细菌生物膜在急性中耳炎大鼠中耳腔的形成特点及意义

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

目的通过观察细菌生物膜在急性中耳炎大鼠中耳腔的形成特点，分析其与急性中耳炎的关系，并探讨该中耳炎模型用于细菌生物膜研究的可行性。方法30只健康雄性SD大鼠，采用随机数字表法分为实验组（24只）和对照组（6只）。麻醉后将50μl肺炎链球菌悬液[1×10^8菌落形成单位（colony forming unit，CFU）／m1]经听泡穿刺注入到实验组大鼠双侧中耳腔，等量生理盐水注入到对照组大鼠双侧中耳腔。分别于注射后1、3、5、7、10、14d各取3只实验组大鼠和1只对照组大鼠的双侧听泡行中耳黏膜扫描电镜观察；另取1只实验组大鼠，显微镜下刮取收集双侧中耳腔表面附着的膜状物，行异硫氰酸荧光素标记的刀豆球蛋白（FITC-ConA）及碘化丙啶（propidium iodide，PI）免疫荧光二重染色，激光扫描共聚焦显微镜观察和革兰染色观察。结果实验组大鼠在炎性反应早期（1d、3d）可见中耳腔大量细菌黏附，定植于局部区域形成微菌落，有吞噬细胞混杂其中，形成初级细菌生物膜；炎性反应中期（5d、7d）可见成熟细菌生物膜散在于黏膜表面，形成特征性的“蘑菇状”、“塔状”三维立体结构；炎性反应后期（10d、14d）部分细菌生物膜呈现衰退迹象。中耳腔膜状物经FITC-ConA、PI二重荧光染色原位标记及革兰染色观察证实由细菌及多糖基质组成。结论细菌生物膜在中耳急性感染的早期即已开始形成，并可能成为日后炎性反应反复发作或迁延不愈的原因；本研究建立的大鼠急性中耳炎模型是研究中耳细菌生物膜较为理想的动物模型。

Objective 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 hiofilm research. Methods 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 × l0^8CFU/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. Results 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 ofrecession. 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. Conclusions 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.