Population Biology of Propionibacterium acnes and Pseudomonas aeruginosa in keratitis and ...

366 Pages·2009·9 MB·English

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A Thesis Submitted for the Degree of PhD at the University of Warwick
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Population biology of
Propionibacterium acnes and
Pseudomonas aeruginosa in ophthalmic
infections and the development of novel
diagnostic tools

by

Anna Gao
BMedSc (Hons.) MBChB

A thesis submitted in fulfilment for the Degree of Doctor of
Philosophy in Microbiology

Department of Biological Sciences
University of Warwick

March 2009
TABLE OF CONTENTS

Table of Contents……………………………………………………………........ i

Figures……….……………………………………………………………….......... viii

Tables………………………………………………………………………………. x

Acknowledgements……….………………………………………………………... xii

Declaration……….……………………………………………………………….... xiii

Abstract……….………………………………………………………………......... xiv

Abbreviations……….……………………………………………………………… xv

Dedication……….……………………………………………………………….....xviii

CHAPTER 1: INTRODUCTION
1.1. Infections of the eye…………………………………………………….. 1
1.1.1. Anatomy of the eye……………………………………………….... 1
1.1.2. Microbial keratitis….…………………………….…………………3
1.1.2.1.Epidemiology….……………………………………………….. 3
1.1.2.2.Clinical findings………………………………………………... 4
1.1.2.3.Causative organisms…………………………………………… 6
1.1.2.4.Conventional Diagnosis ……………………………………….. 7
1.1.2.5.Treatment………………………………………………………. 8
1.1.3. Endophthalmitis……………………………………………………. 10
1.1.3.1.Background…………………………………………………….. 10
1.1.3.2.Clinical findings………………………………………………... 11
1.1.3.3.Causative organisms…………………………………………… 13
1.1.3.4.Risk Factors……………………………………………………. 13
1.1.3.5.Prophylaxis…………………………………………………….. 14
1.1.3.6.Conventional Diagnosis………………………………………... 15
1.1.3.7.Treatment………………………………………………………. 17

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1.2. Propionibacterium acnes……………………………………………….. 20
1.2.1. Background……………………………………………………….... 20
1.2.2. Population biology of P. acnes…….………………………………. 21
1.2.3. Pathogenesis of P. acnes infection....………………………………. 23
1.2.4. P. acnes in postoperative endophthalmitis………………………….25
1.2.5. P. acnes in other clinical diseases………………………………….. 27

1.3. Pseudomonas aeruginosa……………………………………………….. 31
1.3.1. Background……………………………………………………….... 31
1.3.2. Population biology of P. aeruginosa……….……………………… 32
1.3.3. P. aeruginosa in the eye…………………..…………………...…… 33
1.3.4. Pathogenesis of P. aeruginosa infection...…………………………. 34

1.4. Molecular diagnostics………………………………………………...…. 36
1.4.1. Background………………………………………………………… 36
1.4.2. Molecular diagnostics in microbial keratitis……………………….. 37
1.4.3. Molecular diagnostics in endophthalmitis………………………..... 38
1.4.4. P. acnes molecular diagnostics…………………………………….. 39
1.4.5. P. aeruginosa molecular diagnostics……………………………… 40

1.5. Bacterial population biology…………………………………………..... 42
1.5.1. Methods of strain identification……………………………………. 42
1.5.1.1.Genotyping methods…………………………………………… 42
1.5.2. Multilocus sequence typing………..…………………………...….. 45
1.5.2.1.The design of MLST schemes…………………………………. 46
1.5.2.2.Databases and analysis tools…………………………………… 47
1.5.2.3.Epidemiological and clinical applications……………………... 47

1.6. Aims and Objectives…………………………………………………..... 50

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CHAPTER 2: MATERIALS AND METHODS

2.1. Design of an MLST scheme…………………………………………….. 51
2.1.1. Chromosomal mapping and selection of genes……………………. 51
2.1.2. Design of primers for gene amplification and nucleotide
sequencing…………………………………………………………..51

2.2. Bacterial strains…………………………………………………………. 54
2.2.1. P. acnes strains……………………………………………………... 54
2.2.2. P. aeruginosa strains……………………………………………….. 54

2.3. Storage of bacterial strains……………………………………………… 55

2.4. DNA preparation………………………………………………………... 55
2.4.1. P. acnes DNA preparation…………………………………………. 55
2.4.2. P. aeruginosa DNA preparation………………………………….... 56

2.5. Analysis of DNA by agarose gel electrophoresis……………………….. 57

2.6. Quantification of DNA………………………………………………….. 57

2.7. Amplification of DNA using the polymerase chain reaction…………… 57
2.7.1. Optimisation of PCR conditions…………………………………… 57
2.7.2. PCR reactions (P. acnes)…………………………………………... 58
2.7.3. PCR reactions (P. aeruginosa)…………………………………….. 59

