Epidemiology and Ecology of Microbial Communities of the Upper Respiratory Tract

268 Pages·2015·3.73 MB·English

by  Abigail Lois Coughtrie

UNIVERSITY OF SOUTHAMPTON

FACULTY OF MEDICINE
Clinical and Experimental Sciences

Epidemiology and Ecology of Microbial Communities of the Upper Respiratory
Tract
Volume 1 of 1
Abigail Lois Coughtrie
Thesis for the degree of Doctor of Philosophy
August 2015

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UNIVERSITY OF SOUTHAMPTON
ABSTRACT
FACULTY OF MEDICINE
Clinical and Experimental Sciences
Doctor of Philosophy
EPIDEMIOLOGY AND ECOLOGY OF MICROBIAL COMMUNITIES OF THE UPPER
RESPIRATORY TRACT
by Abigail Lois Coughtrie

Respiratory tract infections (RTI) are responsible for over 4 million deaths per year
worldwide. Microbial carriage in the upper respiratory tract is a precursor to respiratory
infection and facilitates person-to-person transmission. A large community-based
swabbing study was conducted, enabling the collection of a large number of swab
samples that would provide key information concerning the epidemiology and ecology
of respiratory tract communities. Traditional culture-based techniques, molecular
methods and ecological and mathematical modelling methods were used.
Participation of members of the community within the swabbing study was shown to
be greater within the self-swabbing group, in older individuals and in less deprived
locations. Carriage of bacterial and viral species within the respiratory tract was shown
to vary with participant age, recent RTI and the presence of other species. Self-taken
swabs were largely non-inferior to healthcare professional (HCP)-taken swabs in
assessing carriage of the targeted bacteria, offering a cheaper and more flexible
alternative to HCP swabbing. Large numbers of capsular types (serotypes), sequence
types and low levels of vaccine-targeted types demonstrate the genetic diversity of
respiratory bacteria as well as their evolution in response to immunisation.
Microbial respiratory community structure was shown to be highly variable with less
nested communities and facilitative relationships between species within young
individuals and those with recent RTI potentially enhancing transmission and survival of
carried species. Neutral and niche processes were both found to be important in
respiratory tract community assembly. These insights into respiratory tract communities
will allow predictions of microbial variation as a result of infection, varying age and
season. Future work will involve 16S rDNA community analysis, further development of
ecological methods and the conduction of larger multi-centre carriage studies.
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Table of Contents

