【作者】 王东；

【导师】 刘又宁；

【作者基本信息】 中国人民解放军军医进修学院 ， 内科学， 2009， 博士

【摘要】 【目的】观察流感嗜血杆菌体外生物被膜的形成,并测定氟喹诺酮类药物莫西沙星、加替沙星、左氧氟沙星和环丙沙星对流感嗜血杆菌生物被膜的影响。在体外实验的基础上,依次建立金黄地鼠肺气肿模型和慢性阻塞性肺病急性发作模型,检测体内生物被膜的形成,并进一步观察莫西沙星对慢性阻塞性肺病急性发作动物模型体内流感嗜血杆菌生物被膜的影响。【方法】（1）从慢性阻塞性肺病急性发作患者痰液中分离流感嗜血杆菌,采用结晶紫法和扫描电镜观察流感嗜血杆菌体外生物被膜的形成。（2）微量肉汤稀释法测定不同药物对流感嗜血杆菌的最低抑菌浓度,观察氟喹诺酮类药物对流感嗜血杆菌生物被膜形成和破坏的影响,以及莫西沙星对生物被膜内细菌的杀菌作用。（3）采用气管内注入弹性蛋白酶法建立金黄地鼠肺气肿模型,制作流感嗜血杆菌琼脂小珠,将其注入肺气肿动物肺内建立慢性阻塞性肺病急性发作的动物模型,测定随时间延长肺组织炎症程度和菌落计数的变化,利用扫描电镜观察气道表面生物被膜的形成。（4）气管内注入流感嗜血杆菌建立慢性阻塞性肺病急性发作动物模型,观察莫西沙星治疗后,肺组织病理和气道表面生物被膜的变化,同时测定炎症因子和菌落计数的改变。【结果】（1）从慢性阻塞性肺病急性发作患者痰液中分离得到流感嗜血杆菌临床株可在体外培养24-48小时形成成熟、致密、典型的生物被膜,不同菌株形成生物被膜的能力有所不同,扫描电镜可在固相支持物表面见到由大量细菌和其分泌的胞外粘质形成的模样结构,并可见到粘液丝和孔道结构。（2）流感嗜血杆菌对氟喹诺酮类、阿奇霉素、氨苄西林、头孢噻肟和利福平敏感,氟喹诺酮药物可在体外抑制流感嗜血杆菌生物被膜的形成,并有一定的破坏作用;其中莫西沙星随药物浓度的升高,生物被膜吸光度值逐渐降低,4mg/L以上浓度的药物可明显减轻生物被膜程度;另外莫西沙星对处于生物被膜内的细菌仍有强大的清除作用,在0.25mg/L以上浓度膜内活菌数明显减少,细菌清除作用呈剂量效应关系。扫描电镜也证实莫西沙星可明显减轻生物被膜的程度。（3）成功建立肺气肿动物模型,病理学检查可见到肺泡结构紊乱,肺泡大小不等,肺泡管、肺泡囊和肺泡明显扩张,肺泡壁变薄,肺泡间隔不同程度的断裂,肺泡腔融合增大,部分融合成肺大泡,符合全小叶型肺气肿,与正常组相比,模型组的肺泡数/视野明显减少,平均肺泡面积和平均内衬间隔明显增大（P＜0.01）。在此基础上,经气管注入流感嗜血杆菌构建慢性阻塞性肺病急性发作的动物模型,随时间延长,肺气肿动物肺内炎症持续存在;肺泡灌洗液中活菌计数结果显示,与正常动物感染相比,感染后1-3周内,灌洗液中细菌数明显较高(10^4.14±0.26 vs.10^2.47±0.31,P＜0.05);肺组织匀浆活菌计数结果显示,正常动物注入细菌后细菌数逐渐减少,3周后没有分离到细菌,而模型动物感染后,细菌得不到清除,1-3周内均可分离出较多的细菌（P＜0.05）;扫描电镜观察,3周后在气道表面形成生物被膜样结构。（4）莫西沙星可有效治疗流感嗜血杆菌感染所致的肺气肿动物的急性发作,镜下可见到炎症程度减轻;1周后血浆中炎性因子水平明显降低（P＜0.05）,支气管肺泡灌洗液中炎性因子水平也有不同程度降低,其中IL-8水平早期就有明显减少;菌落计数结果显示,1周内灌洗液中没有分离到活菌,肺组织匀浆中活菌数也明显下降（P＜0.05）,3周后,细菌数量均再次恢复;但扫描电镜没有发现生物被膜样结构。此外,莫西沙星对气管内放置的硅胶管表面形成的生物被膜也有抑制作用,表现为用药后硅胶管表面分离的细菌数较少（P＜0.05）,扫描电镜也发现在莫西沙星作用下硅胶管表面无法形成成熟的生物被膜,仅见散在的细菌粘附于管壁上,个别有细菌的散在团状聚集,但尚未融合呈膜状。【结论】从慢性阻塞性肺病急性发作患者痰中分离的流感嗜血杆菌可在体外形成典型的生物被膜;通过细菌感染肺气肿动物模型可建立慢性阻塞性肺病急性发作的动物模型,并且随时间延长,细菌数量仍维持在较高水平,同时在气道表面可找到体内生物被膜形成的证据。结合体内外实验的结果,可认为下呼吸道流感嗜血杆菌生物被膜的形成与慢性阻塞性肺病的急性发作密切相关,是导致感染反复出现、病情迁延、治疗效果不完全的重要机制之一。氟喹诺酮类药物对流感嗜血杆菌浮游菌以及生物被膜内的细菌均具有强大的杀菌作用,体外及动物体内研究提示,莫西沙星可作为对抗流感嗜血杆菌生物被膜感染的有效抗菌药物应用于慢性阻塞性肺病急性发作的治疗。更多还原

【Abstract】 【Objective】To detect the biofilms formed by Haemophilus influenzae in vitro, and determine the effect of fluoroquinolones,such as moxifloxacin,gatifloxacin, levofloxacin and ciprofloxacin,on Haemophilus influenzae biofilms.On the basis of in vitro studies,golden hamster’s models of pulmonary emphysema and acute exacerbation of chronic obstructive pulmonary diseases(AECOPD) were established sequentially to probe the formation of biofilms in vivo.In the meantime,the effect of moxifloxacin on Haemophilus influenzae biofilms formed in animal model of AECOPD in vivo was primarily studied.【Methods】(1) Clinical strains of haemophilus influenzae were isolated from AECOPD patients.Biofilms formed by Haemophilus influenzae in vitro were observed by crystal violet assay and scanning electronic microscope(SEM).(2) Minimal inhibitory concentrations of antibiotics for Haemophilus influenzae were measured by microdilution method.Effect of fluoroquinolones on formation and disruption of biofilms was determined,and elimination of bacteria in biofilms by moxifloxacin was also studied.