An Experimental Study On Pathogenic Role Of Bacterial Biofilm In Respiratory System

[Background]: The chronic respiratory infection caused by mucoid Pseudomonas.aeruginosa (PA) is a very common intractable infection. The airway PA are resistant to most antibiotics because they are often existed in forms of biofilm. In clinical treatment, there are no other antibiotics that proved to be effective to respiratory biofilm infection except for macrolides combined with levofloxacin. Some research showed that clindamycin or some Chinese herbs also have the inhibitory effect on PA biofilm, but the related reports are rare. The more important, alginate is the main constituent of PA biofilm, and it plays an important role in the pathogenesis of airway biofilm disease .Alginate not only can bother the body immune clearance but also can be a strong allergen that can cause inflammatory reaction and lung tissue damage. Yet up to now, little is known about the relationship between alginate immune and respiratory biofilm infections.[Objective]: To investigate the influence of Houttuynia Cordata Thunb or Clindamycm combined with levofloxacin on the mucoid PA biofilm. To establish models of alginate immune pneumonia in rabbits. The pathogenic role of alginate on lung tissue was studied. To explore the pathogenesis of biofilm on respiratory system and the merchanism of the airway biofilm disease.[Methods]: (1)Routine plate culture method was used to establish the mucoid PA biofilm models in vitro. The viable bacterial counts in bilfilm were determined by Methylthiazaolyldiphenyltetrazolium(MTT) methods after drug challenge ; The destructive action of drugs on biofilm morphology were observed under SEM .(2) .Bacterial alginate from mucoid PA was extracted with ethanol precipitation method. Rabbits of group A were challenged by saline and can be the control group. Rabbitsof group B and C were immunized with seaweed alginate and bacterial alginate respectively. Rabbits of group D were immunized with seaweed alginate and then inhaled with PA suspension of the same strain. The body weight, temperature and peripheral blood cells were measured after challenge. The serum level of circulating immune complex (CIC) was detected with polythylene glycol (PEG) precipitation assay. Bacteria in bronchoalveolar lavage fluid (BALF) were incubated and the colonies were counted .The cell count and differential count of BALF were detected. The pathogenic action of alginate on lung tissue was observed under microscopy..[Results]:(1). At the 3rd day of incubation, the biofilms of mucoid PA were already formed. The viable bacterial counts of HCT CM or LFX single groups had no significant difference compared to the control group (P>0.05), and the biofilm morphology have little change.(2) The viable counts in HCT or CM combined LFX group were significantly smaller than LFX single group (P<0.05); SEM showed the mucoid material was reduced, and the bacterial numbers in biofilm were decreased. With the increasing of LFX concentration, the biofilm structures were destructed, the remained several bacteria were separated into single bacillus.(3) Be compared to the saline control group and self-control group, the serum CIC level, the cell count and the differential count of BALF were all elevated significantly after challenge, the elevation was more obvious in rabbits of group D. Additionally, bacterial colonies in BALF of group D were increased significantly compared to the other three groups. And rabbits in group D showed obvious inflammatory symptoms and the body weight decreased significantly.(4)Under light microscope, we observed the lymphocyte accumulation and lymphoid follicles around the bronchiolus in all alginate immunized groups (include B,C,Dgroup),and the narrowing of bronchiolus was also observed. In D group, the early pathologic changes were leukocyte dominated inflammation, and there were several small lung abscesses formed .In later stage, chronic lung inflammation was dominated, the proliferation of alveolar septum and pulmonary emphysema were observed.[Conclusion]: (1).Houttuynia cordata thunb has synergistic k...