Molecular Methods for the Detection of TEM- and SHV Related Beta Lactamase Genes in Members of the Enterobacteriaceae
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Molecular Methods for the Detection of TEM- and SHV Related Beta Lactamase Genes in Members of the Enterobacteriaceae Town Andrew Whitelaw Cape of Thesis submitted in fulfilment of the requirements for the degree Master of Science in the Faculty of Medicine, University of Cape Town. UniversityMay 1999 The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgementTown of the source. The thesis is to be used for private study or non- commercial research purposes only. Cape Published by the University ofof Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University CONTENTS Part I Dedication ...............................•.................•.••...•.................•• i Declaration .................................................•.........................ii Acknowledgements ...• ~ •••...........................•....•...........•....•....... iii List of Figures .....•....•.•.••...........................................•..•.•••...• iv List of Tables •........•....................................•......................... vii Abbreviations....................................................................... ix Amino Acid Abbreviations .•••..•.................•.....•........•........•..••... xii Abstract ...........•.••... ~ .••.......................•...••...•...........•...•...••... xiii Chapter One: Literature Review ....................••......•.................•. 1 1.1 Introduction ...................................................................... 1 1.2 Beta Lactams ..................................................................... 1 1.2.1 Structure of Beta Lactams ........................................... 3 1.2.2 Mechanism of Action of Beta Lactams ........................... 5 1.3 Beta Lactamases .................................................................. 6 1.3.1 Mechanism of Action ofBeta Lactamases ........................ 7 1. 3 .1.1 Beta Lactamases in Gram Positive Bacteria............ 8 1.3.1.2 Beta Lactamases in Gram Negative Bacteria ......... 8 1.3.2 Beta Lactamase Inhibitors .................................. 9 1.3.3 Classification ofBeta Lactamases ................................. 10 1. 4 TEM- and SHV -Related Beta Lactamases .................................... 13 1. 4.1 Structure and Regulation of TEM- and SHV-Related ............ 13 Beta Lactamases 1.4.2 Location ofTEM- and SHV-Related Beta Lactamase ........... 18 Resistance Genes 1.4.3 Incidence and Spread ofBeta Lactamases ........................ 19 1. 4. 3.1 Incidence of Beta Lactamase Mediated ................ 19 Resistance 1.4.3.2 Spread of Beta Lactam Resistance ...................... 21 1.4.4. Detection and Characterisation ofBeta Lactarnase ............. 24 Mediated Resistance 1.4.4.1 Phenotypic Methods ...................................... 26 i) Double Disc Diffusion Test ........................... 26 ii) Vitek ESBL Test ....................................... 27 iii) Etest ESBL Screen .................................... 28 iv) Other Tests for Detection ofESBLs ................ 28 1.4.4.1 Molecular Techniques .................................... 29 i) DNA-DNA Hybridisation ............................. 29 ii) Polymerase Chain Reaction ........................... 29 1.5 Aim Of Study .................................................................... 30 Chapter Two: Bacterial Isolates and Antibiotic Sensitivity Testing ..... 31 2.1 Identification of Bacterial Isolates ............................................. 31 2.2 Antibiotic Sensitivity Testing ................................................... 31 2.3 Detection ofESBL Production ................................................ 33 2.4 Results ............................................................................. 34 2.4.1 Identification and Antibiotic Susceptibility ...... ." ................ 34 2.4.2 Detection ofESBL Activity ......................................... 37 2.5 Discussion ................................ ·........................................ 40 Chapter Three: DNA-DNA Hybridisation Studies .......................... 43 3.1 Introduction ...................................................................... 43 3.2 Materials and Methods ......................................................... 44 3.2.1 Bacterial Isolates and Plasmids ..................................... 44 3 .2.1.1 Preparation of Competent Cells ......................... 44 3.2.1.2 Transformation ............................................ 45 3 .2.1.3 Extraction of Genomic DNA............................ 45 3 .2.2 Transfer of DNA to a Stable Matrix ............................... 46 3.2.2.1 Slot Blots .................................................. 