Discovery And Functional Study Of Tolerance-related Genes Of Fluoroquinolone Drugs About Bovine Pasteurella Multocida

Pasteurella multocida type A has become one of the main pathogens of bovine respiratory diseases,and broad-spectrum antibiotics such as fluoroquinolones are mainly used to treat the disease.However,during the treatment,it was found that the bacterium was prone to develop resistance to fluoroquinolones,causing huge economic losses to the animal husbandry.At the same time,it has also seriously shortened the service life of such drugs,posing a threat to the public health safety of humans and animals.In view of this practical clinical problem,it is urgent to study the tolerance of fluoroquinolones,screen potential drug resistance inhibitor targets of fluoroquinolones,and accelerate the research and development of drug resistance inhibitors of fluoroquinolones.In this study,in order to further study the targets related to the tolerance of Pasteurella multocida type A to fluoroquinolones,through continuous passage in vitro of the isolated bovine capsule type A strain,the drug resistant strain was obtained,then the whole genome and transcriptome were sequenced and analyzed,and the differentially expressed genes were screened,the deletion and complement strains of differentially expressed genes were constructed,the phenotype,drug resistance,tolerance and other functions of the deletion strain were preliminarily confirmed.The research results are as follows:Chapter 1:Whole genome sequencing analysis of bovine capsule type A Pm fluoroquinolones resistant and sensitive strainsThe highly resistant strain Pm64 was successfully obtained in vitro,and the growth rate of Pm3 was significantly lower than that of Pm64,the median lethal dose of Pm3 to mice is 3.532×10⁸ CFU/m L,whereas the median lethal dose of Pm64 to mice is 3.682×10¹³CFU/m L);the whole genome of Pm3 and Pm64 was successfully measured,with the length of 2386471 bp and2424216 bp respectively;the total gene length obtained by annotation was 2124156 bp and2159325 bp respectively;the encoding genes of Pm3 and Pm64 have a high degree of collinearity,with only partial gene island sequence differences,which are mainly related to DNA binding,trehalose metabolism,material transport,capsule synthesis,amino acid metabolism and other functions.Chapter 2:Sequence and analysis of transcriptome of bovine capsule type A Pm fluoroquinolones resistant and sensitive strainsIn order to further explore the potential genes related to the resistance of bovine capsule type A Pm to fluoroquinolones,on the basis of genome sequencing,take the whole genome as the reference sequence to analyze its m RNA expression,and the results showed that 1126differentially expressed genes were obtained,including 558 up-regulated genes and 568 down-regulated genes,by further enrichment analysis,10 differentially expressed genes were screened and their expression levels were verified.Some genes significantly expressed before and after the occurrence of type A Pm resistance were screened,such as dna K,grp E,fxs A,adh E,clp B,fts H,rpo,kat A,dusB,etc,and these genes mainly encode chaperones,transcriptional regulators and metabolic enzymes.Chapter 3:Construction of a strain with deletion and supplementation of differentially expressed genes of bovine type A PmTo explore the role of the above differentially expressed genes in the generation of adaptive costs of fluoroquinolone resistance of bovine type A Pm,the homologous recombination mediated by p RE112 and Ng Ago genetic operating system was used to complete partial gene deletion of differentially expressed genes of bovine type A Pm sensitive strain,and construct a constitutive type strong promoter restocking vector to restock the genes of the missing strain of bovine type A Pm,and the results showed that p SHK5（TS）-Ng Ago-ΔdusB recombinant plasmid and p SHK5（TS）-Ng Ago-Δkat A recombinant plasmid were successfully constructed based on Ng Ago genetic operating system,finally,the bovine type A Pm fluoroquinolone sensitive strain P3ΔdusB,Δkat A deletion strain and the corresponding complementary strain Pm were successfully constructed-Δkat A::kat A,Pm-ΔdusB::dusB.Chapter 4:Preliminary Functional Analysis of Bovine Type A Pm dusB and kat A Gene Deletion StrainsIn order to confirm the function of bovine type A Pm dusB and kat A,the growth curve,MIC,MBC,MPC,MSW,time of drug resistance formation and tolerance of deletion strain and complement strain were measured respectively,the results showed that the growth rate of dusB gene deletion strain was faster than that of wild strain,but dusB and kat A gene deletion had no effect on MIC,MPC,MSW and reversal of drug resistance.When using enrofloxacin to induce drug resistance in vitro,the drug resistance of Pm-Δkat A is slower than that of wild strain Pm,moreover,the deletion of kat A gene leads to the decrease of its tolerance to fluoroquinolones,which has the potential to become a fluoroquinolone drug resistance inhibitor.