Screening And Functional Study Of Genes Related To Fluoroquinolone Tolerance Of Bovine Pasteurella Multocida

Pasteurella multocida（Pm）is a broadly infectious pathogen capable of infecting a wide range of domestic animals,poultry,wild mammals and birds,reptiles and humans.In China,with the rapid development of beef cattle farming,bovine respiratory diseases caused by Pm have posed huge economic losses to the beef cattle industry.Fluoroquinolones are widely used in the treatment of diseases caused by Pm because of their broad antibacterial spectrum,strong antibacterial activity and no cross-resistance with other classes of antibacterial drugs.However,with the widespread use of fluoroquinolones,some of the bacterial strains have developed resistance to fluoroquinolones,so it is imperative to investigate the mechanism of tolerance in Pm to fluoroquinolones,in order to screen the potential resistance inhibitor targets and to extend the service life of fluoroquinolones.This study was intended to analyze the potential roles of napA,napB,glgP and foc A genes of bovine Pm in the formation of Pm fluoroquinolone resistance based on the results of the our earlier research group,and to lay a foundation for studying the mechanism of tolerance in Pm.The main experiments were as follows:（1）First,the NgAgo gene editing system was used for deletion of the relevant genes in Pm.Using the Pm genome as a template,PCR amplification of the upstream and downstream homologous arms of the gene to be deleted was performed.The upstream and downstream homologous arms were constructed by overlapping PCR and seamlessly cloned to the linearized knockout vector p SHK5Ts-NgAgo to construct the knockout vector for the gene to be deleted.The constructed knockout vector for the gene to be deleted was electro transformed into Pm,and after homologous recombination,the gene deletion strains for the napA,napB,glgP and foc A genes were successfully screened.In addition,back-complemented strains for the napA,napB,glgP and foc A genes were constructed using the same procedure.（2）Secondly,the biological characteristics and resistance and tolerance-related phenotypes of the gene deletion strains（P3-ΔnapA,P3-ΔnapB,P3-ΔglgP and P3-Δfoc A）and the back-complemented strains（C3-ΔnapA,C3-ΔnapB,C3-ΔglgP and C3-Δfoc A）were determined.The results showed that the deletion of the napA gene not only reduced the growth rate and biofilm formation ability of Pm and prolonged its resistance formation time to enrofloxacin,but also significantly reduced the bactericidal ability of enrofloxacin against Pm.The deletion of the napB,glgP and foc A genes had certain effects on the ability of peritheciam formation,but their effects on the bactericidal ability of enrofloxacin were weaker than those of the napA.The deletion of napB,glgP and foc A genes had some effect on the ability of enrofloxacin to form membranes.（3）Finally,in vivo and ex vivo tests were further used to investigate the effect of the napA gene deletion on the tolerance in Pm to fluoroquinolones.The results showed that the napA gene deletion increased the tolerance in Pm to enrofloxacin and the addition of different concentrations of nitrite to the exogenous source restored the bactericidal ability of enrofloxacin against the napA gene deficient strain.In addition,the napA gene deletion increased the LD₅₀ of the strain in mice but reduced the therapeutic ability of enrofloxacin in a mouse infection model.In summary,we found that the napA gene deletion significantly increased the tolerance in Pm to enrofloxacin,but the increased tolerance could be gradually reduced by exogenous addition of different concentrations of nitrite.The results of this study provides a basis for further studies on the mechanism of tolerance in Pm to fluoroquinolones.