Investigation of the effects of polymicrobial infection on the induction of otitis media

Bacterial otitis media (OM) is a well known paediatric condition predominantly caused by Streptococcus pneumoniae, nontypeable Haemophilus influenzae and Moraxella catarrhalis. The polymicrobial etiology and the mechanisms and pathogenesis of the disease caused by single bacterium, synergistic bacterial-viral interactions, and inflammatory processes have been studied using different animal models. The complexities associated with polymicrobial OM, mechanisms of host-bacterial relationships, inflammatory responses, and microbial interactions would be better understood in an experimental model comprising of the predominant bacteria involved. These issues were explored in this thesis in the context of investigating the effects of polybacterial infection on the incidence and severity of OM. An experimental polymicrobial infection murine (BALB/c) model was established involving different combinations of the above mentioned bacteria along with a respiratory virus (Sendai). This model was used to demonstrate for the first time that the presence of M. catarrhalis as a co-colonising agent of the nasopharynx significantly exacerbates pneumococcal OM. Another significant finding was that the inflammatory response generated, was due to the synergistic bacterial-viral infection and not by the respiratory virus alone (Chapter 2). This model was next used to show that the incidence of lower respiratory and middle ear infections caused by M. catarrhalis and NTHi was not affected by bacterial dosage and pre-colonisation of the nasopharynx (Chapter 3 & 4 respectively). In contrast, pre-viral infection and increased bacterial numbers facilitated greater incidence of pneumococcal OM and lower respiratory tract infections (Chapter 3). The incidence of pneumococcal OM was also evident in the presence of a precoloniser (M. catarrhalis) in the nasopharynx (Chapter 4). The impact of the polymicrobial environment in the nasopharynx on the incidence of OM could be better understood with real-time monitoring of infection progression by biophotonic imaging. Following intranasal infection, co-colonisation patterns and its effect on OM of luminescent NTHi and S. pneumoniae were measured (Chapter 5). The data not only showed rapid ascension of S. pneumoniae when pre-infected with M. catarrhalis and coinfected with NTHi and M. catarrhalis, but also showed the ability of bacteria to colonise different niches. The factors associated with microbial interactions, including adherence, with the host were identified using in vitro cell cultures as a model because of their ease of manipulation and cost-effectiveness (Chapters 6 and 7). This was performed using the above mentioned bacterial combinations and adenovirus on lung (A549) and bronchial (BEAS 2B) epithelial cell lines. When infected alone, adherence of M. catarrhalis and S. pneumoniae to adenovirus-infected BEAS-2B cells was greater than virus-infected A549 cells, but was reduced on BEAS-2B cells in a co-infection with M. catarrhalis and S. pneumoniae. In contrast, this co-infection increased the adherence to virus-infected A549 cells (Chapter 6). This observation supports the findings of the in vivo studies (Chapter 2) indicating a positive association between these two bacteria to cause infections. The pulmonary burden caused by bacterial products could be different in the various respiratory compartments, as seen with different adherence levels to epithelial cells, suggesting the possibility of different cytokine responses. This was demonstrated for IL-6, with significant differences in the release of IL-6 by BEAS 2B and A549 cells in response to bacterial infection, and a synergistic effect of M. catarrhalis and S. pneumoniae, and, S. pneumoniae and NTHi co-infections on IL-6 production (Chapter 7). The overall finding of this thesis was that the nasopharyngeal M. catarrhalis infection not only predisposes pneumococcal otitis media, but also promotes pneumococcal colonisation at a site distal from its initial inoculation site. Furthermore, this association also enhanced the adherence to alveolar epithelial cells releasing high levels of the inflammatory cytokine IL-6. This finding contributes to better understanding of the early onset of OM observed evidently at an alarming rate, particularly in Australian Aboriginal children, and will assist strategies to for preventing and managing infections. -- Abstract.