Functional Study Of RIA₁614 Gene Of ABC Efflux Pump In Riemerella Anatipestifer

Riemerella anatipestifer?R.anatipestifer?is a Gram-negative bacterium that can infects ducks,geese,turkeys and other poultry birds.It severely affects the production and causes significant economic losses of the duck industry.Up-to-date R.anatipestifer consists of 21 serogroups with no cross protection,that remains as a challenge for prevention of the disease using vaccine.Therefore,antibiotics are currently mainly used to prevent and treat R.anatipestiferinfection.Widespread use of antibiotics lead to the growing microbial resistance and resistant strains are constantly being produced.Keeping ii view the foregoing,exploring the resistance mechanism of R.anatipestifer has become one of the current research directions,in order to provide a theoretical basis for reducing the drug resistance of the bacteria and improving the application effect of antibiotics.Until now,little is known about the resistance mechanisms of R.anatipestifer.Preliminary studies shown that R.anatipestifer resistance to aminoglycoside groups mediated by inactivating enzyme.The mutations in the resistance-determining region of the gyrA gene of R.anatipestifer is one of the mechanisms that lead to anti-quinolone resistance.Theefflux pump is anothermain mechanism of resistance to multiple antibiotics in R.anatipestifer.R.anatipestifer carries two types of integrons?class1 and 2?and multiple drug resistance gene cassettes,which are associated with their drug resistance profiles.Also,integrons is the mechanism that mediates resistance in R.anatipestifer.This study identified the biological function of the ABC efflux pump gene RIA₁614 of R.anatipestifer in the three aspects of drug efflux,strain growth and strain virulence.The RIA₁614 gene deletion strain?RA-GD?RIA₁614?and the reverting strain RA-GDc?RIA₁614 of RA-GD strain were constructed by co-culture of donor bacteria and recipient bacteria by conjugative transfer and homologous recombination,and the biological function of RIA₁614 gene in R.anatipestifer RA-GD strain was studied.The antibiotic susceptibility test showed that the RA-GD?RIA₁614 was more sensitive to aminoglycosides and organic solvents than The strain RA-GD and the restorer strain RA-GDc?RIA₁614 of RA-GD.When the strain RA-GD and the restorer strain RA-GDc?RIA₁614was exposed to the sub-inhibition level of aminoglycosides and organic solvents,the transcription level of the RIA₁614 gene was significantly up-regulated.The streptomycin accumulation test showed that the RIA₁614 gene was able to efflux streptomycin,which further indicated that the gene was an efflux pump gene.The growth curve and in vitro competition assay showed that the growth rate of the RA-GD?RIA₁614 was not significantly changed compared with the strain RA-GD and the restorer strain RA-GDc?RIA₁614,but it's in vitro competition test showed that the in vitro competition ability of the RA-GD?RIA₁614 was 5.5 times lower than that of the strain RA-GD,andwas 1.8 times lower than that of the restorer RA-GDc?RIA₁614.The LD50 assay showed that the virulence of the RA-GD?RIA₁614 was 43 times lower than that of the strain RA-GD,and 12 times lower than the recovery strain RA-GDc?RIA₁614.The results of this study indicate that the RIA₁614 gene confers resistance to aminoglycosides and organic solvents in R.anatipestifer,and is also one of the virulencefactors of R.anatipestifer.Based on the results of this study and amino acid sequence analysis,we confirmed that the RIA₁614 gene belongs to the ABC efflux pump,and its encoded protein is named as the MarB protein of the ABC efflux pump of R.anatipestifer.In this study,the RA-GD?RIA₁614 and the recovery strain RA-GDc?RIA₁614 of RA-GD strain were successfully constructed,and the deletion strain and the recovery strain were stably passaged.The ABC efflux pump gene RIA₁614 was identified to confer resistance to aminoglycosides and organic solvents to R.anatipestifer,and it was also one of the virulence factors of R.anatipestifer.