Antibiotic resistance of Flavobacterium and related genera

357 Pages·2015·9.83 MB·English

ANTIBIOTIC RESISTANCE OF
FLAVOBACTERIUM AND RELATED GENERA
by
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K.S.THOMSON, B.Ag.Sc., M.Sc., Tas., M.A.S.M.
Submitted in fulfilment of the requirements for the Degree of
Doctor of Philosophy
University of Tasmania
Hobart
March 1988
,J,
This thesis contains no material which has been accepted for
the award of any other degree or diploma in any university and
to the best of my knowledge contains no copy or paraphrase of
any material published or written by any other person, except
where due reference is made in the text of the thesis.
K.S. THOMSON
University of Tasmania
Hobart
March 1988
TABLE OF CONTENTS
Page
Acknowledgements i
Summary v
I. LI,TERATURE REVIEW 1
a. Introduction 2
b. Beta-lactam antibiotics 9
1. Penicillin binding proteins 11
2. Penams 14
-3. Cephems 20
4. 7-alpha-methoxycephems and oxacephems 23
5. Carbapenems and penems 25
6. Monobactams 28
7. Beta-lactamase inhibitors 31
c. R_esistance of Gram-negative bacteria to
beta-lactam antibiotics 34
1. The clinical problem 34
2. Resistance mechanisms - introduction 36
3. Permeability 37
4. PBP mutations 44
d. Beta-lactamases 45
1. Some methods used to characterize
beta-lactamases 46
2. Beta-lactamases of Gram-_positive bacter_ia 57
3. Richmond and Sykes Classification of beta-
lactamases of bacteria 58
Gram-negat~ve
4. Chromosomally-mediated beta-lactamases 62
5. Beta-lactamases mediated by plasmids and
transposons 67
TABLE OF CONTENTS (Continued)
6. Beta-lactamases and antibiotic resistance
of Gram-negative bacteria 72
e. The Genus Flavobacterium 74
1. Introduction 74
2. Taxonomic history 75
3. Testing for special characters 77
4. Flavobacterium aquatile 82
5. Flavobacterium breve 83
6. Flavobacterium balustinum (and Group IIb) 86
7. Flavobacterium gleum 90
8. Flavobacterium meningosepticum 91
9. Flavobacterium odoratum 102
10. Flavobacterium multivorum 106
11. Flavobacterium spiritivorum 110
12. Flavobacterium thalpophilum 110
13. Flavobacterium incertae sedis 115
s~ecies
14. Genus Weeksella 119
f. Taxonomy and antibiotic resistance of the genus
Flavobacterium: current and future status 120
1. Heterogeneity within species 122
2. Heterogeneity within the genus 123
3. The Flavobacterium-Cytophaga
boundary 125
4. Bacteroides 126
5. The significance of antibiotic resistance
in Flavobacterium 128
II. MATERIALS AND METHODS 131
a. Bacterial strains 132
TABLE OF CONTENTS (Continued)
b. Characterisation tests 132
c. Antibiotics 132
d. Disc diffusion antibiotic susceptibility tests 140
e. 3-dimensional testing 142
f. 3-dimensional and clover leaf (3D+CL) testing 143
g. Minimum inhibitory concentration (MIC) testing 145
h. Enzyme preparations 147
i. Beta-lactamase assays 149
j. Isoelectric focussing and inhibition studies 150
k. Tests with augmentin discs 153
1. %G+C determination 153
III. RESULTS 155
a. Presumptive identifications 156
b. Antibiotic susceptibility tests 157
c. Routine 3-dimensional tests 184
d. Special 3-dimensional tests (including 3D+CL
technique) 186
e. Hydrolysis assays, inhibitor studies and
isoelectric focussing patterns 201
f. Investigations of mechanisms 220
IV. DISCUSSION 226
a. Susceptibility testing 233
b. Beta-lactamases 237
c. F. aquatile 245
d. F. breve 248
e. F. gleum 252
f. F. meningosepticum 254
TABLE OF CONTENTS (Continued)
g. Flavobacterium species belonging to the proposed
genus Sphingobacterium (!_. multivorum,
!_. spiritivorum and F. thalpophilum) 261
h. F. odoratum 264
i. Group IIb 270
j. Taxonomic implications for the Flavobacterium/
Cytophaga complex 273
V. CONCLUSIONS 278
VI. BIBLIOGRAPHY 281
VII. ADDENDUM 329
VIII. APPENDICES 331
Appendix 1: Abbreviations - names of
organisms 333
Appendix 2: Other abbreviations 335
ACKNOWLEDGEMENTS
ii
During the last three years I have been very fortunate to
have been helped and encouraged by an elite group of
microbiologists. Without their help I would not have been
able to complete this work. I now express my gratitude to
them.
I extend special thanks to Dr T.A. McMeekin, my
supervisor, whose direction,. criticism, and provision of
strains and reference materials was vital. This was a project
in which certain technical aspects appeared unduly difficult
because of a lack of locally available expertise or equipment.
On each occasion when it appeared that the work might be
delayed Dr McMeekin somehow very quickly found a way to
overcome the problem. All this was done when he had a
considerably increased workload because of his additional
responsibilities as Dean of the Faculty of Agricultural
Science.
In the eleven weeks I spent at Creighton University,
Nebraska I received special tuition in the theoretical
O~aha,
and laboratory aspects of antimicrobial from PrQfessors
age~ts
C.C. and W.E. Sanders and Associate Professor R.V. Goering.
Since then the assistance and stimulation have continued
across thousands of miles and I thank them for the advice,
experience and ideas which made it possible to undertake this
work.
iii
Isoelectric focussing and photographic facilities were not
available in my own laboratory. I thank Mr J. Presser of the
Hobart Forensic Laboratory, Dr J. Sallis of the University of
Tasmania Biochemistry Department and Bio Rad, Melbourne, for
making their isoelectric focussing equipment available at
various times. I also thank Mr Presser and Mr R. Cruickshank,
Department of Agricultural Science, for help with the
photographs of the isoelectric focussing gels, and Mr D. Lees,
University of Tasmania Clinical School, for the photographs of
the antibiotic susceptibility tests. I am also most grateful
to Miss A. McGuire, University of Tasmania, Department of
Agricultural Science, who performed DNA base ratio
determinations on three strains of Flavobacterium.
Various people provided bacterial strains, either through
Dr McMeekin or directly. Special thanks are extended to Dr R.
Clark, St Josephs Hospital, Omaha, Dr. B. Holmes, National
Collection of Type Cultures, Colindale, London, Dr M. Peel,
Melbourne University, Melbourne, Prof. M.J. Pickett,
University of California, Los Angeles, Dr H. Reichenbach,
Gesellschaft fur Biotechnologische Forschung, Braunschweig,
Prof. C.C. Sanders, Creighton University, Omaha,- and Dr J.
Shewan, Aberdeen.
I also thank my employer, the Department of Veterans
Affairs, and the Fulbright Scholarship Program for my study
visit to the United States. Thanks are also extended to the
pharmaceutical companies which provided the free antibiotic
iv
powders.
Finally I thank my wife, Susan, and my children, Diana,
Stephen and Bert, whose love, understanding, support and
encouragement was a greater inspiration to me than I can
express in words.