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The Phylogeny of Staphylococcus Aureus Clonal Complex 398 and Its Interaction with Various Hosts

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The Phylogeny of Staphylococcus Aureus Clonal Complex 398 and Its Interaction with Various Hosts The phylogeny of Staphylococcus aureus clonal complex 398 and its interaction with various hosts Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Mohamed Mostafa Hefny Abdelbary aus Giza / Ägypten 1. Referent: Professor Dr. Ulrich Nübel 2. Referent: Professor Dr. Michael Steinert eingereicht am: 17.09.2014 mündliche Prüfung (Disputation) am: 10.12.2014 Druckjahr 2015 Vorveröffentlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Publikationen Abdelbary MMH, Wittenberg A, Cuny C, Layer F, Wieler L, Skov R, Larsen J, Hasman H, Fitz- gerald JR, Wagenaar JA, Böse R, Nübel U & Witte W. (2014) Phylogenetic Analysis of Staphylococcus aureus CC398 Reveals a Sub-Lineage Epidemiologically Associated with Infec- tions in Horses. PLoS ONE 9(2): e88083. Tagungsbeiträge Abdelbary MMH, Wittenberg A, Cuny C, Layer F, Wieler L, Skov R, Larsen J, Hasman H, Fitz- gerald JR, Wagenaar JA, Böse R, Nübel U & Witte W (2013). The population structure of ST398 and the emergence of a horse-associated sub-clone. (Vortrag) 3rd ASM-ESCMID Confer- ence on Methicillin-resistant Staphylococci in Animals; in Copenhagen, Denmark. Posterbeiträge Abdelbary MMH, Wittenberg A, Cuny C, Layer F, Wieler L, Skov R, Larsen J, Hasman H, Fitz- gerald JR, Wagenaar JA, Böse R, Nübel U & Witte W (2013). The population structure of ST398 and the emergence of a horse-associated sub-clone. German Symposium on Zoonoses Research; in Berlin, Germany. Cuny C, Adlhoch C, Abdelbary MMH, Layer F, Witte W (2013). Acquisition of the immune evasion gene cluster by livestock-associated MRSA CC398. German Symposium on Zoonoses Research; in Berlin, Germany. Abdelbary MMH, Wittenberg A, Cuny C, Layer F, Wieler L, Skov R, Larsen J, Hasman H, Fitz- gerald JR, Wagenaar JA, Böse R, Nübel U & Witte W (2013). The population structure of ST398 based on mutation discovery. Congress of the European Society for Clinical Microbiology and Infectious Diseases (ECCMID); in Berlin, Germany. Cuny C, Köck R, Abdelbary MMH, Witte W (2013). Rare occurrence of methicillin-susceptible Staphylococcus aureus of spa type t571 (clonal complex CC398) in Germany. 65. Annual Meeting of German Society for Hygiene and Microbiology (DGHM); in Rostock, Germany. Abdelbary MMH, Wittenberg A, Cuny C, Layer F, Wieler L, Skov R, Larsen J, Hasman H, Fitz- gerald JR, Wagenaar JA, Böse R, Nübel U & Witte W (2012). Population structure of ST398 based on mutation discovery. 64. Annual Meeting of German Society for Hygiene and Microbiology (DGHM); in Hamburg, Germany. Abdelbary MMH, Wittenberg A, Cuny C, Layer F, Wieler L, Skov R, Larsen J, Hasman H, Fitz- gerald JR, Wagenaar JA, Böse R, Nübel U & Witte W (2012). Population structure of ST398 based on mutation discovery. German Symposium on Zoonoses Research; in Berlin, Germany. Table of contents TABLE OF CONTENTS ZUSAMMENFASSUNG ............................................................................................................................... IV SUMMARY ..................................................................................................................................................... V 1 INTRODUCTION .................................................................................................................................... 1 1.1 STAPHYLOCOCCI ................................................................................................................................... 1 1.2 STAPHYLOCOCCUS AUREUS ................................................................................................................... 1 1.3 STAPHYLOCOCCUS AUREUS AND THE WAVES OF ANTIBIOTIC RESISTANCE ....................................... 2 1.4 STAPHYLOCOCCUS AUREUS GENOME STRUCTURE ............................................................................... 3 1.4.1 CORE GENOME ................................................................................................................................................... 3 1.4.2 ACCESSORY GENOME ........................................................................................................................................ 4 1.5 STAPHYLOCOCCUS AUREUS VIRULENCE FACTORS ............................................................................... 8 1.6 STAPHYLOCOCCUS AUREUS AND THE HOST’S INNATE IMMUNE SYSTEM .......................................... 10 1.7 MOLECULAR TYPING OF STAPHYLOCOCCUS AUREUS ........................................................................ 