Aeromonas hydrophila isolates from clinical cases （n=43） were tested against 8 antimicrobial agents and typed by outer membrane protein （OMP） pattern by using sodium dodecyl sulfate gel electrophoresis. All isolat...Aeromonas hydrophila isolates from clinical cases （n=43） were tested against 8 antimicrobial agents and typed by outer membrane protein （OMP） pattern by using sodium dodecyl sulfate gel electrophoresis. All isolates were resistant to ampicillin （MICs, ≥16 μg mL-1） and sulfamonomethoxine （MICs≥64 μg mLl）, but susceptible to norfloxacin （MICs,≤0.5 μg mL-1）. There was a high incidence of resistance to erythromycin （90.70%） and tylosin （93.02%）, while a low incidences of resistance to ciprofloxacin （2.33%）, enrofloxacin （2.33%） and florfenicol （4.65%）. Six different outer membrane protein patterns were found among 34 isolates by analyzing proteins in the range of 22 to 50 kDa, other than 9 isolates with their respective profiles. The strains with the similar OMP profiles had similar resistances. Compared with the other strains from the same OMP patterns, NB-1, A.Pun and MR-1 had lacked the proteins in the range of 30 to 45 kDa and their resistance to florfenicol substantially increased. It is speculated that the outer membrane protein changes might correlate with decreased susceptibility to florfenicol in the three strains. Some strains which showed completely identical OMP types had a little difference in their resistance to fluoroquinolones, indicating that there might be other factors that were involved in the antimicrobial resistance of A. hydrophila.展开更多
A pair of primers were designed according to the published nucleotide sequence of a putative outer membrane protein gene(omp) of Aeromonas hydrophila.With the specific primers,a target fragment about 1.1 kb was amplif...A pair of primers were designed according to the published nucleotide sequence of a putative outer membrane protein gene(omp) of Aeromonas hydrophila.With the specific primers,a target fragment about 1.1 kb was amplified from Aeromonas hydrophila ML316 via PCR.The target fragment was inserted into the linearized pGEM-T easy vector.After enzyme restriction and sequencing analysis,the nucleotide data had been further analyzed by DNAman and ClutalW software.The analysis results showed that the cloned DNA fragment had a longest open reading frame(ORF) of 1035 nt,it predicted to be encoded a 344-aa protein with the molecular weight of 36 kD.Hydrophobicity analysis suggested that the protein was highly hydrophilic,especialy at the first 24 amino-acid,this region could function as signal peptide.The homologious comparison proved the cloned gene had 96% homology to the sequence of the omp gene,and the alignment of the amino acid sequence was 98%.The recombinant plasmid was constructed with the target gene and the expressing vector pGEX-4T-1 and then was transformed into E.coli BL21(DE3)by BamH and Sal I.The fusion protein was expressed under the IPTG inducing condition,and exhibited about 62 kD in size,very close to the predicted molecular weight of GST-MOMP,furthermore,the fusion protein was specifically recognized by anti-serum which raised against the major outer membrane protein of AHML316.Considering all these together,it proved that the cloned gene represented the major outer membrane protein gene of AHML316,and the expressed gene products shared identical antigenicity with the natural main outer membrane protein,and also provided technical support for developing an advanced gene engineering vaccine against Aeromonas hydrophila.展开更多
基金the National Natural Science Foundation of China(31072151)the Specialized Research Fund for the Doctoral Program of Higher Education,China(20090097110007)the Priority Academic Program Development of Jiangsu Higher Education Institutions,China(PAPD)
文摘Aeromonas hydrophila isolates from clinical cases （n=43） were tested against 8 antimicrobial agents and typed by outer membrane protein （OMP） pattern by using sodium dodecyl sulfate gel electrophoresis. All isolates were resistant to ampicillin （MICs, ≥16 μg mL-1） and sulfamonomethoxine （MICs≥64 μg mLl）, but susceptible to norfloxacin （MICs,≤0.5 μg mL-1）. There was a high incidence of resistance to erythromycin （90.70%） and tylosin （93.02%）, while a low incidences of resistance to ciprofloxacin （2.33%）, enrofloxacin （2.33%） and florfenicol （4.65%）. Six different outer membrane protein patterns were found among 34 isolates by analyzing proteins in the range of 22 to 50 kDa, other than 9 isolates with their respective profiles. The strains with the similar OMP profiles had similar resistances. Compared with the other strains from the same OMP patterns, NB-1, A.Pun and MR-1 had lacked the proteins in the range of 30 to 45 kDa and their resistance to florfenicol substantially increased. It is speculated that the outer membrane protein changes might correlate with decreased susceptibility to florfenicol in the three strains. Some strains which showed completely identical OMP types had a little difference in their resistance to fluoroquinolones, indicating that there might be other factors that were involved in the antimicrobial resistance of A. hydrophila.
文摘A pair of primers were designed according to the published nucleotide sequence of a putative outer membrane protein gene(omp) of Aeromonas hydrophila.With the specific primers,a target fragment about 1.1 kb was amplified from Aeromonas hydrophila ML316 via PCR.The target fragment was inserted into the linearized pGEM-T easy vector.After enzyme restriction and sequencing analysis,the nucleotide data had been further analyzed by DNAman and ClutalW software.The analysis results showed that the cloned DNA fragment had a longest open reading frame(ORF) of 1035 nt,it predicted to be encoded a 344-aa protein with the molecular weight of 36 kD.Hydrophobicity analysis suggested that the protein was highly hydrophilic,especialy at the first 24 amino-acid,this region could function as signal peptide.The homologious comparison proved the cloned gene had 96% homology to the sequence of the omp gene,and the alignment of the amino acid sequence was 98%.The recombinant plasmid was constructed with the target gene and the expressing vector pGEX-4T-1 and then was transformed into E.coli BL21(DE3)by BamH and Sal I.The fusion protein was expressed under the IPTG inducing condition,and exhibited about 62 kD in size,very close to the predicted molecular weight of GST-MOMP,furthermore,the fusion protein was specifically recognized by anti-serum which raised against the major outer membrane protein of AHML316.Considering all these together,it proved that the cloned gene represented the major outer membrane protein gene of AHML316,and the expressed gene products shared identical antigenicity with the natural main outer membrane protein,and also provided technical support for developing an advanced gene engineering vaccine against Aeromonas hydrophila.
