| Previously, five isolates of Aeromonas hydrophila (Ah638, Ah640, Ah563, Ah541and Ah465) were characterized by biochemical tests and conserved in our laboratory, Aeromonas hydrophila ATCC7966were used as control strains. In this study, artificial induction of resistant to levofloxacin resistance A. hydrophila were obtained with sub-inhibitory concentration subculture method in order to investigate mechanisms of fluoroquinolone resistance and cross sensitivity to quinolones and other drugs. Cloning, prokaryotic expression and polyclonal antiserum preparation of Aeromonas hydrophila aerolysin was obtained by immunizing rabbits. To analysis the immunogenicity of polyclonal antiserum, Crucian carp (Carassius aumtus) were injected intraperitoneally with antiserum and challenged with a virulent strain (Ah541) of Ahydrophila24h post-immunization. The results were summarized as follows:1. The minimum inhibitory concentration (MIC) of5pathogenic A.hydrophila strains was interpreted by the Clinical and Laboratory Standards Institute breakpoints for levofloxacin respectively. And Aeromonas hydrophila ATCC7966were used as control strains. In addition, the MIC to levofloxacin were that Ah465of as an intermediary strains,5strains to levofloxacin were sensitive. A.hydrophila was grown at28℃for72h and used it to test for the development of resistance after9sequential subcultures in sub-inhibitory concentrations of levofloxacin. After9subcultures the MIC to levofloxacin was500-3906times greater than initial values. The expected fragments of gyrA QRDR and the parC QRDR were amplified and obtained from all mutants and wild type strains. The nucleotide sequences were determined and compared. Sequence analysis showed that all mutants carried a point mutation in the GyrA QRDR at codon83, leading to the substitution Ser-83→Ile. In addition, one strains harbored a mutation in the GyrA QRDR at position92, generating a Leu-92→Met change. The predicted peptide GyrA fragment differed at only four positions outside the QRDR, at positions50(Phe or Tyr),116(Asn or Ser),165(Thr or Ser), and168(Val or Ile), and no amino acid alterations were discovered in parC QRDRs. The reverse mutations were observed at position83,116,165in induced mutants which showed resistance to fluoroquinolone resistance. For these levofloxacin-resistant mutants, the activity of levofloxacin against all mutants but one was increased two or fourfold in the presence of reserpin. And all mutants showed increased resistance to each of the other three quinolones (norfloxacin, enrofloxacin and ofloxacin).2. Aerolysin (Aer) gene of Aeromonas hydrophila strain Ah15isolated in HuBei, China was amplified by PCR and sequenced. Phylogenetic analysis revealed that the strains isolated from China for a together, foreign strains for a together in phylogenetic tree. The Aer of Ah15was cloned into the vector pET-32a(+) and expressed in Escherichia coli BL21(DE3) pLysS. A band of72.2kD fusion protein of Aer was confirmed as a inclusion body protein by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Western-blotting shows the polyclonal antiserum obtained by immunizing rabbits can recognize the recombinant fusion protein, showing that the recombinant protein has immunogenicity, and can be used for candidate genes of genetic engineering vaccine.3. Crucian carp (Carassius aumtus) were injected intraperitoneally with antiserum and challenged with a virulent strain (Ah541) of A.hydrophila24h post-immunization. Significantly higher mortality was noted in crucian carp passively immunized with physiological saline (87%), in comparison with mortalities of40%in the fish passively immunized with antiserum14days after A.hydrophila infection by i.p. injection (P<0.05). The results suggested that immunity provided by antiserum because antibody titres against A.hydrophila in the fish injected with antiserum is same to the control in agglutination test. |
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