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Download (7MB) 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] THE CHARACTERISATION OF THE GENUS MICROCOCCUS AND THE GENUS STOMATOCOCCUS USING PHENOTYPIC AND GENOTYPIC TECHNIQUES. ALISTAIR THOMAS LEANORD B.Sc, MB.ChB. A thesis submitted in fulfilment of the requirements for the degree of Doctor of Medicine at the University of Glasgow. The work for this thesis was undertaken at:- THE DEPARTMENT OF MEDICAL MICROBIOLOGY/ ABERDEEN UNIVERSITY MEDICAL SCHOOL/ ABERDEEN. SUBMITTED 1992 ALISTAIR LEANORD © ProQuest Number: 11011446 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 com 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 ProQuest 11011446 Published by ProQuest LLC(2018). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 2 SUMMARY This study shows that the biochemical inertness of the genus Micrococcus makes the API microtitre strip an unsuitable technique by which to identify species within the genus and that colony pigmentation is an invalid character with which to speciate the genus Micrococcus. The genus Micrococcus forms a heterogeneous group of organisms when ribotype group and pulsed-field gel electrophoresis (PFGE) is considered. The use of two restriction enzymes forms different ribotype groupings within the genus. In this study M.kristinae, M.varians and the majority of M.sedentarius formed homogeneous core ribotype profiles. M.luteus and M.lylae formed heterogeneous ribotype and PFGE groups. The ability of M.lylae and M.sedentarius to belong to M.luteus ribotype groups demonstrates a genetic relationship between these species and M. luteus. Restriction enzyme analysis (REA) and PFGE were the most useful techniques for typing the micrococci. By PFGE M.roseus has a genome size of 3,387 kilobases (kb) which is approximately 30% larger than other members of the genus, which have genomes of between 1,823 and 2,598 kb, and may warrant generic status. By REA and ribotyping it was shown that people carry a clonal population of M.luteus over a period of time. 3 1) INTRODUCTION 1.1) The genus Micrococcus............................ 14 1.2) The present taxonomic status of the family Micrococcaceae and the genus Micrococcus.............. 16 1.3) Skin carriage of the genus Micrococcus............ 21 1.4) Micrococci isolated from clinical sources........ 22 1.5) The separation of staphylococci from micrococci.............................................. 30 1.5.1) Cell wall structure............................. 33 1.5.2) Cellular enzymes................................ 37 1.6) Molecular methods of bacterial characterisation. 38 1.7) The clone model of population structure........... 45 1.8) The Antarctic base..................... 47 1.9) The aims of the study............................ 48 2) MATERIALS AND METHODS 2.1) Sampling.......................................... 53 2.2) Differentiation of staphylococci from micrococci. 56 2.3) Identification to the species level............. 57 4 2.4) Antibiotic sensitivities.......................... 57 2.5) Sodium-dodecyl-sulphate polyacrylamide gel electrophoresis........................................ 58 2.6) Protein estimation................................. 60 2.7) DNA extraction.................................... 60 2.8) Calculation of theoretical cutting frequencies. 62 2.9) Restriction enzyme digestion..................... 63 2.10) Agarose gel electrophoresis...................... 64 2.11) Estimation of molar percentage guanine/cytosine content.................................................. 64 2.12) Southern blotting................................ 65 2.13) 5'-end labelling of 16S and 23Sribosomal RNA. 66 2.14) Ribosomal RNA probing............................ 67 2.15) Preparation of DNA inserts for pulsed field gel electrophoresis......................................... 68 2.16) Digestion and electrophoresis ofDNA inserts. 69 3) RESULTS 3.1) Revival of isolates in the UK .................... 72 5 3.2) Identification of isolates to the genus level. 73 3.3) Identification to the species level.............. 74 3.4) Sodium-dodecyl-sulphate polyacrylamide gel elctrophoresis.......................................... 84 3.5) Restriction enzyme analysis (REA)................ 88 3.5.1) Choice of restriction enzyme for REA............ 88 3.5.2) Analysis of restriction enzyme profiles......... 91 3.5.3) Differentiation of the genus Staphylococcus and the genus Micrococcus by the use of restriction enzymes................................................ 107 3.6) Identification of micrococci using 16S and 23S rRNA probing................................................ Ill 3.7) Characterising presumptive Stomatococcus isolates............................................... 131 3.8) Choice of restriction enzyme for PFGE........... 138 3.9) Micrococcal genome size as determined by PFGE. 141 3.10) Characterisation of micrococci and stomatococci isolated from clinical material...................... 147 3.11) The skin carriage and prevalence of the micrococcal ribotypes.............................................. 149 6 4) DISCUSSION 4.1) Skin sampling and isolate survival............... 159 4.2) Assignation of isolates to the genus Micrococcus............................................ 162 4.3) Identification of isolates to the species level using the API microtitre strip........................ 162 4.4) Differentiation of staphylococci and micrococci by restriction enzymes.................................... 167 4.5) Analysis of restriction enzyme profiles.......... 171 4.6) Analysis of 16S and 23S rRNA probing............ 173 4.7) Analysis of Stomatococcus spp. isolated from skin.................................................... 182 4.8) Pulsed-field gel electrophoresis to size genomes and determine genomic heterogeneity of members of the genus Micrococcus............................................ 185 4.9) Micrococci isolated from clinical material. 188 4.10) The temporal skin carriage of micrococci in an isolated community..................................... 189 4.11) Future work...................................... 191 5) Bibliography ....................................... 194 6) Appendices ......................................... 230 7 LIST OF TABLES AND FIGURES FIGURES 1) Map of Antarctic Peninsula showing the position of Faraday Base........................................... 50 2) Faraday base....................................... 51 3) SDS-PAGE of whole-cell protein profiles of 13 micrococci............................................. 86 4) SDS-PAGE of whole-cell protein profiles of 13 micrococci showing heterogeneity within the genus. 87 5) M.luteus DNA digested with the restriction enzymes BamEl, Sail, Bglll, Hindlll, EcoRl and FcoRV........... 97 6. a, b/ c, d) Digestion of M.luteus DNA with BamEl, Pstl, Hindlll and EcoRI respectively.............. 98-101 7) EcoRI REA groups 2 and 3 ....................... 104 8) DNA of isolates identified as M.luteus by the API microtitre strip, digested with EcoRI............. 105 9) DNA of isolates identified as M.sedentarius digested with EcoRI.......................................... 106 10) The use of restriction enzyme digestion to estimate the % mol GC content............................... 110 11) BamHI ribotype profiles of the genus Micrococcus. 126 12) BamEl ribotype profiles of M.luteus............. 127 13) Sail ribotype profiles of the genusMicrococcus. 128 14) Sail ribotype profiles of M. sedentarius............. 129 15) M.varians DNA digested with Sail.............. 130 16) DNA of presumptive Stomatococcus isolates digested with BamEl............................................. 136 17) PFGE patterns of restriction enzyme digests of genomic DNA of M.luteus isolate 972.................. 143 18) Pulsed field gel electrophoresis of Hindlll digested genomic DNA extracted from M.lylae, M.luteus, M.roseus, M.varians, M.kristinae and M. sedentarius............. 144 19) PFGE patterns of Hindlll restriction enzyme digests of genomic DNA of BamEl ribotype groups 1 and 2 ........ 145 20) The Sail ribotype profiles of 13 micrococci isolated from clinical material................................ 153 TABLES 1) The separation of 201 Gram-positive catalase-positive cocci to the genus level.............................. 81 2) The identification of type strains by the API microtitre biochemical test strip.................... 82
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