Effects Of Macrolides On Biofilm Formation Induced By Nontypeable Haemophilus Influenzae

Bacteria biofilm(BBF) can attenuates bacteria sensitivity to antibiotics, reduce the antibiotics concentration in biofilm,and leads to incomplete elimination of bacteria and disease recurrence.Chronic obstructive pulmonary disease(COPD) is a respiratory tract disease commonly seen in clinics,which is the fourth cause of death on the planet.Infectious characteristics of acute exacerbations of COPD are chronicity of disease and acute attacks repeatedly, which is similar with biofilm-associated infections.Nontypeable Haemophilus influenzae is the major pathogenic bacteria of many chronic recurrent diseases, including acute Explosion of chronic obstructive pulmonary diseases(AECOPD). It was reported that Haemophilus influenza can form BBF in vitro and escape the attack of antibiotics and immune system,which suggested that Haemophilus influenza BBF might be associated with the progress of AECOPD.Ventilation associated pneumonia(VAP) is commonly seen during mechanical ventilation, which can result in high morbidity and mortality,especially in patients with chronic obstructive pulmonary diseases.Biofilm formed on endotracheal tube is one of important causes of VAP.Objective:To determine the activity of the macrolides in the treatment of infection associated with Nontypeable Haemophilus influenzae Biofilm Formation in vivo,vitro and on endotracheal tubes.Methods:Formation of bacterial biofilm was examined by crystal violet assay and scanning electron microscope.Alterations of biofilms were measured under varying concentrations of macrolides In vitro.We evaluate the destruction of different concentrations azithromycin on Haemophilus Influenzae biofilms through crystal violet assay and scanning electron microscope,viable cells counting in biofilms was also carried out.Rat models of chronic bronchitis were established by instillation of LPS and smoking,which were infected by Haemophilus influenzae coated by agar beads to build the rat models of acute exacerbation of chronic bronchitis.Formation of biofilms was determined by viable cells count in BALF and lung tissue homogenate,and pathology under SEM and light microscope.Levels of TNF-α,IL-6,IL-8 in serum and BALF and quantity of bacteria in BALF and homogenate were measured to evaluate the effects of azithromycin administered orally on model rats infections by Haemophilus influenzae,and to judge the role of inflammatory factors in pathophysiology of AECOPD.Chronic bronchitis rats were intubated with a silicone tube,and then infected by bacteria to establish the animal models of endotracheal tube infections.Formation of biofilms was detected by SEM.Viable cells counting was carried out to evaluate the effect of azithromycin orally on biofilms on endotracheal tubes.Results:Through crystal violet assay,Biofilm synthesis was observed at subMIC and stronger in Roxithromycin and Erythromycin at 1/8 MIC,inhibited by Azithromycin at higher concentrations.Disruption of mature biofilms could be achieved at relatively higher concentration,Azithromycin displayed more powerful activity.Optical density of biofilms and viable cell counting were significantly decreased at the concentration of 8 folds of MIC for azithromycin, and biofilm structure was destroyed by azithromycin at concentration of 10 folds of MIC.at subMIC,azithromycin could damage the biofilm formation of Nontypeable Haemophilus influenzae and reduce the viable cell counting. Pathology of respiratory tract tissue of rats with chronic bronchitis showed lodging of cilia,exuviation,increase of secretion in lumens,infiltration of inflammatory cells,proliferation of goblet cells,rupture of smooth muscles. Comparatively,intactness of mucosal epithelium,smooth muscles were shown in control group.Quantity of bacteria in BALF and homogenate on day 1 after inoculation was higher than 10~7 both in control and chronic bronchitis groups.A lot of inflammatory cells,infiltrated into alveolus,parabronchial area and interstitium,accompanied with lung tissue consolidation and more exudations in chronic bronchitis rats.Viable cells count in homogenate of chronic bronchitis rats dropped to the lowest level after 2 weeks,and maintained at 10~2 after 1 month with an increasing trend.Biofilm-like structure was found in infected chronic bronchitis rats lung tissue under SEM,which entrapped the bodies of bacteria. Bacteria in BALF of infected chronic bronchitis rats were eliminated more quickly after azithromycin administration.Bacteria in homogenate could be detected even after 1 month with or without drug therapy,but,viable cells count was significantly lower in azithromycin group.Pathological results also showcased that infiltration of inflammatory cells and exudations in alveolus were conspicuously alleviated after azithromycin therapy.Levels of 3 inflammatory factors were markedly reduced in azithromycin group.Inflammatory factors held on to a relatively higher level when more bacteria survived in BALF,and decreased with the falling of viable cells count.When bacteria colonized in lung tissue,levels of inflammatory factors were higher than that in control group,but lower than that in infections group.Levels of IL-6 and IL-8 in BALF were lower after 1 week in drug group than that in control group.Uneven biofilms were formed on the surface of endotracheal tubes under SEM,and slim bodied of bacteria enclosed by a lot of extracellular matrix.Viable cells counting was reduced by azithromycin compared with that without drug,but there was no difference after 1 month.Conclusions:Haemophilus Influenzae is capable to form biofilm in vitro,in vivo of chronic bronchitis rats and on endotracheal tubes,Haemophilus Influenzae is capable to form biofilm in subconcentration of AZI,ROX and ERY. Sufficient dosage might control early formation of biofilms.Azithromycin exerts better effects on breakdown of biofilm than other macrolides.Azithromycin was able to interfere with adhesion,damage the structure of biofilm,and reduce viable cells counting at high concentrations.Rat models of chronic bronchitis and acute infection of chronic bronchitis by Haemophilus influenzae were established. Azithromycin exerts excellent therapeutic effects on acute exacerbations of chronic bronchitis infected by Haemophilus influenzae,and eradication of bacteria in biofilms.Inflammatory factors can be regarded as measurement of evaluating the degree of infections.Azithromycin can reduce the level of IL-6 and IL-8. Azithromycin can exert inhibitory effect on Haemophilus influenzae biofilms formed on endotracheal tubes.