Construction Of ABC Efflux Pump GE296-RS03245 Gene Deletion Mutant Of Riemerella Anatipestifer And Its Contribution To Organic Solvent Resistance

Riemerella anatipestifer(RA)is widespread worldwide,is highly contagious,and has a high mortality rate,causing serious damage to the economic income of farmers.Pathogens have many serotypes,and vaccine prevention is difficult.The use of antibiotics is currently recognized as a feasible preventive measure for the disease.However,the unrestricted use of antibacterial drugs has caused an endless emergence of multi-drug resistant strains,which has increased the difficulty of effective prevention and treatment.Therefore,understanding its resistance mechanism is extremely important for the prevention and control of Riemerella anatipestifer.The efflux pump is one of the main drug resistance mechanisms of drug-resistant strains.Among them,the ABC efflux pump is an important efflux mechanism of the efflux pump family.Its basic structure consists of two TMD domains and two NBD domains.Rely on ATP hydrolysis to provide energy efflux drugs.At present,there are few studies on the drug resistance system of this bacterium,and further research on its drug resistance can accumulate theoretical knowledge for the effective prevention,control and treatment of Riemerella anatipestifer.In this study,the RA-LZ01 strain GE296-RS03245 gene deletion strain RA?3245 was constructed by homologous recombination and combined transfer methods,and growth curve determination,in vitro competition determination,MIC value determination,organic solvent sensitivity determination and real-time fluorescence quantification were performed.PCR assay to determine the effect of GE296-RS03245 gene on the biological function of RA-LZ01 strain.Through growth curve data analysis,it is found that the growth rate of the GE296-RS03245 gene deletion mutant has no significant difference compared with the parent strain RA-LZ01;in vitro competition experiments show that there is no significant difference in the competitiveness of the deletion strain and the parent strain;antibiotic sensitivity experiments show that the deletion strain RA?3245 There is no significant change in susceptibility to aminoglycosides,sulfonamides,?-lactamases,peptides,quinolones,and tetracyclines;the organic solvent sensitivity experiment found that the missing strains are less sensitive to organic solvents such as methanol,ethanol and isobutanol.Sensitivity was significantly improved;through real-time fluorescent quantitative PCR experiments,it was found that the parental strain was completely exposed to the organic solvent methanol,ethanol,and isobutanol,and the transcription and expression levels of the GE296-RS03245 gene increased by 3.1times,6.7 times,and 3.5 times,respectively.The results of the above studies show that the GE296-RS03245 gene does not affect the growth rate and in vitro competitiveness of the RA-LZ01 strain,and has nothing to do with the resistance of several common antibiotics.The GE296-RS03245 gene mediates the resistance of R.anatipestifer to organic solvent methanol.,Ethanol and isobutanol resistance.Sequence analysis revealed that the GE296-RS03245 gene encodes the protein containing the ATP-binding cassette domain of the ABC efflux pump,which is the NBD domain of the ABC efflux pump.