Feature and significance of bacterial biofilm formation in middle-ear mucosa in the rat model of acute otitis media

Abstract

To investigate the relationship between bacterial biofilm and acute otitis media by observing the feature of bacterial biofilm formation in middle-ear mucosa in the rat model of acute otitis media and to study the possibility of application this rat model in bacterial biofilm research. A total of 30 healthy, male SD rats were studied, 24 animals served as experimental group were bilaterally injected with 50 µl of Streptococcus pneumoniae suspension (1 × 10(8) CFU/ml) via a transbullar approach into the middle ear cavity after anesthesia and six animals were bilaterally inoculated equivalent saline account for control group. At day 1, 3, 5, 7, 10 and 14 after inoculation, bilateral middle-ear mucosal specimens were collected from three infected animals and one control animal for scanning electron microscopy (SME). Membranoid substance attached the bilateral middle ear mucosa were collected under the microscope from the other one infected animals, which were prepared for confocal laser scanning microscope (CLSM) with immunofluorescence in situ labeling technique and light microscopy using Gram staining. At the early stage of infection (1 day, 3 days), lots of bacterial adhesion, permanent planting in the local regions of the middle ear cavity and microcolonies formation were found, with mixed phagocytic cells, showing a primary bacterial biofilms formation. In the middle term of infection (5 days, 7 days), mature bacterial biofilm scattered on the mucosal surface, formed characteristic three-dimensional structure of "mushroom-shaped" towers. At the late inflammatory period (10 days, 14 days), the bacterial biofilms presented signs of recession. CLSM with FITC-ConA and PI double staining in situ labeling and light microscopy using Gram staining indicated that bacteria and polysaccharide matrix within the biofilms were viable. These preliminary findings provide evidence that bacterial biofilms form at the early phase of acute middle ear infection and it may be an important factor in the development of recurrent or persistent otitis media. The rat model of AOM established in this study may be an ideal animal model facilitating the bacterial biofilms research.