11 1.7.1 PULSED-FIELD GEL ELECTROPHORESIS ..................................................................................................... 12 1.7.2 MULTILOCUS SEQUENCE TYPING................................................................................................................. 12 1.7.3 STAPHYLOCOCCAL PROTEIN A TYPING ...................................................................................................... 13 1.7.4 SCCMEC TYPING............................................................................................................................................. 13 1.8 POPULATION STRUCTURE OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS ..................... 14 1.8.1 HEALTHCARE-ASSOCIATED MRSA ............................................................................................................ 15 1.8.2 COMMUNITY-ASSOCIATED MRSA .............................................................................................................. 16 1.9 MRSA IN ANIMALS ............................................................................................................................. 17 1.10 CLONAL COMPLEX 398 ................................................................................................................... 19 1.11 AIM OF THIS STUDY ......................................................................................................................... 20 1.12 STUDY DESIGN ................................................................................................................................. 20 2 MATERIALS AND METHODS ........................................................................................................... 22 2.1 MATERIALS ......................................................................................................................................... 22 2.1.1 STAPHYLOCOCCUS AUREUS CC398 ISOLATE COLLECTION ...................................................................... 22 2.1.2 CHEMICALS, ENZYMES AND PROTEINS ....................................................................................................... 23 2.1.3 EQUIPMENT .................................................................................................................................................... 24 2.1.4 NUTRIENT MEDIA .......................................................................................................................................... 25 2.1.5 COMMERCIAL KITS ........................................................................................................................................ 26 I Table of contents 2.1.6 STANDARD SOLUTIONS ................................................................................................................................. 26 2.1.7 SOFTWARE AND INTERNET TOOLS ............................................................................................................. 27 2.2 MICROBIOLOGICAL METHODS ........................................................................................................... 29 2.2.1 BACTERIAL GROWTH .................................................................................................................................... 29 2.2.2 ANTIMICROBIAL SUSCEPTIBILITY TESTING ............................................................................................... 29 2.2.3 EXTRACTION OF THE CHROMOSOMAL DNA ............................................................................................. 29 2.2.4 QUANTIFICATION OF THE EXTRACTED CHROMOSOMAL DNA ............................................................... 30 2.2.5 MOLECULAR TYPING OF S. AUREUS ............................................................................................................. 30 2.2.6 DETECTION OF VARIOUS MOBILE GENETIC ELEMENTS ............................................................................ 32 2.2.7 POLYMERASE CHAIN REACTION APPROACH .............................................................................................. 32 2.3 MUTATION DISCOVERY ...................................................................................................................... 33 2.3.1 PCR CONDITIONS FOR THE MUTATION DISCOVERY ANALYSIS ............................................................... 34 2.3.2 CAPILLARY-BASED SANGER SEQUENCING ................................................................................................. 34 2.3.3 SINGLE-NUCLEOTIDE POLYMORPHISMS ANALYSIS................................................................................... 35 2.3.4 PHYLOGENETIC ANALYSIS OF CC398 ........................................................................................................ 37 2.3.5 MOLECULAR EVOLUTION OF
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