Post J C
Department of Pediatric Otolaryngology, Allegheny General Hospital, Pittsburgh, Pennsylvania 15212-4722, USA.
Laryngoscope. 2001 Dec;111(12):2083-94. doi: 10.1097/00005537-200112000-00001.
OBJECTIVES/HYPOTHESIS: Bacteriologic studies of otitis media with effusion (OME) using highly sensitive techniques of molecular biology such as the polymerase chain reaction have demonstrated that traditional culturing methods are inadequate to detect many viable bacteria present in OME. The presence of pathogens attached to the middle-ear mucosa as a bacterial biofilm, rather than as free-floating organisms in a middle-ear effusion, has previously been suggested to explain these observations. The suggestion has been speculative, however, because no visual evidence of such biofilms on middle-ear mucosa has heretofore been collected. The hypotheses motivating the current study were: 1) biofilms of nontypable Hemophilus influenzae will form on the middle-ear mucosa of chinchillas in an experimental model of OME, 2) these biofilms will exhibit changes in density or structure over time, and 3) biofilms are also present on tympanostomy tubes in children with refractory post-tympanostomy otorrhea. The objective of this study was to collect visual evidence of the formation of bacterial biofilms in these situations.
Laboratory study of bacteriology in an animal model and on medical devices removed from pediatric patients.
Experimental otitis media was induced in chinchillas by transbullar injection of nontypable H. influenzae. Animals were killed in a time series and the surface of the middle-ear mucosa was examined by scanning electron microscopy (SEM) for the presence of bacterial biofilms. Adult and fetal chinchilla uninfected controls were similarly examined for comparison. In addition, tympanostomy tubes that had been placed in children's ears to treat OME and removed after onset of refractory otorrhea or other problems were examined by SEM and by confocal scanning laser microscopy for bacterial biofilms, and compared with unused control tubes.
Bacterial biofilms were visually detected by SEM on the middle-ear mucosa of multiple chinchillas in which H. influenzae otitis media had been induced. Qualitative evaluation indicated that the density and thickness of the biofilm might increase until at least 96 hours after injection. The appearance of the middle-ear mucosa of experimental animals contrasted with that of uninjected control animals. Robust bacterial biofilms were also visually detected on tympanostomy tubes removed from children's ears for clinical reasons, in contrast with unused control tubes.
Bacterial biofilms form on the middle-ear mucosa of chinchillas in experimentally induced H. influenzae otitis media and can form on tympanostomy tubes placed in children's ears. Such biofilms can be directly observed by microscopy. These results reinforce the hypothesis that the bacterial aggregates called biofilms, resistant to treatment by antibiotics and to detection by standard culture techniques, may play a major etiologic role in OME and in one of its frequent complications, post-tympanostomy otorrhea.
目的/假设：采用聚合酶链反应等高灵敏度分子生物学技术对分泌性中耳炎（OME）进行细菌学研究表明，传统培养方法不足以检测出OME中存在的许多活细菌。此前有人提出，病原体以细菌生物膜的形式附着于中耳黏膜，而非以中耳积液中自由漂浮的生物体形式存在，以此来解释这些观察结果。然而，这一观点一直是推测性的，因为迄今为止尚未收集到中耳黏膜上此类生物膜的视觉证据。本研究的假设如下：1）在OME实验模型中，不可分型流感嗜血杆菌生物膜将在龙猫的中耳黏膜上形成；2）这些生物膜的密度或结构会随时间发生变化；3）难治性鼓膜造口术后耳漏患儿的鼓膜造口管上也存在生物膜。本研究的目的是收集这些情况下细菌生物膜形成的视觉证据。
在动物模型以及从儿科患者身上取出的医疗设备上进行细菌学实验室研究。
通过经鼓膜注射不可分型流感嗜血杆菌在龙猫身上诱发实验性中耳炎。按时间序列处死动物，通过扫描电子显微镜（SEM）检查中耳黏膜表面是否存在细菌生物膜。对未感染的成年和胎儿龙猫对照组进行同样检查以作比较。此外，对为治疗OME而置入儿童耳内、在出现难治性耳漏或其他问题后取出的鼓膜造口管，通过SEM和共聚焦扫描激光显微镜检查细菌生物膜，并与未使用的对照管进行比较。
通过SEM在多只诱发了流感嗜血杆菌性中耳炎的龙猫的中耳黏膜上肉眼检测到细菌生物膜。定性评估表明，生物膜的密度和厚度可能会增加，至少在注射后96小时内如此。实验动物的中耳黏膜外观与未注射的对照动物形成对比。出于临床原因从儿童耳中取出的鼓膜造口管上也肉眼检测到了强大的细菌生物膜，这与未使用的对照管形成对比。
在实验诱发的流感嗜血杆菌性中耳炎中，细菌生物膜在龙猫的中耳黏膜上形成，并且可在置入儿童耳内的鼓膜造口管上形成。此类生物膜可通过显微镜直接观察到。这些结果强化了以下假设，即被称为生物膜的细菌聚集体对抗生素治疗具有抗性且难以通过标准培养技术检测到，可能在OME及其常见并发症之一鼓膜造口术后耳漏中起主要病因作用。