LIST OF TABLES ................................................................................................................................ 9
LIST OF FIGURES ............................................................................................................................ 11
DECLARATION OF AUTHORSHIP ................................................................................................. 13
ACKNOWLEDGMENTS ................................................................................................................... 15
ABBREVIATIONS ............................................................................................................................. 17
CHAPTER 1. INTRODUCTION ........................................................................................................ 19
1.1 RESPIRATORY TRACT INFECTIONS .......................................................................................... 19
1.1.1 Upper Respiratory Tract Infections ............................................................................. 19
1.1.2 Lower Respiratory Tract Infections ............................................................................. 20
1.2 INVASIVE DISEASE .................................................................................................................... 21
1.3 EPIDEMICS AND PANDEMICS OF RESPIRATORY DISEASE ....................................................... 21
1.3.1 Epidemics and Pandemics of Bacterial Infection...................................................... 21
1.3.2 Epidemics and Pandemics of Viral Infection ............................................................. 22
1.3.3 Secondary Bacterial Infections .................................................................................... 23
1.4 MICROBIAL CARRIAGE IN THE RESPIRATORY TRACT .............................................................. 24
1.4.1 The Biology of Respiratory Carriage .......................................................................... 24
1.4.2 Carriage Study Methodology ....................................................................................... 26
1.5 CARRIAGE STUDIES FOR THE EVALUATION OF VACCINATION STRATEGIES ........................... 27
1.5.1 Methods for Assessing Vaccine Effectiveness ......................................................... 27
1.5.2 Relationship between Carriage and Disease ............................................................ 27
1.5.3 Elucidating Respiratory Epidemiology ........................................................................ 29
1.5.4 Evaluating Vaccine Effectiveness in Disease and Carriage ................................... 30
1.6 THE RESPIRATORY TRACT ECOSYSTEM .................................................................................. 31
1.6.1 The Role of Diversity in Health and Disease ............................................................. 31
1.6.2 Assessing Diversity of an Ecological Niche............................................................... 33
1.7 BIOFILMS AND RESPIRATORY TRACT INFECTIONS ................................................................... 33
1.8 DYNAMICS OF THE RESPIRATORY TRACT ECOSYSTEM ........................................................... 35
1.8.1 Strain Mutation and Recombination ........................................................................... 35
1.8.2 Bacterial-Viral Interactions ........................................................................................... 36
1.8.3 Bacterial-Bacterial Interactions.................................................................................... 37
1.8.4 Host-Pathogen Interactions ......................................................................................... 38
1.9 VACCINATION STRATEGIES ...................................................................................................... 39
1.9.1 Non-Conjugate Vaccines against Respiratory Pathogens ...................................... 39
1.9.2 H. influenzae Type b Conjugate Vaccine................................................................... 39
1.9.3 Pneumococcal Conjugate Vaccines ........................................................................... 40
1.9.4 Meningococcal Vaccines .............................................................................................. 41
1.9.5 Vaccine Development Difficulties................................................................................ 42
1.10 SELECTION PRESSURES OF IMMUNISATION PROGRAMMES .................................................... 42
1.11 ADVANCES IN MICROBIAL EPIDEMIOLOGY RESEARCH METHODS ........................................... 45
1.11.1 Quantitative and Real-time PCR ................................................................................. 45
1.11.2 Microbial Typing ............................................................................................................ 46
1.11.3 Genome Sequencing .................................................................................................... 47
1.11.4 Statistical Methods in Cross-sectional Studies ......................................................... 49
1.12 AIMS AND OBJECTIVES ............................................................................................................. 50
CHAPTER 2. MATERIALS AND METHODS .................................................................................. 53
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2.1 PARTICIPANT RECRUITMENT AND SAMPLE COLLECTION ......................................................... 53
2.1.1 Study Rationale .............................................................................................................. 53
2.1.2 Duration and Timing of Swabbing Time-points ......................................................... 54
2.1.3 Participating General Practitioner Practices .............................................................. 54
2.1.4 Sample Size .................................................................................................................... 56
2.1.5 Participant Recruitment ................................................................................................. 56
2.1.6 Study Groups .................................................................................................................. 58
2.1.7 Swabbing ........................................................................................................................ 59
2.2 SAMPLE PROCESSING ............................................................................................................... 59
2.3 IDENTIFICATION OF RESPIRATORY BACTERIA USING CULTURE ............................................... 60
2.3.1 Microbial Identification ................................................................................................... 60
