The microbiome of otitis media with effusion and the influence of Alloiococcus otitidis on Haemophilus influenzae in polymicrobial biofilm Chun L Chan Bachelor of Radiography and Medical Imaging (Honours) Bachelor of Medicine, Bachelor of Surgery Department of Otolaryngology, Head and Neck Surgery University of Adelaide, Adelaide, Australia Submitted for the title of Doctor of Philosophy November 2016 C L Chan i This thesis is dedicated to those who have sacrificed the most during my scientific endeavours My amazing family Flora, Aidan and Benjamin C L Chan ii Table of Contents TABLE OF CONTENTS .............................................................................................................................. III THESIS DECLARATION ............................................................................................................................. VII ACKNOWLEDGEMENTS ........................................................................................................................... VIII THESIS SUMMARY ................................................................................................................................... X PUBLICATIONS ARISING FROM THIS THESIS .................................................................................................. XII PRESENTATIONS ARISING FROM THIS THESIS ............................................................................................... XIII ABBREVIATIONS .................................................................................................................................... XV LIST OF TABLES .................................................................................................................................... XVII LIST OF FIGURES ................................................................................................................................. XVIII CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW .................................................................. 1 1.1 OTITIS MEDIA WITH EFFUSION ....................................................................................................... 2 1.1.1 Background ..................................................................................................................... 2 1.1.2 Epidemiology and natural history ................................................................................... 3 1.1.3 Aetiology and pathogenesis ........................................................................................... 3 1.1.3 Clinical symptoms and diagnosis of OME ..................................................................... 13 1.1.4 Management of otitis media with effusion .................................................................. 14 1.2 THE ROLE OF BACTERIA IN OTITIS MEDIA ........................................................................................ 23 1.2.1 Detection of Bacteria and its advances ........................................................................ 23 1.2.2 The microbiota of the middle ear ................................................................................. 31 1.2.3 The nasopharynx as a reservoir for otitis media ........................................................... 33 1.2.4 The external ear canal as a potential reservoir for otitis media ................................... 35 1.2.5 Bacteria associated with otitis media ........................................................................... 37 C L Chan iii 1.2.6 The role of biofilm in otitis media ................................................................................. 51 1.3 SUMMARY OF LITERATURE REVIEW ............................................................................................... 55 1.4 AIMS ...................................................................................................................................... 57 CHAPTER 2: THE MICROBIOME OF OTITIS MEDIA WITH EFFUSION ................................................ 58 2.1 ABSTRACT ................................................................................................................................ 61 2.2 INTRODUCTION ......................................................................................................................... 62 2.3 MATERIALS AND METHODS ......................................................................................................... 63 2.3.1 Selection Criteria ........................................................................................................... 63 2.3.2 Sample collection .......................................................................................................... 63 2.3.3 DNA extraction ............................................................................................................. 64 2.3.4 PCR amplification of the 16S rRNA gene and sequencing ............................................ 64 2.3.5 Bioinformatics pipeline ................................................................................................. 65 2.3.6 Biostatistical analysis ................................................................................................... 65 2.4 RESULTS .................................................................................................................................. 67 2.4.1 Demographic data: ....................................................................................................... 67 2.4.2 Middle ear microBiome ................................................................................................ 67 2.4.3 Adenoid microBiome versus middle ear microBiome .................................................... 72 2.4.4 MEF adenoid microBiome versus control ..................................................................... 74 2.5 DISCUSSION ............................................................................................................................. 77 2.6 CONCLUSION ............................................................................................................................ 81 CHAPTER 3: THE EXTERNAL EAR CANAL AS A RESERVOIR FOR THE MIDDLE EAR: A 16S PHYLOGENETIC ANALYSIS ............................................................................................................. 82 3.1 ABSTRACT ................................................................................................................................ 85 3.2 INTRODUCTION ......................................................................................................................... 86 C L Chan iv 3.3 METHODS ............................................................................................................................... 87 3.3.1 Study group .................................................................................................................. 87 3.3.2 Sample collection .......................................................................................................... 87 3.3.3 DNA extraction ............................................................................................................. 88 3.3.4 PCR amplification of the 16S rRNA gene and sequencing ............................................ 88 3.3.5 Bioinformatics pipeline ................................................................................................. 88 3.3.6 Biostatistical analysis ................................................................................................... 89 3.4 RESULTS .................................................................................................................................. 91 3.4.1 MicroBiome of middle ear with effusion....................................................................... 91 3.4.2 MicroBiome of the external ear canal .......................................................................... 91 3.4.3 Comparing the microbiome of the middle ear to the external canal ............................ 94 3.4.4 Bilateral effusions ......................................................................................................... 95 3.4.5 Combined with previous data set ................................................................................. 95 3.5 DISCUSSION ............................................................................................................................. 98 3.6 CONCLUSION .......................................................................................................................... 102 CHAPTER 4: INDIRECT PATHOGENICITY OF ALLOIOCOCCUS OTITIDIS IN MULTISPECIES BIOFILM WITH HAEMOPHILUS INFLUENZAE: EFFECTS ON ANTIBIOTIC SENSITIVITY AND PERSISTENCE ..... 103 4.1 ABSTRACT .............................................................................................................................. 106 4.2 BACKGROUND ........................................................................................................................ 107 4.3 METHODS ............................................................................................................................. 109 4.3.1 Bacterial strains and handling .................................................................................... 109 4.3.2 Planktonic cell assays ................................................................................................. 109 4.3.3 Biofilm assays ............................................................................................................