https://theses.gla.ac.uk/ Theses Digitisation: https://www.gla.ac.uk/myglasgow/research/enlighten/theses/digitisation/ This is a digitised version of the original print thesis. Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten: Theses https://theses.gla.ac.uk/ [email protected] Molecular investigation of an epidemic MRS A: a comparative study of its recognition, introduction and spread in the West of Scotland Alistair W. Brown BSc. A thesis submitted for the Degree of Master of Science Faculty of Medicine, University of Glasgow December 1998 University Department of Bacteriology Royal Infirmary Castle Street Glasgow G4 OSF ProQuest Number: 10390908 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a co m plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQ uest 10390908 Published by ProQuest LLO (2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLO. ProQuest LLO. 789 East Eisenhower Parkway P.Q. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 t o R A R Y (I I 9^ DECLARATION This thesis is the original work of the author and the information, ideas and opinions expressed herein are my own unless otherwise stated. Alistair W, Brown S u • * ‘•*- «; m i I would like to thank Dr David J. Platt, Dr Dugald R. Baird and Dr Giles F.S. Edwards for their advice and patience, which must have been stretched on several occasions during the course of this study. Thanks also to Dr Tony Girdwood for allowing me the opportunity to undertake this course of study. Special thanks to Mr George Donnelly for extraordinary computing skills used in the presentation of this thesis. Thank you also to staff at Microbiology, Stobhill NHS Trust and particularly to Shelley and Linda for help, advice and shoulders to moan on when the going got tough. Thanks to all family and friends for encouragement and for having faith and finally, very special thanks to my wife Heather for continued support and incredible amounts of patience during some very difficult times. Ill Thesis Title......................................................................................................... i Declaration........................................................................................................ ü Acknowledgements......................................................................................... iü Table of Contents............................................................................................ iv Index of Figures............................................................................................ viii Index of Tables.................................................................................................xi ABSTRACT...........................................................................1 1. INTRODUCTION................................................................4 1.1 The genus Staphylococcus..................................................................5 1.2 Virulence of Staph, aureus.................................................................6 1.3 Development of resistance................................................................. 9 1.4 Prevalence of MRS A infection........................................................10 1.5 Epidemiology of MRSA infection.................................................. 12 (a) Sources and transmission of MRSA......................................................................./ 2 (b) Control o f MRSA..........................................................................................................14 1.6 Mechanism of methicillin resistance............................................. 17 (a) mecA and mec associated DNA.............................................................................. / 7 (b) Expression of methicillin resistance......................................................................19 IV 1.7 Strain identification and epidemiological typing of MRSA 20 1.8 MRSA in the West of Scotland .............................................. 26 1.9 Aims of the investigation and study design....................................30 (a) Aims of the investigation...........................................................................................30 (b) Techniques used to characterise the "Lisbon strain ” ........................................31 2. MATERIALS AND METHODS .............................34 2.1 Collection of isolates.......................................................................... 35 (a) Lisbon isolates.............................................................................................................. 35 (b) Control g rou ps...............................................................................................................37 2.2 Storage of isolates................................................................................38 2.3 Antimicrobial susceptibility testing...................... 38 2.4 Bacteriophage typing........................................................................ 46 2.5 Bio typing....................................................................................... 46 (a) Urease production......................................................................................................... 47 (b) Tween 80 hydrolysis..................................................................................................... 47 2.6 Genomic fingerprinting..................................................................... 47 (a) Buffers and Reagents....................................................................................................47 (b) Centrifugation................................................................................................................ 47 (a) Extraction and purification of genomic DNA......................................................48 (d) Restriction enzyme digestion of genomic DNA................................................... 49 (e) Horizontal gel electrophoresis.................................................................................50 (f) Visualisation of DNA fragments............................................................................... 50 2.7 Plasmid Profiling................................................................................. 51 (a) Preparation of cell lysates..........................................................................................51 (b) Vertical gel electrophoresis........................................................................................52 2.8 Plasmid fingerprinting......................................................................52 (a) Extraction and purification ofplasmid DNA...................................................... 52 (b) Horizontal gel electrophoresis................................................................................ 54 2.9 Computer aided analysis of gels.....................................................54 (a) Calculation of similarity coefficients..................................................................... 54 (b) Construction of dendrogi'ams................................................................................... 55 3. RESULTS.......................................................................... 56 3.1 Genotyping of Staph, aureus control strains with the restriction endonuclease H h a l....................................................... 57 (a) Methicillin-sensitive Staph aureus (MSSA)...................................................... 57 (b) Methicillin-resistant Staph, aureus (MRSA).......................................................61 (i) EMRSA-1........................................................................................................ 61 (ii) EMRSA-15 ......................................................................................................64 (iii) EMRSA-16 ...................................................................................................... 69 (iv) “Other” M RSA............................................................................................... 74 (a) “Stobhill" type....................................................................................... 74 (b) Sporadic MRSA...................................................................................... 77 3.2 Genotyping of Staph, aureus control strains with the restriction endonuclease Sau 3A \....................................................81 3.3 Genotyping of Lisbon strain MRSA with H h a\........................84 3.4 “French strain” MRSA................................................................. 92 3.5 Genotyping of Lisbon strain MRSA with Sau3A \......................98