2.3.2 Statistical Analysis ......................................................................................................... 62
2.4 IDENTIFICATION OF RESPIRATORY BACTERIA AND VIRUSES USING REAL-TIME PCR ............. 64
2.4.1 Selection of Samples ..................................................................................................... 64
2.4.2 Nucleic Acid Extraction ................................................................................................. 65
2.4.3 Real-time PCR ............................................................................................................... 65
2.4.4 Standard Curves for Quantification of Bacterial Species ......................................... 70
2.4.5 Statistical Analysis ......................................................................................................... 70
2.5 TYPING OF BACTERIAL ISOLATES ............................................................................................. 71
2.5.1 Selection of Isolates ...................................................................................................... 71
2.5.2 PCR Serotyping of S. pneumoniae and H. influenzae Isolates .............................. 72
2.5.3 MRSA Typing of S. aureus ........................................................................................... 73
2.6 WHOLE GENOME SEQUENCING ................................................................................................ 74
2.6.1 Selection of Isolates ...................................................................................................... 74
2.6.2 DNA Extraction ............................................................................................................... 74
2.6.3 DNA Quantification ........................................................................................................ 75
2.6.4 Genomic DNA Tagmentation ....................................................................................... 75
2.6.5 PCR Amplification of Tagmented DNA ....................................................................... 75
2.6.6 PCR Clean-up ................................................................................................................ 76
2.6.7 Library Normalisation .................................................................................................... 76
2.6.8 Library Pooling and Loading the MiSeq Cartridge .................................................... 76
2.6.9 De novo Sequencing on the MiSeq............................................................................. 77
2.7 WHOLE GENOME SEQUENCE ANALYSIS .................................................................................. 77
2.7.1 Trimming of Sequencing Adaptors .............................................................................. 77
2.7.2 Determining the Quality of Sequences ....................................................................... 78
2.7.3 Genome Assembly using Velvet .................................................................................. 78
2.7.4 Assembly Improvement ................................................................................................ 78
2.7.5 Determining the Quality of Assemblies ....................................................................... 79
2.7.6 Serotyping ....................................................................................................................... 79
2.7.7 Sequence Typing ........................................................................................................... 80
2.7.8 Antibiotic Resistance Analysis ..................................................................................... 81
2.7.9 Vaccine Candidates and Antigens .............................................................................. 82
2.8 ECOLOGICAL ANALYSIS ............................................................................................................ 83
2.8.1 Nestedness ..................................................................................................................... 83
2.8.2 Species Distribution ....................................................................................................... 84
2.8.3 Application of Ecological Theory ................................................................................. 85
CHAPTER 3. PARTICIPATION IN A COMMUNITY SWABBING STUDY .................................. 87
3.1 INTRODUCTION .......................................................................................................................... 87
3.2 ANALYSIS OF COMMUNITY PARTICIPATION .............................................................................. 88
3.2.1 Characteristics of Participants ..................................................................................... 88
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3.2.2 Participation of the Study Groups ............................................................................... 89
3.2.3 Age Distribution of Participants ................................................................................... 91
3.3 THE IMPACT OF DEPRIVATION ON PARTICIPATION .................................................................. 93
3.4 THE IMPACT OF POPULATION DENSITY ON PARTICIPATION .................................................... 95
3.5 DISCUSSION .............................................................................................................................. 97
CHAPTER 4. MICROBIAL CARRIAGE IN THE UPPER RESPIRATORY TRACT .................. 101
4.1 INTRODUCTION ....................................................................................................................... 101
4.2 SAMPLE INFORMATION ........................................................................................................... 102
4.3 ANALYSIS METHODOLOGIES .................................................................................................. 103
4.3.1 Bacterial Carriage ........................................................................................................ 103
4.3.2 Viral Prevalence .......................................................................................................... 104
4.3.3 Co-carriage ................................................................................................................... 105
4.4 CARRIAGE OF BACTERIAL SPECIES IN THE RESPIRATORY TRACT ........................................ 106
4.4.1 Analysis of Swabbing Methodologies for Detecting the Prevalence of Bacterial
Carriage 106
4.4.2 Patterns of Carriage according to Participant Demographics............................... 114
4.5 PREVALENCE OF VIRAL SPECIES IN THE RESPIRATORY TRACT ............................................ 124
4.5.1 Analysis of Swabbing Methodologies for Detecting the Prevalence of Viral
Species 124
