Identification And Antimicrobial Resistance Mechanism Of Riemerella Anatipestifer

The aim of this study is to identifiy clinical isolates of Riemerella anatipestifer from diseased ducks and to detect the antimicrobial resistance trends as well as detection of the resistance gene of the clinical isolates. In this study, Riemerella anatipestifer strains were isolated and identified by morphological observation, cultural characteristics, biochemical tests and PCR methods of16S rRNA, and the susceptibilities of clinical isolates of Riemerella anatipestifer to15antimicrobial were detected by microdilution method with TSB broth supplemented with5%bovin serum. PCR methods were used to detect resistance genes of the clinical isolates to chloramphenicol, aminoglycoside and rifampicin.The cat gene of chloramphenicol was located whether on genome DNA or plasmid by southern hybridization. Mutation point in rpoB gene of rifampicin, aac and aadA genes which are responsible for aminoglycosides resistance were analyzed by sequencing. Four parent strains of Riemerella anatipestifer were chosen by step wise exposuring to increasing levels of florfenicol followed by selection of resistant mutants. PCR method was used to detect the florfenicol resistance gene floR and to amplify23S rRNA and ribosomal protein L4and L22of Riemerella anatipestifer resistant strains selected in vitro by florfenicol passage.Through the results of morphological observation, biochemical tests and PCR detection, the clinical isolates were identified as Riemerella anatipestifer..The MIC results showed that the clinical isolates were highly resistant to chloramphenicol, rifampicin, colistin, cephalosporin antibiotics and aminoglycosides, but in some degree, sensitive to florfenicol, tetracycline and quinolone antibiotics. PCR results showed that cat gene was mainly involved in chloramphenicol resistance and cat gene was located on genome DNA by southern hybridization. The aminoglycosides gene was detected as aac and aadA, which have a high degree homology of98%compared with the gene sequence in NCBI. In four rifampicin resistant strains, the sequences of rpoB were compared between resistant strains and their parental strains by sequencing. There were some different mutations of rpoB in five rifampicin resistant strains. One strain mutated at position of357and358with inserting the nucleotide C and G and at the position of360with an insertion of nucleotide A resulting in Glu-Asp and Asp-His mutations in sequences. One strain existed a mutation at position of517of rpoB (T-C) resulting in Thr mutated to Cys. The other two strains had a mutation (G-A) at position of383and resulted in Arg-Lys change. There is no report on the sites of these mutations, it need be further studied whether these mutations is mediated resistant to rifampin. Riemerella anatipestifer sensitive strains could be successfully induced into florfenicol resistant strains by stepwise increasing concentration of florfenicol. Multiple mutants with significantly increased MICs (64～128mg·L-1) were generated from each parent strain. Meanwhile these mutants were resistant to erythromycin with a high MIC value. The growth curve of parental and mutants showed that the induced-resistant strain growed slower than the parent strain. The florfenicol resistant genes were not detected in all induced-resistant strains. None of the parental strain had23S rRNA and ribosomal proteins L4and L22genes mutations. The florfenicol resistant genes were not detected in all induced-resistant strains. No strains had changes in ribosomal protein L4and L22. However, one strain mutated at position of A2078G, A2304T and G2083T in23S rRNA. One strain mutated at position of A2058T, A2126G and A2353T in23S rRNA. And the third mutant had mutations at position of A2045T and A2577G in23S rRNA. All results indicated that Riemerella anatipestifer strains isolated from diseased ducks were resistant to chloramphenicol, aminoglycoside and rifampicin, but sensitive to florfenicol and some fluroquinolones.