In Vitro And In Vivo Studies Of Correlation Between Biofilm Formation By Haemophilus Influenzae And Acute Exacerbations Of Chronic Obstructive Pulmonary Disease

【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.