(3) Intratracheal instillation of porcine pancreatic elastase developed pulmonary emphysema in golden hamsters,which were infected by agar beads enclosing Haemophilus influenzae to establish animal model of AECOPD.Temporal alterations of inflammation and viable cell counting in lung tissue were determined,and formation of biofilms on surface of respiratory tracts was observed by SEM.(4) Moxifloxacin was administered to AECOPD golden hamsters infected by Haemophilus influenzae,and pathology of lung tissue,inflammatory factors level,viable cell counting and biofilms in vivo were measured,compared with infected animals without drug therapy.【Results】(1) Clinical strains isolated from patients with AECOPD were capable of forming mature,tight and typical biofilms in 24-48 hours in vitro. Striking differences were observed among strains with regard to the ability to form biofilm.Typical membrane-like structure formed by bacterial cells and extracellular matrix was detected,and slime silk and porous channels were also found.(2) Haemophilus influenzae is sensitive to fluoroquinolones,azithromycin, ampicillin,Cefotaxime and rifampicin.Fluoroquinolone,as one of the most powerful drug to eradicate Haemophilus influenzae,could inhibit initial biofilm synthesis at concentrations higher than two times of minimal inhibitory concentration.Disruption of mature biofilms could be achieved at relatively higher concentrations.Among fluoroquinolones,moxifloxacin decreased optical density values of biofilms significantly at the concentrations higher than 4mg/L. The similar results were obtained for viable cell counting.Bacteria in biofilms were eliminated partly or completely at concentrations higher than 0.25mg/L. Alleviation of biofilms was also corroborated by SEM.(3) Animal model of pulmonary emphysema was successfully established,whose pathological characteristics include disorder of alveolar structure,diverse sizes of alveoli, attenuation of alveolar walls,etc.Rupture and fusion of alveoli led to obvious enlargement of alveolar duct,alveolar sacs and alveoli and formation of bullae, which matches the features of panacinar emphysema.Compared with normal animals,number of alveoli per field was significantly decreased;mean alveolar area and mean linear intercept were significantly increased(P＜0.05).Animals of pulmonary emphysema were infected by Haemophilus influenzae to construct AECOPD animal models,whose inflammation was longer and more severe than that in normal animals.Viable bacteria counting(VBC) in bronchoalveolar lavage fluid(BALF) of emphysema animals 1-3 weeks after infection was significantly higher than that in normal animals(P＜0.05).VBC in lung tissue homogenate of normal animals after infection reduced