46 3 .2.2.2 Colony Blots ............................................... 46 3.2.3 Methods Used in the Preparation of the TEM Probe ............ 47 3.2.3.1 PCR Assay ................................................. 47 3.2.3.2 Agarose Gel Electrophoresis ............................. 50 3.2.3.3 DNA Extraction from Agarose Gels ............................. 51 3.2.3.4 Cloning ............................................................... 51 i) Preparation of Insert ............................................ :51 ii) Preparation of Vector. .......................................... 52 iii) Ligation .......................................................... 52 iv) Transformation ................................................. 52 v) Isolation and Characterisation of Recombinants ............ 52 vi) Plasmid DNA Extraction ...................................... 53 vii) Restriction Endonuclease Digestion ........................ 54 3.2.3.5 DNA Sequencing ................................................... 54 3.2.4 Methods Used in the Preparation of the SHV Probes ........... 56 3.2.5 Preparation ofProbes ................................................ 59 3.2.5.1 ECL Chemiluminescent Labelling ...................... 59 3.2.5.2 32P Radioloabelling ofProbe ............................ 59 3.2.6- DNA-DNA Hybridisation Using TEM and SHV Probes ..... 60 3.2.6.1 ECL Labelled Probes ..................................... 60 3.2.6.2 32Plabelled Probe....................................... 61 3.3 Results ............................................................................ 61 3.3.1 Preparation ofTEM Probe .......................................... 61 3.3.2 Results ofHybridisation with the TEM Probe ................... 62 3.3.2.1 Slot Blots ................................................... 62 3.3.2.2 Colony Blots ............................................... 64 3.3.3 Results ofHybridisation with the SHV Probes .................. 67 3.3.3.1 Slot Blots ................................................... 67 3.3.3.2 Colony Blot. ............................................... 73 3.4 Discussion ........................................................................ 75 DEDICATION To my parents DECLARATION I, Andrew Christopher Whitelaw, hereby declare that the work on which this thesis is based is my own original work (except where acknowledgements indicate otherwise), and neither the whole work, or any part thereof, has been, is being or will be submitted for another degree in this or any other university. I empower the University of Cape Town to reproduce for the purposes of research, the whole or any part of this work in any form whatsoever. Signed: ii ACKNOWLEDGEMENTS I would like to thank everyone in the clinical laboratory at Groote Schuur Hospital, especially Pam Derby, for patience and help when it came to teaching me the rudiments of bacterial identification procedures and sensitivity testing. An additional thank you to everyone in the media kitchen, for supplying me with what seemed like endless amounts of molten MacConkey agar (among other things), often at very short notice. To the staff at the Medical Graphics department at the hospital for all their assistance and advice on photography, as well as their patience with my never-ending requests. When I first started working on this project I had little to no experience in molecular biological techniques, and I must express my sincere appreciation to everyone who put up with my questions, and patiently explained and demonstrated things to me. I must thank Professor Lafras Steyn and Dr Harold Zappe in particular for introducing me to the world of computer analysis as well as for introducing me to the fundamentals of molecular biology. My friends and family have been a constant source of support and encouragement, especially when things were not progressing as smoothly as one may have wished, and for this I will always be grateful. Finally, to my supervisor Dr Gay Elisha. Without her, I never would have started this project, and all along she has assisted and guided me, but also let me go my own way which allowed me to learn from my mistakes. I am indebted to her for all this, but probably most of all for her unbelievably patient advice on how to write scientifically. 111 LIST OF FIGURES Fig 1.1 Basic structure of the beta lactam ring. 3 Fig 1.2 Basic Structure of the four major classes of beta 4 lactam (Condemi & Sheehan, 1996). Fig 1.3 Hydrolysis of the beta lactam ring (Livermore, 7 1993). Fig 1.4 Structure of Gram positive and Gram negative cell 9 walls (Grieco, 1982). Fig 1.5 Double disc test on an ESBL producing isolate of K. 27 pneumoniae Fig 2.1 MIC determination usmg a ceftazidime- 33 ceftazidime/clavulanate E-strip Fig 3.1 PCR consists of repeated cycles