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Molecular Population and Colonisation Factor Analysis of The Molecular population and colonisation factor analysis of the Staphylococcus intermedius group Jeanette Bannoehr Thesis presented for the degree of Doctor of Philosophy The University of Edinburgh 2009 Declaration The research presented in this thesis is entirely my own work, except where otherwise stated. No part of this thesis has been submitted in any other application for a degree or professional qualification. Jeanette Bannoehr September 2009 ii Acknowledgements I would like to thank my supervisors Dr. J. Ross Fitzgerald, Professor Keith L. Thoday, and Professor Adri H. M. van den Broek for their support, guidance, and advice throughout the course of this study. I am also grateful to Keith and Adri for encouraging my interest in small animal dermatology. I also would like to thank Dr. Nouri L. Ben Zakour for invaluable help and patience with all the bioinformatics, and to Dr. Caitriona Guinane for sharing her molecular knowledge and technical expertise. I am thankful to many people at The University of Edinburgh for technical assistance, including Dr. Jeremy Brown for helping with the computerised image analysis, Robyn Cartwright for the work with CK10, Dr. Even Fossum and Professor Juergen Haas for introducing me to Gateway cloning, Lorna Hume for help with the moisture chambers, Dr. Arvind Mahajan and Edith Paxton for the introduction to cell culture work, and Dr. Darren Shaw for support and advice with the statistical analysis. I am very grateful to Professor Magnus Hook, Texas A & M University, USA for inviting me and to Dr. Sabitha Prabhakaran for fantastic technical support during my stay in Texas. I would like to acknowledge the Royal (Dick) School of Veterinary Studies for funding this research. Special thanks must go to the original LBEP girls- Caitriona, Nouri, Bethan, Gill, and Shruti- you all were a pleasure to work with and have become great friends! I would like to thank Allen- you have been a fantastic friend from the very start! Thanks to James and Makrina for being there in first year, and thanks to all members, past and present, of LBEP, ZAP, and MPRL. I would like to thank Christa, Ana, Carla, and Marcel for introducing me to life as dermatology residents. I like to say thanks to the dog owners who volunteered their pets for corneocyte collection, and to all the other people who have been inspiring and helpful throughout the past few years. I am thankful to my partner Ray and to my family for their love and endless moral support. iii Content Page Title page i Declaration ii Acknowledgements iii Contents iv List of commonly used abbreviations xiv List of figures xviii List of tables xxiii Abstract xxvi Chapter 1. Introduction 1 1.1 Introduction to staphylococci 2 1.2 Clinical relevance of Staphylococcus species 2 1.2.1 Staphylococcal antimicrobial resistance 3 1.2.2 Canine skin and keratinocyte migration and differentiation- anatomical and physiological features 5 1.2.3 Classification of the canine cutaneous bacterial flora 8 1.2.4 Clinical aspects of S. intermedius-host interactions 9 1.2.4.1 S. intermedius in healthy dogs 9 1.2.4.2 Classification of canine bacterial skin infections 9 1.2.4.3 Prevalence and treatment of canine pyoderma in veterinary practice 11 1.2.4.4 S. intermedius adherence to canine corneocytes 13 1.2.4.5 Canine atopic dermatitis (AD) and staphylococcal adherence 15 1.3 Pathogenesis of staphylococcal disease 16 1.3.1 Virulence factors of S. aureus 16 1.3.1.1 Enzymes 16 iv Page 1.3.1.2 Biofilm formation 16 1.3.1.3 Capsule 17 1.3.1.4 Lipoteichoic acid and peptidoglycan 17 1.3.1.5 Cytotoxins 18 1.3.1.6 Exfoliative Toxins 19 1.3.1.7 Staphylococcal superantigens 20 1.3.1.7.1 Superantigen activity 20 1.3.1.7.2 Enterotoxins 21 1.3.1.7.3 Toxic Shock Syndrome Toxin-1 (TSST-1) 22 1.3.1.7.4 Superantigen-like proteins 23 1.3.1.8 Chemotaxis inhibitory protein of staphylococci 23 1.3.1.9 S. aureus adherence-mediating surface proteins 23 1.3.1.9.1 Secretable expanded repertoire adhesive molecules (SERAMs) 24 1.3.1.9.2 Microbial surface components recognising adhesive matrix molecules (MSCRAMMs) 25 1.3.1.9.2.1 Clumping Factor A (ClfA) 28 1.3.1.9.2.2 Clumping Factor B (ClfB) 35 1.3.1.9.2.3 The fibronectin-binding MSCRAMMs FnbpA and FnbpB 38 1.3.1.9.2.4 The collagen-binding MSCRAMM Cna 43 1.3.1.9.2.5 The elastin-binding MSCRAMM EbpS 45 1.3.1.9.2.6 Protein A (SpA) 45 1.3.1.9.2.7 The serine-aspartate dipeptide