Genome And Plasmid Analysis Of Riemerella Anatipestife Rifampicin Resistant Strain RA-WH9

Riemerella anatipestifer?R.anatipestifer?is a gram-negative bacterium,which belongs to the family Flavobacteriaceae.It can cause a severe acute or chronic sepsis in ducks,geese,chickens,turkeys and other birds.R.anatipestifer is ubiquitous in the environment and has been reported worldwide.Due to its high mortality and low feed conversion rate,R.anatipestifer infection leads to great economic losses.Currently,antibiotics are still the most widely used strategy to control R.anatipestifer infection,because of weak cross-protection between different serotypes.Rifampicin is one of the most effective broad-spectrum antibiotics against bacterial pathogens,mainly used in the treatment of tuberculosis,leprosy,tubercular meningitis and other diseases,it's also used in many veterinary clinical drug prescriptions.In this study,a strain of Riemerella anatipestifer was isolated and purified from the tissues and organs of diseased duck,consequently named RA-WH9.K-B diffusion assay was applied to determine the antibiotic susceptibility of RA-WH9.The results showed that RA-WH9 was sensitive to ceftizoxime,chloramphenicol,florfenicol,doxycycline,erythromycin and lincomycin,intermediate sensitive to enrofloxacin,spectinomycin and tetracycline,and resistant to streptomycin,polymyxin B,ampicillin,neomycin,penicillin,norfloxacin and rifampicin.The rifampicin MIC of RA-WH9 was 50??g/m L determined by microdilution method according to CLSI2018.Using the third-generation sequencing technology of bacterial genome and bioinformatics platform,we constructed and analyzed the genomic complete map of RA-WH9.The results indicated that RA-WH9 contained one chromosome of 2488018 bp and GC content is 34.58%.A total of 2563 open reading frame were identified,including 88 drug-resistant genes,consistent with the drug-resistant phenotypes of RA-WH9.Furthermore,RA-WH9 carried a plasmid,we named p RA-59,which contains a replicator of type Inc.The skeleton structure of p RA-59 was similar to plasmid p JX,p CFC1 and p CFC2.13 genes were predicted in p RA-59,including three copies of the ISRa1 transposase.The experiments of bacterial conjugation and transduction confirmed that p RA-59 could increase the rifampicin resistance of DH5? and BL21.It could also significantly improve the rifampicin MIC about 60 times of CH3,Yb2 and HXb2.Nevertheless,we completed bacterial genome sequencing of CH3-59?CH3 conjugator?.Comparing the genome sequence of CH3 and CH3-59,we found that the Ton B-dependent receptor gene?M949₁173?was interrupted by ISRa1 transposase and its upstream promoterof p RA-59 in CH3-59.Therefore,we hypothesized that the mechanism of p RA-59 mediated drug resistance might be by inserting host bacterial chromosomes with the aid of upstream promoter,leading to the mutation of host bacteria rpo B gene,thus causing changes in rifampicin susceptibility of host bacteria.