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Isolation and Characterisation of Methicillin-Resistant Staphylococci

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Isolation and Characterisation of Methicillin-Resistant Staphylococci Cover illustration: © Jan Ranson - www.janranson.be Printing: University Press, Zelzate ISBN 978-90-5864-303-2 CODA/CERVA Isolation and Characterisation of Methicillin-Resistant Staphylococci from Domestic Animals Wannes Vanderhaeghen Thesis submitted in fullfillment of the requirements for the degree of Doctor (PhD) in Veterinary Sciences 2012 Department of Pathology, Bacteriology and Avian Diseases Faculty of Veterinary Medicine Ghent University Promoters: Prof. dr. Patrick Butaye, Prof. dr. Katleen Hermans, Prof. dr. Freddy Haesebrouck Members of the examination committee: Prof. dr. dr. h. c. A. de Kruif Chairman - Faculty of Veterinary Medicine, Ghent University Prof. dr. S. De Vliegher Faculty of Veterinary Medicine, Ghent University Prof. dr. O. Denis Hôpital Erasme, Université libre de Bruxelles Prof. dr. J.A. Wagenaar Faculty of Veterinary Medicine, Utrecht University Prof. dr. A. Martens Faculty of Veterinary Medicine, Ghent University Prof. dr. S. Daminet Faculty of Veterinary Medicine, Ghent University This doctoral research was supported by the Federal Public Service of Health, Food Chain Safety and Environment, project nr. RF-6189. The study on MRSA in bovine mastitis was partly supported by the Belgian Antibiotic Policy Coordinating Committee (BAPCOC). TABLE OF CONTENTS Table of Contents ......................................................................................................... 7 List of Abbreviations ................................................................................................... 11 Preface .......................................................................................................................... 13 I - REVIEW OF THE LITERATURE .................................................................................... 15 Chapter 1 The staphylococci ...................................................................................... 17 1.1 Microbiology of staphylococci .............................................................................. 17 1.1.1 Taxonomy ......................................................................................................... 17 1.1.2 General characteristics of staphylococci .......................................................... 17 1.1.2.1 Phenotypic characteristics ........................................................................... 17 1.1.2.2 Genotypic characteristics ............................................................................ 19 1.1.3 Ecology and pathogenicity of staphylococci .................................................... 19 1.2 Identification and typing of staphylococci ............................................................ 22 1.2.1 Phenotypical identification ............................................................................... 22 1.2.2 Genotypical identification ................................................................................ 23 1.2.3 Phenotyping ...................................................................................................... 23 1.2.4 Genotyping ....................................................................................................... 24 1.2.4.1 Restriction Fragment Length Polymorphism (RFLP) - Pulsed Field Gel Electrophoresis (PFGE) ............................................................................... 24 1.2.4.2 Multiple-Locus Variable-number tandem-repeat Analysis (MLVA) ......... 25 1.2.4.3 Multilocus Sequence typing (MLST) .......................................................... 25 1.2.4.4 spa typing .................................................................................................... 26 Chapter 2 Methicillin resistance in staphylococci ..................................................... 27 2.1 Cell wall synthesis, β-lactam antimicrobials and methicillin resistance ................ 27 2.2 Structure and characteristics of SCCmec ............................................................... 28 2.2.1 SCCmec as a mobile genetic element ............................................................... 28 2.2.2 SCCmec essential regions: mec complex and ccr complex .............................. 29 2.2.3 SCCmec non-essential regions: joining regions J1, J2 and J3 .......................... 31 2.2.4 SCCmec types and subtypes ............................................................................. 31 2.2.5 SCCmec typing ................................................................................................. 32 2.2.6 SCC non-mec .................................................................................................... 33 2.3 Variants of mecA .................................................................................................... 34 2.4 Origin of mecA and SCCmec in MRSA ................................................................. 34 7 TABLE OF CONTENTS 2.5 Transfer of SCCmec among staphylococci ............................................................ 35 2.6 Factors affecting methicillin resistance – Hetero-resistance .................................. 36 2.7 Testing for methicillin resistance ........................................................................... 36 Chapter 3 Methicillin-resistant staphylococci in animals .......................................... 39 3.1 MRSA .................................................................................................................... 39 3.1.1 A short history of MRSA: from human hospitals to livestock ......................... 39 3.1.2 Occurrence and characteristics of MRSA in dogs, pigs and bovines ............... 41 3.1.2.1 Characteristics of MRSA CC398 ................................................................ 41 3.1.2.2 MRSA in dogs ............................................................................................. 42 3.1.2.2.1 MRSA CC398 ........................................................................................ 43 3.1.2.2.2 Non-CC398 MRSA ................................................................................ 43 3.1.2.3 MRSA in pigs .............................................................................................. 44 3.1.2.3.1 MRSA CC398 ........................................................................................ 44 3.1.2.3.2 Non-CC398 MRSA ................................................................................ 45 3.1.2.4 MRSA in bovines ........................................................................................ 47 3.1.2.4.1 MRSA CC398 ........................................................................................ 47 3.1.2.4.2 Non-CC398 MRSA ................................................................................ 48 3.2 MRNAS .................................................................................................................. 50 3.2.1 Occurrence, species diversity and characteristics of MRNAS in dogs ............. 50 3.2.2 Occurrence, species diversity and characteristics of MRNAS in pigs .............. 51 3.2.3 Occurrence, species diversity and characteristics of MRNAS in bovines ........ 53 3.3 Methicillin-resistant staphylococci in animals – Conclusions ................................ 55 II - AIMS OF THE STUDY ................................................................................................ 57 III - EXPERIMENTAL STUDIES ........................................................................................ 61 Chapter 4 Methicillin-resistant Staphylococcus aureus ST398 associated with subclinical and clinical mastitis in Belgian cows ........................................ 63 Chapter 5 Screening for methicillin-resistant staphylococci in dogs admitted to a veterinary teaching hospital ......................................................................... 77 Chapter 6 Characterisation of methicillin-resistant non-Staphylococcus aureus staphylococci carriage isolates from pigs .................................................... 87 Chapter 7 Characterisation of methicillin-resistant non-Staphylococcus aureus staphylococci carriage isolates from different bovine populations ............. 105 ΙV - GENERAL DISCUSSION ........................................................................................... 127 V - REFERENCES ........................................................................................................... 141 8 TABLE OF CONTENTS Summary - Samenvatting ............................................................................................. 179 Dankwoord ................................................................................................................... 189 Curriculum Vitae Wannes Vanderhaeghen .................................................................. 197 9 LIST OF ABBREVIATIONS ACME arginine catabolic mobile element AFLP amplified fragment length polymorphism BURST based upon related sequence types CA-MRSA community-associated methicillin-resistant Staphylococcus aureus CC clonal complex ccr cassette chromosome recombinase CLSI clinical and laboratory standards institute CNS coagulase-negative staphylococci CPS coagulase-positive staphylococci CV core-variable DLV double locus variant DNA deoxyribonucleic acid dru direct repeat unit EARSS European Antimicrobial Resistance Surveillance System EMRSA epidemic methicillin-resistant Staphylococcus aureus HA-MRSA hospital-associated methicillin-resistant Staphylococcus aureus IS insertion sequence ISS integration site sequence IWG-SCC international
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