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