 90% unlustiger gewesen. Auch für viele fachliche Diskussionen - vielen Dank! (Fast) last, but not least: Ein riesiggroßer Dank geht an Herrn Dr. Martin Kaase für seine zu jeder Zeit freundschaftliche Unterstützung, die zahllosen fruchtbaren fachlichen Diskussionen, das kritische Korrekturlesen von Postern, Manuskripten und dieser Arbeit und als wandelndes Lexikon für alle Fragen bezüglich der medizinischen Mikrobiologie. Vielen Dank! Ein Dank, der so groß ist, dass ich ihn nicht in Worten auszudrücken vermag, gebührt zu guter Letzt meinen Eltern, meiner Schwester und meiner Frau Freya, die mich zu jeder Zeit bedingungslos unterstützt, ermutigt und aufgebaut haben. Vielen, vielen Dank! Contents I Contents Contents ................................................................................................................................................ I List of Figures ................................................................................................................................... IV List of Tables ...................................................................................................................................... V Abbreviations .................................................................................................................................. VI 1 Introduction ............................................................................................................................... 1 1.1 -lactam antibiotics ....................................................................................................................................... 1 1.2 β -lactam antibiotics: The bacterial cell wall synthesis ......................... 5 1.3 TargetMechanisms structures of antibiotic of β resistance ...................................................................................................... 8 1.4 -lactamases .................................................................................................................................................. 10 1.4.1β -lactamases ......................................................................................................................... 12 1.4.2 Class A β-lactamases ......................................................................................................................... 13 1.4.3 Class BC β-lactamases ......................................................................................................................... 14 1.4.4 β-lactamases ........................................................................................................................ 14 1.5 CarbapenemasesClass D β and their distribution ............................................................................................ 15 1.6 -lactamase genes ................................................................................................................ 16 1.7 PseudomonasMobility of β aeruginosa .......................................................................................................................... 19 1.8 Citrobacter freundii ..................................................................................................................................... 19 1.9 Objectives of this work .............................................................................................................................. 20 2 Material and Methods .......................................................................................................... 22 2.1 Material ............................................................................................................................................................ 22 2.1.1 Instruments .......................................................................................................................................... 22 2.1.2 Disposable material .......................................................................................................................... 23 2.1.3 Chemicals .............................................................................................................................................. 24 2.1.4 Antibiotics ............................................................................................................................................. 25 2.1.5 Wafers containing antibiotics ....................................................................................................... 26 2.1.6 Antibiotic gradient test strips ....................................................................................................... 26 Contents II 2.1.7 Kits und standards ............................................................................................................................ 26 2.1.8 Enzymes ................................................................................................................................................. 27 2.1.9 Antibodies ............................................................................................................................................. 27 2.2 Microbial strains, plasmids and oligonuclotides ............................................................................ 28 2.2.1 Microbial strains ................................................................................................................................ 28 2.2.2 Plasmids ................................................................................................................................................. 28 2.2.3 Oligonucleotides ................................................................................................................................. 29 2.3 Methods ........................................................................................................................................................... 37 2.3.1 Microbiological methods ................................................................................................................ 37 2.3.2 Phenotypic methods for antibiotic resistance analysis ..................................................... 39 2.3.3 Molecular biology methods ........................................................................................................... 40 2.3.4 Biochemical methods ....................................................................................................................... 46 2.3.5 In silico methods ................................................................................................................................. 50 3 Results ....................................................................................................................................... 51 3.1 The search for novel carbapenemases ............................................................................................... 51 3.1.1 Identification of IMP-31 in Pseudomonas aeruginosa NRZ-00156 ................................ 51 3.1.2 Identification of OXA-233 in Citrobacter freundii NRZ-02127 ........................................ 55 3.1.3 Identification of KHM-2 in Pseudomonas aeruginosa NRZ-03096 ................................. 58 3.2 Analysis of the genetic environment of blaIMP-31,
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