| Pathogenic Aeromonas hydrophila is one of the important pathogens which causes livestock sepsis, human diarrhea and wound infection. It not only brings aboutsignificant losses in aquaculture, but also threatens thepublic health of humans as well as animals. Freshwater fish is susceptible toAeromonas hydrophila, which increasing the chances of disease outbreaks. This disease is usually controlled by antibiotics, however, the overuse of antibiotics has caused a rising bacteria resistancerate and multiple resistance phenotype, which will not only affectthe development of the aquaculture industry, but also bring great difficulties for the prevention and treatment of this disease. At the same time, it also has serious impacts on the quality of aquatic products and ecological environment, which posing a great challenge to food safety and public health.Therefore,masteringthe distribution and drug resistance of aquatic pathogenic Aeromonas hydrophila will lay the foundation for finding a safe and effective prevention and control method.In this research, a total of 369 samples including of gill swabs, various fish intestinal contents and fish pond waterwere collected in Changchun area from 2014 to 2015. First, selective culture media were used to isolate Aeromonas hydrophila, polymerase chain reaction(PCR) were used to detect virulence genes.Then, ordinary PCR, 16 SrRNA sequencing, the National standard method and biochemical identification were used to identify the strains of virulence genes-carrying Aeromonas hydrophila. Finally, MLST identification assay was conducted. A total of 363 strains were isolated from 369 samples, 40 strains of which carried virulence genes. 32%(15/50)of them isolated from sick fish, while 7.7%(24/313) of them isolated from healthy fish. All these 40 strains were proved positive forAeromonas hydrophila identification in polymerase chain reaction(PCR). 16 SrRNA sequencing was not able to distinguish species of aeromonaseffectively. The National standard method and biochemical identification provided basicallyconsistent results(24 and 25 strains respectively).The results varied with different appraisal method, and for a better identification, growth characteristics and biochemical parameters were needed.In this study, 2 sets of primers and probes were designed based on the sequences of the 16 S rDNA and aerolysin(aer A) gene, and duplex real-time TaqMan polymerase chain reaction as say were established for the detection of Aeromoonas hydrophila. Different concentrations of standard plasmid were used to investigate thesensitivity of the newly-built system against the ordinary PCR. The specificity of the established method was evaluated by using 12 other pathogens as contrast. Isolation and identification methods were used as control t o test the reliability in the application in clincial sample. The results showed the sensitivit y of the duplex real-time TaqMan PCR assay for testing of recombinant plasmids was 10 copie s per reaction, which was 100 times higher than the ordinary PCR. There were no specific amp lificationwhen the 12 other pathogenic bacteria were tested. Consistent results of the clincial samples were acquiredfrom routine methods and this newly-built identification methods. So the duplex real-time TaqMan polymerase chain reaction(PCR) assay will provide supplem entary means for the rapid diagnosis and epidemiological investigationof pathogenicAerom onas hydrophila.In this study, for investgated the pathogenicity of Aeromonas hydrophila, The genes ofaerolysin and hemolysin were detected. Carriage ratio of the two virulence factors wa s detected by PCR. Cytotoxicity tests and animal experiment were used to explore the t oxicities of the isolates carrying virulence genes. The results showed that the two virule nce genes were widely existed in the pathogenic isolates. The carriage rate of aerolysin and hemolysin were 97.5%(39/40) and 92.5%(37/40), respectively. Furthermore, 36 isol ates carried both virulence genes. Cytotoxicity tests demonstrated that all 40 virulence ge ne-positive isolates showed varying degrees of cytopathic effect. The toxicity of positive isolates was higher than that of negative isolates. All isolates were lethal to mice, and t he difference in deaths and the lethal time showed diverse virulence of these strains, wh ich demonstrated the reliabilityof cytotoxicity tests. Our study also demonstrated that viru lence strength have no direct relation with the carriage ofaerolysin gene or hemolysin ge ne.In order to explore the drug-resistant spectrum and resistance feature of Aeromonashydrophila isolates, K-B disk diffusion method and BD Phoenix?-100 Automated Microbiology System were used to detect the sensitivity ofthe virulence genes-carrying isolates to 26 kinds ofcommonly used antibiotics. And PCR assay were used to detect 10 related drug resistance genes. The results showed that 54 strains were sensitive to five kinds of antibiotics including imipenem, cefotaxime and aztreonamand. Both of them were resistant to five kinds of antibiotics, including streptomycin, clindamycin and penicillin. All strains showed multi-drug resistance pattern. All of pathogenic Aeromonas hydrophila isolates were resistant to at least 3 kinds of antibiotics, and some of them resistant to 6 kinds at most.The resistance of strains from diseased fish were much lower compared with those from healthy fish. A strain, named AH24, presented the most severe drug resistance and showed a significant difference with others.Among all the resistance genes detected, 10 were poorly detected in these strains. The investigation obtained the basic epidemiological data of aquatic products in Changchunarea, which may provide scientific basis for further study about the formation and transmission of drug resistance in aquatic product, as well as the clinical treatment of Aeromonas hydrophila. |
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