The microbiome of otitis media with effusion and the influence of Alloiococcus otitidis on Haemophilus influenzae in polymicrobial biofilm

Abstract

The research described herein follows an extensive review of the literature on the role of bacteria in the aetiology of otitis media with effusion. Otitis media and otitis media with effusion have been classically associated with three main bacteria, Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis. However, with advancements in culture-independent molecular techniques, previously un-culturable bacteria have been identified. Moreover, using these technologies researchers have been able to characterise the entire microbial populations of many body sites, which has allowed for greater insight into the host-pathogen relationship. The initial investigation focused on characterising the microbiome of the middle ear in children with otitis media with effusion and is detailed in chapter two. This study formed the foundation for the thesis and was critical in confirming whether the adenoid pad is a bacterial reservoir for the middle ear. We collected middle ear fluid and adenoid pad swabs from children undergoing ventilation tube insertion at the Women’s and Children’s Hospital, Adelaide, Australia and used state of the art 16S rRNA molecular gene sequencing to characterise the microbiome of the middle ear in these patients. We found that the microbial communities within the middle ear and on the adenoid pad shared some common bacteria; however, we also discovered the presence of bacteria not found in the adenoid pad. Namely, these bacteria were Alloiococcus, Corynebacterium spp. and Staphylococcus spp. These bacteria are more commonly associated with colonisation of the outer ear canal, which lead us to believe that the external auditory canal could play a part in seeding the middle ear with bacteria. The follow-on study is detailed in chapter three and addresses the question of whether or not the external auditory canal was a reservoir for the middle ear. We again returned to the Women’s and Children’s Hospital to collect samples. For this study, we collected middle ear fluid and also external auditory canal lavages. Upon comparisons of the microbiome, we found that both the external ear canal and adenoid pad contribute to the microbiome of the middle ear. Additionally, upon analysis of the bacterial relationships within the middle ear effusion, we recognised that there was an inverse correlation relationship between H. influenzae and A. otitidis. The final study addressed the question of the nature of the relationship between H. influenzae and A. otitidis and is the subject of chapter 4. Otitis media with effusion has been suggested to be a biofilm driven disease. Therefore, we sought to discover whether A. otitidis forms biofilm. We found that not only does A. otitidis form single species biofilm, but that it also forms polymicrobial biofilm with H. influenzae. The subsequent polymicrobial biofilm allowed the bacteria to express a new phenotype, that altered the persistence and antibiotic resistance profile of these bacteria. The findings of this study suggest that A. otitidis plays a previously undescribed role in the pathogenesis of otitis media with effusion.