Rimary Research On The Mechanism Of Ambroxol Against Haemophilus Infiuenzae Biofilm

Bacterial biofilm(BF) is a kind of growth way of the bacteria corresponding to the free floating bacteria; it is a dynamic, mixed and continuously varying community; there is maybe only one kind of bacteria or fungus within its interior but more often there are many different kinds of microbes. It can be described as that the bacteria is embedded in a thick and sticky barrier consisting of sugar and protein. This kind of biofilm can protect the microbe from the threat of outside, which is considered as an important factor causing drug resistance in persistent infected diseases.Biofilm theory was proposed in 1978 for the first time,and had confirmed some diseases had something to do with the biofilm formation,including tooth decay, periodontitis, endocarditis, pulmonary cystic fibrosis etc.In recent years, the relationship between biofilm and clinical diseases was studied broadly. So far, it has been confirmed that many kinds of clinical diseases have relationship with the formation of Haemophilus influenzae biofilm, such as acute episode of chronic obstructive pulmonary disease(COPD), pulmonary cystic fibrosis(CF), chronic rhinosinusitis(CRS), secretory otitis media(OMA), adenoid hypertrophy. Ambroxol(AMB), as a kind of apophlegmatisant, can clear and damage the biofilm of Staphylococcus aureus, Pseudomonas aeruginosa etc. AMB can reduce the expression of related gene-algD, algR and algU, which encode the synthe-sis of alginate, the main component of extracellular lipopolysaccharide of Staphylococcus aureus and raise the expression of supressor gene mucA,thus inhibits the biofilm formation; it can also reduce the signal molecule AHL by inhibiting the expression of gene las I and rhl I, which encode the AHL synthetase of quorum sensing(QS) system of Pseudomonas aerugino-sa, to inhibit the BF formation. The previous study of this laboratory also discovered that AMB could damage and remove the BF of Hemophilus influenza in vitro, and could improvethe antibacterial effect obviously combined with the antibiotics. However, the removing and damaging mechanism is currently not well known, and there is no correlative report.This thesis explored the biofilm inhibition mechanism of AMB on Haemophilus influenzae by three aspects that the planktonic state of Haemophilus influenzae, the early stage of adhesion of BF and mature biofilm.In this experiment, the standard strain of Haemophilus influenzae ATCC24479 and the experimental strain with stronger film forming ability Hi23 were selected.First of all, the effect of AMB of different concentration on Haemophilus influenzae of planktonic state without BF was oberved continuously and dynamically, comparing the growth of Haemophilus influenzae in culture medium, and the growth curve was drawn.And then, the effect of AMB of different concentration to the early stage of adhesion of BF was tested by semi quantitative experiment,the value of A570 was tested by multifunctional enzyme mark instrument after dyeing and bleaching, and the data were analyzed. Afer that, the mature biofilm of Haemophilus influenzae was constructed in vitro, the formation was confirmed by scanning electron microscope,the total RNA of mature biofilm of Haemophilus influenzae was extracted by the kit method, Reverse Transcription PCR was performed after the purity and concentration were appraised by micro-spectrophotometer, real-time fluorescence quantitative reaction was carried out and finally the mRNA expression of Haemophilus influenzae biofilm related gene-luxS, pil A and licD was compared under the influence of different concentration AMB.It could be discovered that AMB of different concentration had little effect to the growth of the both two strains ATCC24479 and Hi23 of planktonic state by Haemophilus influenzae growth curve, and there was nodifference. However, after the ATCC24479 and Hi23 were cultivated for different time, the 96 hole plates were stained by crystal violet and tested by enzyme mark instrument, the statistical analysis of value A showed that there was early adhesion biofilm formation of different degree afer the 6groups of 2 strains of Haemophilus influenzae were cultivated,comparing with the control group, AMB of different concentration could all reduce the early adhesion of Haemophilus influenzae(P<0.05), thus inhibiting the BF further formation, and AMB in high concentration had more obvious effect,exhibiting concentration dependence(P<0.05).The Haemophilus influenzae biofilm model was constructed in vitro successfully, the total RNA of Haemophilus influenzae biofilm was extracted, Reverse Transcription PCR and fluorescence quantitative PCR were performed, and the data were analyzed, which showed that the expression of luxS, pilA and licD was the the highest in the blank control group, comparing the two experimental groups with the control group, the expression of luxS, pilA and licD reduced obviously, and the difference had statistical significance(P<0.05).Comparing the AMB group(3.75mg/l) with the AMB group(1.875mg/l),the expression of luxS, pilA and licD reduced more obviously, and the difference had statistical significance(P<0.05).From the above experiments: AMB has no obvious effect to Haemophilus influenzae of planktonic state; AMB can reduce the early adhesion of Haemophilus influenzae biofilm formation and inhibit the BF further formation; AMB can reduce the expression of Haemophilus influenzae biofilm formation related gene-luxS, pilA and lic D, and then inhibit the formation and maturity. This thesis provides theoretical basis for the clinical application of AMB and benifical medication guide of the treatment of Haemophilus influenzae biofilm infection in clinical.