Construction And Phenotypic Analysis Of RecO Gene Deletion Strain Of Bovine Pasteurella Multocida Capsular Type A

Pasteurella multocida capsular type A(Pm)is one of the main pathogens of bovine respiratory disease(BRD)in China.In recent years,it has been widely spread across different regions in China under the breeding and transportation mode of "Cattle transported from north to south and from west to east".The traditional prevention and control measures against Pm are still based on broad-spectrum antibiotics,but the overuse of antibiotics certainly will lead to the increase of bacterial resistance.Previous studies have found that under the impact of lowconcentration antibiotics(such as fluoroquinolones and macrolide drugs),Pm is easy to form resistance and tolerance mutations,which also bring difficulties to the prevention and control of Pm.Therefore,while standardizing the use of antibiotics,it is of great significance to develop new antibiotic resistance inhibitors to slow down the formation of antibiotic resistance.Related studies have found that deletion mutations of key molecules in the SOS repair system can affect the resistance and tolerance of bacteria to fluoroquinolones to a certain extent,and are independent of the known target mutations to mediate the generation of resistance.Some researchers have proposed that by targeting the inhibition of SOS repair pathway related molecules,antibiotic tolerance of bacteria can be reduced and antibiotic resistance can be inhibited to a certain extent,so as enhancing the antibacterial effect of antibiotics.For this reason,the highly pathogenic Pm fluoroquinolone sensitive strain P3 selected by our research group in the previous stage and the fluoroquinolone resistant strain P32 induced by enrofloxacin at sub-inhibitory concentration of P3 were used as the object in this study.Functional verification was performed on the SOS pathway differential gene recO screened from the omics analysis results of P3 and P32 treated with fluoroquinolones.The main contents of the experiment were as follows:(1)Using Pasteurella multocida capsular type A fluoroquinolone sensitive strain P3 as template,the upstream and downstream homologous arm genes of recO were amplified respectively.The homologous arm genes of recO were linked by overlapping PCR and introduced into the suicide plasmid p RE112 to construct the recombinant suicide plasmid p RE112-(?)recO.The plasmid was transformed into the strain P3 and P32 by conjugation transfer,and the gene deletion strains were constructed by two homologous recombination processes.Hence the recO gene deletion strains O3 and O32 were obtained finally.At the same time,the Pm promoter enhanced expression vector p UC18-Ptpi AT was constructed,and the recO coding gene was introduced into the vector for the recO gene deletion repaired of O3 and O32 strains,and finally the deletion repaired strains C3 and C32 were obtained.(2)Biological characteristics of fluoroquinolone sensitive strain P3,drug resistant strain P32,recO gene deletion strains O3 and O32,and deletion repaired strains C3 and C32 were identified,and it was confirmed that the deletion and deletion repaired of recO gene did not affect the basic growth characteristics of Pm.In addition,the effects of recO gene deletion on the resistance and tolerance to fluoroquinolones of Pm were tested by MIC,MBC,resistance formation time,time killing curve,and fluoroquinolone tolerance tests.It was found that the deletion of recO gene did not affect the resistance to fluoroquinolones of Pm.However,tolerance of Pm to fluoroquinolones was reduced and the formation time of fluoroquinolone resistance was effectively prolonged after recO gene deleted.(3)The effect of recO gene deletion on SOS repair response was evaluated by q PCR and construction of fluorescence reporter vector for detecting fluorescence reporter activity.It was found that recO gene deletion significantly reduced the level of SOS repair response of Pm.However,the rec BCD pathway genes makes up the influence of recO gene deletion on SOS repair response to a certain extent.In addition,through Pm infection and fluoroquinolone treatment in mice model,the formation frequency of fluoroquinolone resistance mutations was detected.It was confirmed that under the treatment of 1/2 MIC and 1 MIC fluoroquinolones in vivo,Pm is easier to form fluoroquinolone resistance mutation,and the deletion of recO gene can effectively reduce the drug resistant mutation formation frequency of Pm in vivo.In conclusion,through the construction of recO gene deletions and phenotypic analysis,this study found that inhibition of recO gene of Pm could not only reduce the level of SOS repair response but also reduce the tolerance to fluoroquinolones and prolong the formation time of resistance under the action of fluoroquinolones at low levels.Therefore,it is speculated that recO gene is a potential target of antimicrobial synergist for fluoroquinolones.This study lays a foundation for screening of fluoroquinolones antimicrobial synergist targets of Pm.