Construction Gene Deletion Strain And Function Study Of SatP Gene Of Bovine Pasteurella Multocida

In recent years,the continuous evolution of major pathogenic bacteria in veterinary clinic under the pressure of drugs,the problem of drug resistance of the main pathogens is becoming more and more serious.As one of the main pathogens of bovine respiratory diseases,Pasteurella multocida(Pm)has developed different degrees of drug resistance to commonly used veterinary clinical drugs(fluoroquinolones,macrolides,etc.).More importantly,when the research group tested the drug resistance of bovine capsule type A Pm in veterinary clinic,it was found that,Some strains did not have drug resistance target mutation under the action of antibacterial drugs,but they could not be cured quickly after multiple infections,which indicated that bacteria were resistant to such drugs to a certain extent,but the relationship between tolerance generation and drug resistance formation and the mechanism of tolerance generation were not reported.Therefore,based on the clinical practice of bovine capsular type A Pm tolerant bacteria,this study carried out the research on the main regulation mechanism of its tolerance,and the research results laid a foundation for the further study of veterinary clinical pathogenic bacteria tolerance.The main research contents mainly include the following 3 parts:First of all,transcriptomic analysis of Pm with different drug resistance levels was based on two clinically isolated bovine capsular type A Pm strains with the same PFGE typing and sensitive and resistant to enrofloxacin.Enrofloxacin-resistant bacteria with unchanged genotype were obtained by in vitro induction method,Furthermore,transcriptomics sequencing and analysis of sensitive bacteria,drug-resistant bacteria and highly drug-resistant bacteria treated with sub-inhibitory concentration of enrofloxacin were carried out,and finally five genes whose expression levels were significantly up-regulated with drug resistance were selected,namely satP,foc A,nap F,glg P and nap D,among which satP gene was up-regulated most significantly.It is speculated that it may be related to the formation of.The second,construction and verification of satP gene deletion strain and replenishment strain in order to further study the function of satP gene,PRE 112suicide vector containing homologous arms upstream and downstream of satP gene was constructed,and sensitive strain and drug-resistant strain satP gene deletion strain were constructed by homologous recombination method.Real-time fluorescence quantitative PCR was used to determine the expression of satP in the deletion strain,which proved that the gene deletion was successful;At the same time,the improved PUC-18 vector containing Pasteurella strong promoter was constructed,and the satP gene replenishment strain was constructed,which laid a foundation for the study of satP gene function.The last,preliminary study on the function of satP gene of strains with different drug resistance levels.Compared with wild strains,the MIC,MBC,MPC and drug resistance reversal of satP gene deletion strains have no significant changes,but the rate of drug resistance formation induced by satP gene deletion strains in vitro is significantly slower than that of wild strains.MDK₉₉ experiment,agar diffusion method and drug resistance mutation frequency experiment confirmed that the tolerance of satP gene deletion strain was significantly lower than that of wild strain At the same time,this study also tested the virulence of the gene deletion strain and the wild strain,and the results showed that the virulence of the satP gene deletion strain decreased about 400 times,and the mice infected with the satP gene deletion strain were easier to be treated than the mice infected with the wild-type bovine Pm strain.In a word,this study conducted transcriptomics sequencing and analysis of three bovine type A Pm strains with the same pulsed field molecular typing and different levels of resistance to enrofloxacin,A gene-satP,whose expression level is significantly up-regulated with drug resistance,was screened and obtained.The construction of satP gene deletion strain proved that the deletion of this gene could significantly reduce the tolerance and virulence of the strain.