gradually,and no bacteria was isolated 3 weeks after infection.Comparatively,Haemophilus influenzae stayed in lung tissue of emphysema animals for a relatively longer period of time,VBC in lung tissue homogenate held on to a higher quantity even 3 weeks after infection (P＜0.05).Biofilm-like structure was clearly detected on the tract surface of emphysema animal 3 weeks after infection.Therefore,it is deemed that chronic infection of lower respiratory tracts of emphysema animals is resulted from existence of Haemophihs influenzae biofilm in vivo,which is likely main cause of repeated exacerbations of COPD.(4) Moxifloxacin was able to treat AECOPD infected by Haemophihs influenzae effectively;inflammation in lung tissue was conspicuously improved after administration of moxifloxacin.Levels of inflammatory factors in plasma were significantly decreased 1 week after treatment compared with that without drug therapy(P＜0.05),levels of inflammatory factors in BALF were also reduced,among which IL-8 dropped down noticeably in early stage.No planktonic bacteria was isolated from BALF 1 week after moxifloxacin treatment,and VBC in lung tissue homogenate was declined significantly(P＜0.05);nevertheless,after 3 weeks,VBS in BALF and homogenate increased to a moderately large quantity.No biofilm-like structure was found by SEM.Moreover,moxifloxacin exerted an inhibitory effect on biofilms formed on surface of silicone tube positioned intratracheally.VBC of silicone tube was significantly reduced by moxifloxacin(P＜0.05);and no mature biofilms’ formation was observed on surface by SEM under drug therapy,only scattered bacteria adhered on the surface without fusion as biofilm.【Conclusion】Clinical strains of Haemophilus influenzae from AECOPD patients are able to form typical biofilms in vitro.AECOPD animal models can be established by infecting pulmonary emphysema animals with Haemophilus influenzae.Quantity of bacteria in lung tissue maintains a fairly high level, meanwhile,formation of biofilm is found in vivo on the surface of lower respiratory tracts.Pathophysiology of AECOPD animal model is very similar with clinical manifestations of acute exacerbations and stable phase of COPD. Combined with in vitro and in vivo studies,it is supposed positively that intimate correlation exists between Haemophilus influenzae biofilms formed on lower respiratory tracts and attacks of AECOPD,which is one of the most important mechanisms for occurrence of repeated infections,chronicity of disease and unsatisfactory outcomes of antibiotics therapy.Planktonic Haemophilus influenzae is highly sensitive to fluoroquinolones,and bacteria in biofilms can also be eliminated by them.In vitro and in vivo studies demonstrate that moxifloxacin is an effective drug to antagonize Haemophilus influenzae biofilms, so it is an optimal antibiotics option to treat AECOPD.更多还原