Study On Riemerella Anatipestifer GE296?RS02115 Mediated Tetracycline Tolerance

Riemerella anatipestifer(R.anatipestifer)is the main pathogen that seriously threatens the healthy development of the duck industry in my country,and brings huge economic losses to the duck industry.There are currently 21 serotypes of Riemerella anatipestifer,and there is no cross-protection between different serotypes.At present,antibacterial drugs are mainly used to prevent and treat this bacterial infection.However,with the widespread use of antibacterial drugs,the phenomenon of multi-drug resistance of the bacteria has become more and more serious,and the research on its resistance mechanism is more urgent.The Major Facilitator Superfamily(MFS)transporter is one of the largest known membrane transporter families,and its function is closely related to many life activities.At the same time,it also participates in the efflux of drugs,leading to multiple drug resistance of Riemerella anatipestifer.In this study,Riemerella anatipestifer RA-LZ01 strain was used as the research object to construct GE296?RS02115 gene deletion and reverting strains to study the effect of MFS efflux pump GE296?RS02115 on the resistance of Riemerella anatipestifer.Through homologous recombination mediated by suicide plasmid p RE112 and forward screening with erythromycin resistance marker,gene deletion strain?GE296?RS02115 was obtained,and E.coli-Riemerella anatipestifer shuttle plasmid p CPRA was used as the vector to construct the response strain c?GE296?RS02115.The minimum inhibitory concentration(MIC)of 11 types of commonly used clinical antibiotics against wild strains,missing strains and reverted strains was determined by the micro broth dilution method,and the pathogenicity of the strains to ducks was evaluated by measuring the growth curve.Predict the six key amino acid positions of GE296?RS02115 gene related to drug resistance,and carry out point mutations one by one to verify the resistance of point mutants to tetracycline antibiotics.Express GE296?RS02115 protein and point mutant protein,and perform ITC detection with tetracycline to verify the binding ability of the protein and tetracycline.The results showed that compared with the wild strain,the MIC value of the tetracycline antibiotics of the missing strain was significantly decreased,while the MIC value of other antibiotics did not change significantly;The growth rate of the deleted strain was significantly lower than that of the parent strain;The deletion of the GE296?RS02115 gene significantly reduced(P<0.05)the virulence of R.anatipestifer.The challenge test found that the mortality of the deleted strain was significantly reduced,and the growth rate and virulence of the deleted strain were also restored after gene restoration.Six point mutant strains were successfully constructed,and the drug susceptibility test found that after the double arginine at positions 77-78 was mutated,it was more sensitive to tetracycline antibiotics than the unmutated strain.After protein expression and purification of the point mutant gene and the unmutated gene,the tetracycline ITC test found that the binding ability of the mutant protein at positions 77-78 to tetracycline was significantly reduced.It shows that the 77-78 th double arginine is the key amino acid site involved in the resistance of tetracycline antibacterial drugs.This study successfully constructed LZ-01 strain GE296?RS02115 gene deletion strain ?GE296?RS02115 and gene reverting strain c?GE296?RS02115,and both the deletion strain and the reverting strain can be passaged stably.It was identified that the MFS efflux pump gene GE296?RS02115 mediates the efflux of tetracycline antibiotics,which can significantly affect the growth and virulence of Riemerella anatipestifer.Both the point mutation drug susceptibility test and the ITC test proved that the double arginine at positions 77-78 of the GE296?RS02115 gene is a key site related to drug resistance in this gene.