2.8. Purification of PCR product…………………………………………….. 60
2.8.1. QIAquick PCR purification kit…………………………………….. 60
2.8.2. MiniElute 96 UF PCR purification kit……………………………... 61

2.9. P. acnes recA gene amplification and sequencing…………………...…. 61

2.10. Automated sequencing………………………………………………….. 62
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2.10.1. DNA sequencing reaction………………………………………….. 62
2.10.2. Ethanol precipitation of sequencing reaction………………………. 63
2.10.3. Sequencing…………………………………………………………. 63

2.11. Allele and Sequence Type (ST) assignment…………………………….. 64

2.12. Immunofluorescence Microscopy (IFM) of P. acnes…………………... 64
2.12.1. Production of Monoclonal Antibodies……………………………... 64
2.12.2. Immunofluorescence Microscopy (IFM) ………………………….. 64

2.13. Random amplification of polymorphic DNA (RAPD) analysis……….. 65

2.14. P. aeruginosa exoU and exoS gene amplification……………………... 66

2.15. P. acnes rapid diagnostics………………………………………………. 68
2.15.1. Target gene selection and primer design…………………………... 68
2.15.2. PCR conditions…………………………………………………….. 68
2.15.3. DNA extraction methods…………………………………………... 69

2.16. Bioinformatics………………………………………………………...… 71
2.16.1. Genome sequences………………………………………………... 71
2.16.2. Homology searches……………………………………………...... 71
2.16.3. Sequence analysis tools………………………………………….... 71
2.16.4. START v2………………………………………………................ 71
2.16.5. LIAN v3.5……………….………………………………………... 72
2.16.6. MEGA v4.1……………………………………………………….. 72
2.16.7. SplitsTree v4.1……………………………………………………. 72
2.16.8. eBURST v3……………………………………………………….. 72
2.16.9. ClonalFrame v1.1………………………………………………..... 73
2.16.10. PAUP v4………………………………………………………..… 73
2.16.11. GelCompar II……………………………………………………... 73

2.17. Reagents and Buffers……………………………………………………. 74
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CHAPTER 3: MICROBIAL KERATITIS STUDY

3.1. Introduction……………………………………………………………... 75
3.2. Aims………………………………………………………...…………... 75
3.3. Methods……………………………………………………………..…... 76
3.4. Results and Discussion………………………………………………….. 77
3.4.1. Epidemiology and clinical findings………………………………... 77
3.4.2. Predisposing risk factors………………………………………….... 79
3.4.3. Microbiology results……………………………………………...... 82
3.4.4. Treatment…………………………………………………………... 85
3.4.5. Complications…………………………………………………….... 85
3.5. Future work……………………………………………………………... 85
3.6. Conclusions……………………………………………………………... 86

CHAPTER 4: MLST ANALYSIS OF PROPIONIBACTERIUM ACNES

4.1. Introduction……………………………………………………………... 87
4.2. Aims………………………………………………………...…………... 88
4.3. Results and Discussion………………………………………………….. 89
4.3.1. P. acnes MLST scheme………………………………………..…... 89
4.3.1.1.P. acnes strains and selection of genes………………….……... 89
4.3.1.2.MLST database……………………………………………….... 89
4.3.1.3.Allelic variation………………………………………………... 98
4.3.1.4.Selective pressure of MLST loci……………………………….. 98
4.3.2. Analysis of P. acnes isolates for sequence types (STs) ……..…….. 99
4.3.2.1.Recombination occurring across loci…………………………... 102
4.3.3. Phylogenetic analysis……………………………………………..... 105
4.3.3.1.eBURST analysis of STs……………………………………..... 106
4.3.3.2.Phylogenetic trees…………………………………………….... 113
4.3.3.3.Splits decomposition analysis………………………………….. 120
4.3.3.4.Clonal frame analysis…………………………………………... 123
4.3.3.5.Sequence diversity……………………………………………... 127
4.3.4. Association between STs, clonal complexes and disease..………… 129
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4.3.5. Analysis of recombination…………………………………………. 133
4.3.5.1.Index of association……………………………………………. 133
4.3.5.2.Degree of congruence among loci……………………………... 136
4.3.5.3.Splits decomposition analysis………………………………….. 143
4.3.5.4.Clonal frame analysis…………………………………………... 143
4.3.5.5.Point mutation versus recombination…………………………... 145
4.3.6. Validation of the P. acnes MLST scheme………………………..... 149
4.3.6.1.Comparison of MLST with 16S rRNA gene analysis………..... 149
4.3.6.2.recA gene analysis……………………………………………....152
4.3.6.3.Immunofluorescence microscopy (IFM) analysis……………... 155
4.3.6.4.Random amplification of polymorphic DNA (RAPD) analysis.. 159
4.4. Future work………………………………………………………………... 161
4.5. Conclusions ……………………………………………………………...... 162

