Biofilm and intracellular infection: persistence mechanisms of bacterial otopathogens in chronic and recurrent otitis media

[Truncated abstract] Background and Hypothesis. Almost all children will experience at least one episode of acute otitis media (AOM), however a subset of children experience recurrent episodes of AOM (rAOM) or chronic OME where effusions do not resolve for more than 3 months. Indigenous children experience a disproportionate rate of ear disease and are more likely to develop chronic suppurative OM. While OM is often caused by bacteria, antibiotics are surprisingly ineffective at preventing rAOM or chronic OME. We hypothesise that the recalcitrance to standard treatments is due to the presence and persistence of otopathogenic bacteria either intracellularly or in biofilm on the middle ear mucosa of children with chronic and recurrent OM. Aims: To assess the presence of bacterial biofilm and intracellular persistence of known otopathogens on the middle ear mucosa and in the middle ear effusions of Indigenous and non-Indigenous children undergoing ear surgery for rAOM, chronic OME and CSOM. To assess middle ear effusions from children with rAOM to determine the role of DNA in bacterial persistence and to assess if it represents a target for therapeutic interventions. Methods: Middle ear mucosal biopsies were collected from 45 children, both Indigenous and non- Indigenous, undergoing ventilation tube insertion or tympanoplasty. A further middle ear mucosal biopsy was collected as a healthy tissue control from a child undergoing cochlear implant surgery. The first biopsy set was examined using transmission electron microscopy (TEM). Fluorescence in situ hybridisation methodology with species specific oligonucleotides was established to examine the middle ear mucosa for bacterial biofilm and intracellular infection and to identify otopathogens. Middle ear effusions (MEE) were cultured and assessed using PCR. Furthermore, MEE were examined using LIVE/DEAD viability staining and FISH, these were further examined for the presence of actin by staining with phalloidin.