repeat (Sdr)-family of S. aureus 48 1.3.1.9.2.8 Serine-aspartate dipeptide repeat (Sdr)-proteins of other Staphylococci 49 1.3.2 Virulence factors of S. intermedius 50 1.3.2.1 S. intermedius cytotoxins 51 1.3.2.2 S. intermedius exfoliative toxin 51 1.3.2.3 S. intermedius superantigens 52 1.3.3 Quorum sensing in Staphylococci 52 v Page 1.3.3.1 Global regulation of virulence factors in S. aureus 53 1.3.3.1.1 The accessory gene regulator, agr 53 1.3.3.1.2 The staphylococcal accessory regulator, sarA 55 1.3.3.1.3 Genetic diversity of the accessory gene regulator (agr) locus in S. aureus 56 1.3.3.1.4 Association of accessory gene regulator (agr) types and S. aureus disease 56 1.3.3.1.5 The accessory gene regulator (agr) system in S. intermedius 57 1.4 Population genetics of S. aureus and S. intermedius 58 1.4.1 Typing methods for bacterial isolates 58 1.4.2 Population genetic analysis of S. aureus 60 1.4.3 Population genetic analysis of S. intermedius 62 1.5 Hypothesis of the project 64 1.6 Aims of the project 65 Chapter 2. General Materials and Methods 66 2.1 Bacterial culture conditions and media 67 2.2 Oligonucleotides 67 2.3 DNA isolation, purification and analysis 67 2.3.1 Genomic DNA extraction 67 2.3.2 Purification of PCR products 68 2.3.3 Gel extraction and purification of DNA fragments 68 2.3.4 Plasmid DNA isolation and purification 68 2.3.5 Restriction endonuclease digestion of DNA 68 2.3.6 DNA agarose gel electrophoresis 69 2.3.7 DNA quantification 69 2.3.8 DNA sequencing 69 2.3.9 DNA sequence analysis 70 vi Page 2.4 Protein analysis 70 2.4.1 Protein quantification 70 2.4.2 Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) 70 2.4.3 Western blotting 71 Chapter 3. Population genetic structure of the ‘Staphylococcus intermedius group’ 72 3.1 Introduction 73 3.2 Aims and Strategy 74 3.3 Materials and Methods 75 3.3.1 Bacterial strains used for DNA multilocus sequence analysis 75 3.3.2 Gene loci selected for DNA sequence analysis 84 3.3.3 PCR amplification of gene fragments for DNA sequence analysis 84 3.3.4 DNA sequencing 86 3.3.5 DNA sequence and molecular evolutionary analysis 86 3.3.6 eBURST analysis 87 3.3.7 Nucleotide sequence accession numbers 87 3.3.8 PCR amplification of the partial pta gene for restriction endonuclease digestion 87 3.3.9 Restriction digestion with MboI endonuclease 88 3.4 Results 89 3.4.1 Multilocus sequence analysis of S. intermedius 89 3.4.2 S. intermedius isolates differentiate into three different phylotypes 89 vii Page 3.4.3 The SIG belong to a larger phylogenetic group of animal- associated staphylococcal species 93 3.4.4 Phylogenetic analysis reveals that S. pseudintermedius, and not S. intermedius, is the common cause of canine pyoderma 97 3.4.5 S. pseudintermedius has a largely clonal population structure 97 3.4.6 Successful clones of S. pseudintermedius are identified on different continents 99 3.4.7 Four predicted AIP variants were detected among the SIG strains investigated, including a novel subtype 99 3.4.8 Assortive recombination has contributed to the distribution of agr alleles 101 3.4.9 Sixteen of the S. pseudintermedius strains examined encode the mecA gene, conferring methicillin-resistance 101 3.4.10 Methicillin-resistant S. pseudintermedius (MRSP) have evolved by multiple mecA gene acquisitions by different clones 101 3.4.11 Development of a novel diagnostic test for the identification of S. pseudintermedius, using a simple PCR-restriction fragment length polymorphism (RFLP) approach 102 3.5 Discussion 107 Chapter 4. Genome-wide identification and analysis of novel cell wall- anchored proteins encoded by Staphylococcus pseudintermedius ED99 112 4. 1 Introduction 113 4. 2 Aims and Strategy 114 viii Page 4.3 Materials and Methods 115 4.3.1 Bacterial strains used in this study 115 4.3.2 Genome-wide screen for genes encoding cell wall-anchored proteins 115 4.3.3 In silico structural analysis of cell wall-anchored proteins 117 4.3.4 Southern blot analysis 117 4.3.5 PCR amplification of spsP and spsQ gene fragments 120 4.4 Results 122 4.4.1 Identification of genes encoding 17 putative cell wall- anchored proteins in the S. pseudintermedius ED99 genome 122 4.4.2 Six S. pseudintermedius cell wall-anchored proteins contain 122 predicted tandem repeat regions 4.4.3 SpsD, SpsI, SpsL, and SpsO have two predicted IgG-like folds 128 4.4.4 The putative cell wall-anchored proteins SpsD, SpsL, and SpsO have several typical MSCRAMM features 128 4.4.5 S.
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