Multi-species bacterial biofilm and intracellular infection in otitis media.

BACKGROUND: Bacteria which are metabolically active yet unable to be cultured and eradicated by antibiotic treatment are present in the middle ear effusion of children with chronic otitis media with effusion (COME) and recurrent acute otitis media (rAOM). These observations are suggestive of biofilm presence or intracellular sequestration of bacteria and may play a role in OM pathogenesis. The aim of this project is to provide evidence for the presence of otopathogenic bacteria intracellularly or within biofilm in the middle ear mucosa of children with COME or rAOM. METHODS: Middle ear mucosal biopsies from 20 children with COME or rAOM were examined for otopathogenic bacteria (either in biofilm or located intracellularly) using transmission electron microscopy (TEM) or species specific fluorescent in situ hybridisation (FISH) and confocal laser scanning microscopy (CLSM). One healthy control biopsy from a child undergoing cochlear implant surgery was also examined. RESULTS: No bacteria were observed in the healthy control sample. In 2 of the 3 biopsies imaged using TEM, bacteria were observed in mucus containing vacuoles within epithelial cells. Bacterial species within these could not be identified and biofilm was not observed. Using FISH with CLSM, bacteria were seen in 15 of the 17 otitis media mucosal specimens. In this group, 11 (65%) of the 17 middle ear mucosal biopsies showed evidence of bacterial biofilm and 12 demonstrated intracellular bacteria. 52% of biopsies were positive for both biofilm and intracellular bacteria. At least one otopathogen was identified in 13 of the 15 samples where bacteria were present. No differences were observed between biopsies from children with COME and those with rAOM. CONCLUSION: Using FISH and CLSM, bacterial biofilm and intracellular infection with known otopathogens are demonstrated on/in the middle ear mucosa of children with COME and/or rAOM. While their role in disease pathogenesis remains to be determined, this previously undescribed infection pattern may help explain the ineffectiveness of current treatment strategies at preventing or resolving COME or rAOM.

The role of chronic infection in children with otitis media with effusion: evidence for intracellular persistence of bacteria.

OBJECTIVE: Demonstrate mucosal bacterial infection in children with otitis media with effusion (OME). STUDY DESIGN AND SETTING: Middle ear mucosal biopsies from 11 children with OME were examined for bacteria utilizing transmission electron microscopy. This was correlated with standard culture and polymerase chain reaction (PCR) of middle ear effusions. RESULTS: Gram-positive coccal bacteria were demonstrated in middle ear mucosal epithelial cells of 4 of 11 (36%) children. Morphological appearance of bacteria and detection of pneumolysin DNA by PCR in middle ear fluid suggests a role for persistent intracellular infection with Streptococcus pneumoniae and other gram-positive cocci in some cases of OME. CONCLUSION: Intracellular bacterial infection of middle ear mucosal epithelial cells in children with OME may be an important mechanism for bacterial persistence, and contribute to inflammation and mucus production in the pathogenesis of this condition. SIGNIFICANCE: Persistent intracellular infection is a novel paradigm for OME pathogenesis in children and may influence antibiotic effectiveness in treatment of this condition.