CHAPTER 5: PROPIONIBACTERIUM ACNES MOLECULAR DIAGNOSTICS

5.1. Introduction……………………………………………………………... 164
5.2. Aims…………………………………………………………………….. 165
5.3. Results and Discussion………………………………………………….. 166
5.3.1. Specificity of PCR assay………………………………………….... 166
5.3.2. Melting-curve analysis……………………………………………... 168
5.3.3. Quantitative real-time PCR……………………………………….... 170
5.3.4. Rapid real-time PCR……………………………………………...... 173
5.3.5. Comparison of DNA extraction methods………………………….. 173
5.4. Future work……………………………………………………………... 174
5.5. Conclusions……………………………………………………………... 175

CHAPTER 6: MLST ANALYSIS OF PSEUDOMONAS AERUGINOSA

6.1. Introduction……………………………………………………………... 176
6.2. Aims…………………………………………………………………….. 177
6.3. Results and Discussion………………………………………………...... 178
6.3.1. P. aeruginosa strains and MLST scheme………………………….. 178
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6.3.2. Analysis of P. aeruginosa isolates by ST..……………………….... 193
6.3.3. Phylogenetic analysis…………………………………………..…... 193
6.3.3.1.Phylogenetic trees…………………………………………….... 193
6.3.3.2.eBURST analysis…………………………………………..…... 197
6.3.4. Analysis of recombination. ………………………………………... 201
6.3.4.1.Index of association………………..…………………………... 201
6.3.4.2.Splits decomposition analysis………………………………...... 201
6.3.5. Type III secretion system (TTSS) …………………………………. 206
6.3.5.1.Determination of TTSS effector genotypes…………………..... 206
6.3.5.2.Correlation between MLST, TTSS genotyping and serotyping.. 208
6.4. Future work……………………………………………………………... 211
6.5. Conclusions……………………………………………………...…….... 211

CHAPTER 7: GENERAL DISCUSSION

7.1. Main objectives………………………………………………………..... 213
7.2. Microbial keratitis study………………………………………………... 213
7.3. P. acnes MLST………………………………………………………..... 214
7.4. Comparisons of P. acnes MLST with 16S rRNA and
recA gene analyses...………………….……………………………….... 216
7.5. Comparisons of P. acnes MLST with P acnes IFM analysis ………….. 218
7.6. Comparisons of P. acnes MLST with RAPD analysis………………….. 218
7.7. P. acnes molecular diagnostics ……………………………………….... 219
7.8. P. aeruginosa MLST……………………………………………………. 220
7.9. P. aeruginosa exoU and exoS genotyping ……………………………... 221
7.10. Conclusions……………………………………………………………... 222

REFERENCES…………………………………………………………………...... 223

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FIGURES

1.1 Anatomy of the eyeball………………………………………………….…. 2

1.2 Pseudomonas corneal ulcer……………………………………………….... 5

1.3 Bacterial corneal ulcer with hypopyon…………………………………….. 5

1.4 Postoperative endophthalmitis…………………………………...………… 12

1.5 Serratia marcescence endophthalmitis…………………………………….. 12

1.6 Comparison of the steps involved in molecular typing methods…………... 48

1.7 Flow diagram of the steps in the MLST protocol…………………….……. 49

3.1 Number of cases of suspected microbial keratitis and
Central England Temperature during each month of the study……….…… 78

3.2 Microbial keratitis cases by age groups……………………………….…… 81

3.3 Spectrum of isolated organisms cultured from corneal scrapes
in microbial keratitis cases………………………………………...……….. 84

4.1 Gene map of the P. acnes chromosome with locations of the seven
housekeeping genes in the MLST scheme……………………………...….. 90

4.2 Screen shots of online database for P. acnes MLST scheme……………91-92

4.3 Frequency of isolates within the study population of 125 P. acnes isolates.. 101

4.4 ST-35 clonal frame shot showing aroE recombination……………………. 104

4.5 eBURST diagram showing the clusters of related STs and individual
unlinked STs of 125 P. acnes isolates (SLV)…………………………..…. 111

4.6 eBURST diagram showing the clusters of related STs and individual
unlinked STs of 125 P. acnes isolates (DLV)……………………………… 112

4.7 Unrooted phylogenetic dendogram of the 38 P. acnes STs, analysed by
Neighbour-Joining method based on concatenated sequences………….…. 115

4.8 Unrooted UPGMA dendrogram, based on allelic profiles of the
38 P. acnes STs with Jukes-Cantor-based algorithm………………….……116

4.9 Unrooted phylogenetic dendrogram, analysed by Neighbour-Joining
method based sequences of each of the seven loci……………………..117-119

4.10 Splits decomposition analysis based on concatenated sequences of
the seven housekeeping genes of the 38 STs……………………………..... 121
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