4.5.2 Patterns of Viral Species Prevalence according to Participant Demographics .. 125
4.6 CO-CARRIAGE OF MICROBIAL SPECIES IN THE RESPIRATORY TRACT .................................. 133
4.6.1 Analysis of Swabbing Methodologies for Detecting Co-carriage ......................... 133
4.6.2 Patterns of Co-carriage according to Participant Demographics ......................... 135
4.7 DISCUSSION ............................................................................................................................ 140
CHAPTER 5. PHENOTYPIC AND MOLECULAR DIVERSITY OF RESPIRATORY BACTERIA147
5.1 INTRODUCTION ....................................................................................................................... 147
5.2 ISOLATES ANALYSED .............................................................................................................. 148
5.3 SEQUENCING QUALITY CONTROL .......................................................................................... 148
5.4 S. PNEUMONIAE ...................................................................................................................... 150
5.4.1 Distribution of Serotypes ............................................................................................ 150
5.4.2 Sequence Type Diversity ........................................................................................... 151
5.4.3 Antibiotic Resistance .................................................................................................. 156
5.4.4 Analysis of Vaccine Candidates and Antigens ....................................................... 157
5.5 H. INFLUENZAE ....................................................................................................................... 158
5.5.1 Distribution of Serotypes ............................................................................................ 158
5.5.2 Sequence Type Diversity ........................................................................................... 158
5.5.3 Antibiotic Resistance .................................................................................................. 162
5.5.4 Analysis of Vaccine Candidates and Antigens ....................................................... 162
5.6 METHICILLIN RESISTANT S. AUREUS ...................................................................................... 163
5.7 DISCUSSION ............................................................................................................................ 164
CHAPTER 6. ECOLOGY OF MICROBIAL RESPIRATORY TRACT COMMUNITIES ............. 169
6.1 INTRODUCTION ....................................................................................................................... 169
6.2 SAMPLES AND COVARIATES USED IN ECOLOGICAL ANALYSES ............................................. 171
6.3 NESTEDNESS OF RESPIRATORY COMMUNITIES .................................................................... 172
6.3.1 Patterns of Nestedness within Communities ........................................................... 174
6.3.2 Swabbing Season ....................................................................................................... 176
6.3.3 Participant Age ............................................................................................................ 178
6.3.4 Recent RTI ................................................................................................................... 180
6.4 SPECIES DISTRIBUTION WITHIN RESPIRATORY COMMUNITIES.............................................. 182
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6.4.1 Patterns of Species Distribution within Individuals ................................................. 182
6.4.2 Swabbing Season ........................................................................................................ 184
6.4.3 Participant Age ............................................................................................................. 184
6.4.4 Recent RTI .................................................................................................................... 185
6.5 BACTERIAL ABUNDANCES WITHIN RESPIRATORY COMMUNITIES ........................................... 185
6.5.1 Bacterial Species Abundances .................................................................................. 185
6.5.2 Ecological Theory an Patterns of Community Assembly ....................................... 188
6.6 DISCUSSION ............................................................................................................................ 192
CHAPTER 7. DISCUSSION ........................................................................................................... 197
7.1 PARTICIPATION IN A COMMUNITY SWABBING STUDY ............................................................. 197
7.2 MICROBIAL CARRIAGE IN THE UPPER RESPIRATORY TRACT ................................................. 199
7.3 PHENOTYPIC AND MOLECULAR DIVERSITY OF RESPIRATORY BACTERIA ............................. 201
7.4 ECOLOGY OF MICROBIAL RESPIRATORY TRACT COMMUNITIES ............................................ 202
7.5 FUTURE WORK ........................................................................................................................ 204
7.6 FINAL CONCLUSION ................................................................................................................ 204
APPENDICES ................................................................................................................................. 207
APPENDIX 1. STUDY QUESTIONNAIRE ................................................................................................. 207
APPENDIX 2. STUDY LABORATORY SAMPLE SHEET ........................................................................... 208
APPENDIX 3. STANDARD CURVES FOR QUANTIFICATION OF BACTERIAL SPECIES ............................ 209
APPENDIX 4. MASTERMIX COMPONENTS FOR PCR SEROTYPING OF S. PNEUMONIAE ..................... 212
APPENDIX 5. PRIMERS FOR IN SILICO SEROTYPING OF S. PNEUMONIAE ............................................ 215
APPENDIX 6. PRIMERS FOR IN SILICO SEROTYPING OF H. INFLUENZAE ............................................. 215
APPENDIX 7. PLOTTEMP.R SCRIPT USED FOR NESTEDNESS ANALYSIS ............................................ 216
APPENDIX 8. ANTIGEN ALLELES PRESENT IN S. PNEUMONIAE ISOLATES ........................................... 217
APPENDIX 9. ANTIGEN ALLELES PRESENT IN H. INFLUENZAE ISOLATES ............................................. 220
APPENDIX 10. CHI-SQUARED TEST RESULTS SHOWING DEVIATIONS FROM THE RANDOM (POISSON)
DISTRIBUTION IN REAL-TIME PCR SAMPLES ....................................................................................... 223
REFERENCES ................................................................................................................................ 225

