| Pasteurella multocida mainly causes pasteurellosis,also known as hemorrhagic septicemia,in a variety of animals.Among them,poultry infection is manifested as avian cholera,pig infection can cause atrophic rhinitis and swine lung disease,cattle and sheep infection is manifested as hemorrhagic septicemia and infectious pneumonia,which has caused serious economic damage to the breeding industry.Quorum Sensing is a widespread bacterial communication mechanism that regulates a variety of physiological phenomena,such as virulence factors,bioluminescence and biofilm formation.And studies have shown that there are quorum sensing related genes in Pm.Therefore,the search for safe and effective Quorum Sensing inhibitors can provide a possible way to control pathogen infection and drug resistance.In this study,QSI of Pm was screened by molecular docking and BB170 bioluminescence method,and its effect on biological characteristics of Pm was investigated.The synergistic antibacterial effect of Azelastine and Kanamycin was verified in the pneumonia model of Pm infected mice,which provided a new idea for the development of clinical antibacterial drugs.The main research contents and results of this paper are as follows:(1)The three-dimensional structure of Lux S receptor protein was obtained through homology modeling,and 17 QSI candidate compounds were virtually screened out from ZINC database by Auto Dock Vina software.The BB170 bioluminescence method determined that 120μM Azelastine had the strongest QS inhibition activity,which significantly inhibited the production of signal molecules without affecting the normal growth of bacteria,and Azelastine had a clear dose-effect relationship on the inhibition of signal molecules.(2)By investigating the effects of Azelastine on the biological characteristics of fluoroquinolone-sensitive strain P3 and drug-resistant strain P32 in bovine capsular type A Pm,it was confirmed that Azelastine could significantly inhibit the biofilm formation content of bovine capsular type A Pm,but Azelastine had no significant effects on the growth curve,colony morphology and capsular content.The results of stress resistance test showed that hypertonic pressure tolerance and oxidative pressure tolerance were enhanced and high temperature tolerance was decreased after nitrogen drostine treatment of bovine capsular type A Pm.(3)The drug sensitivity of Azelastine was determined by microbroth dilution method.The results showed that the minimum inhibitory concentration of Azelastine on bovine capsular type A Pm was 128 μg/m L.Then,the combined drug sensitivity test was conducted to further investigate the effect of the combination of Azelastine and Ennofloxacin,Norfloxacin,Ciprofloxacin,Erythromycin,Streptomycin,Kanamycin,Tetracycline.The results showed that the combination of Azelastine and Kanamycin was synergistic antibacterial effect,and the antibacterial activity of FIC is 0.375,which is 4～8 times higher than that of single drug.(4)A mouse pneumonia model infected with bovine capsular type A Pm was established to further investigate the bacteriostatic effects of Azelastine and Kanamycin in vivo.The results showed that the survival rate of mice in the drug combination group was 100%.Compared with model group,the drug combination significantly reduced the bacterial load in the lungs of mice.After hematoxylin-eosin staining of lung tissue,it was observed that the drug combination also significantly reduced lung inflammation caused by bovine capsular type A Pm in mice,such as lung tissue lesions,congestion and inflammatory cell infiltration. |
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