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List of Tables

TABLE 1. STANDARD MICROBIOLOGICAL IDENTIFICATION TECHNIQUES FOR S. PNEUMONIAE, H.
INFLUENZAE, M. CATARRHALIS, P. AERUGINOSA, S. AUREUS AND N. MENINGITIDIS ....................... 60
TABLE 2. PRIMERS AND PROBES USED IN THE BACTERIAL REAL-TIME PCR REACTIONS ....................... 66
TABLE 3. PRIMERS AND PROBES USED IN THE VIRAL REAL-TIME PCR REACTIONS ............................... 67
TABLE 4. LIMITS OF DETECTION OF THE COMMERCIAL PCR KITS .......................................................... 68
TABLE 5. MLST GENES FOR S. PNEUMONIAE AND H. INFLUENZAE .......................................................... 81
TABLE 6. CHARACTERISTICS OF THE STUDY PARTICIPANTS .................................................................... 88
TABLE 7. REAL-TIME PCR AND CULTURE SAMPLE INFORMATION ......................................................... 102
TABLE 8. COMPARISON OF CULTURE AND REAL-TIME PCR METHODS FOR THE DETECTION OF
BACTERIAL SPECIES USING MCNEMAR’S CHI-SQUARED TEST ...................................................... 113
TABLE 9. LOGISTIC REGRESSION MODEL OF S. PNEUMONIAE CARRIAGE IN SELF-TAKEN NOSE SWABS
......................................................................................................................................................... 115
TABLE 10. LOGISTIC REGRESSION MODEL OF M. CATARRHALIS CARRIAGE IN SELF-TAKEN WHOLE
MOUTH SWABS ................................................................................................................................ 117
TABLE 11. LOGISTIC REGRESSION MODEL OF H. INFLUENZAE CARRIAGE IN SELF-TAKEN NOSE SWABS
......................................................................................................................................................... 119
TABLE 12. SUMMARY STATISTICS OF P. AERUGINOSA CARRIAGE IN SELF-TAKEN WHOLE MOUTH
SWABS ............................................................................................................................................. 121
TABLE 13. LOGISTIC REGRESSION MODEL OF S. AUREUS CARRIAGE IN SELF-TAKEN NOSE SWABS .. 123
TABLE 14. VIRAL CARRIAGE BY SWABBING METHOD ............................................................................. 125
TABLE 15. SUMMARY STATISTICS OF RSV PREVALENCE IN NOSE SWABS ........................................... 126
TABLE 16. SUMMARY STATISTICS OF ADENOVIRUS PREVALENCE IN NOSE SWABS ............................. 128
TABLE 17. SUMMARY STATISTICS OF RHINOVIRUS/ENTEROVIRUS CARRIAGE IN NOSE SWABS ........... 130
TABLE 18. SUMMARY STATISTICS OF CORONAVIRUS CARRIAGE IN NOSE SWABS ............................... 132
TABLE 19. LOGISTIC REGRESSION MODEL FOR BACTERIAL CO-CARRIAGE IN CULTURE-DETECTED
NOSE SWABS .................................................................................................................................. 137
TABLE 20. SUMMARY STATISTICS OF VIRAL CO-CARRIAGE PREVALENCE IN REAL-TIME PCR-DETECTED
NOSE SWABS .................................................................................................................................. 138
TABLE 21. LOGISTIC REGRESSION MODEL FOR BACTERIAL AND VIRAL CO-CARRIAGE IN REAL-TIME
PCR-DETECTED NOSE SWABS ....................................................................................................... 139
TABLE 22. QUALITY CONTROL METRICS FOR WHOLE GENOME SEQUENCES OF S. PNEUMONIAE AND H.
INFLUENZAE ..................................................................................................................................... 149
TABLE 23. FREQUENT ASSOCIATIONS OF PNEUMOCOCCAL SEQUENCE TYPES WITH SEROTYPES ...... 152
TABLE 24. ANTIBIOTIC RESISTANCE AND SUSCEPTIBILITY GENES PRESENT WITHIN S. PNEUMONIAE
ISOLATES (N=232) .......................................................................................................................... 156
TABLE 25. VACCINE CANDIDATES FOUND WITHIN THE S. PNEUMONIAE ISOLATES (N=34) .................... 157
TABLE 26. ANTIBIOTIC RESISTANCE GENES PRESENT WITHIN H. INFLUENZAE ISOLATES (N=80) ........ 162
TABLE 27. VACCINE CANDIDATES FOUND WITHIN THE H. INFLUENZAE ISOLATES (N=32) ..................... 163
TABLE 28. NUMBER (N) OF METHICILLIN-RESISTANT S. AUREUS ISOLATES IDENTIFIED WITHIN EACH
SWAB TYPE AND SWABBING TIME-POINT ......................................................................................... 163
TABLE 29. NESTEDNESS RESULTS FOR NOSE AND NP SAMPLES DETECTED BY REAL-TIME PCR ..... 175
TABLE 30. CHI-SQUARED RESULTS SHOWING SPECIES ABUNDANCE DEVIATIONS FROM MACARTHUR’S
BROKEN STICK MODEL OF NEUTRAL COMMUNITY ASSEMBLY ...................................................... 189

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