| In order to research the epidemic characteristics and antimicrobial resistance status of Vibrio from the important farming areas mairculture source. The Vibrios were isolated and identificated from several main objects(Cynoglossus semilaevis, Penaeus vannamei Boone, Portunus trituberculatus, etc.) in four areas of Jiangsu, Shanghai, Fujian and Hainan. The sensitivity of the Vibrios to 16 kinds of antimicrobial agents were detected, and the preponderant Vibrios separated from this experiment were typed by PFGE to understand the genetic relationship of strains from different sources. Subsequently, we tested the integron-related elements in Vibrio through the establishment of a multi-PCR detection method, and then the drugresistant gene cassette was amplified to explore the integron-related elements mediated molecular mechanism of resistance to Vibrios. Finally, we detected resistance-related qnrVC gene to quinolone, and study the prevalence and propagation mechanism of qnrVC gene in Vibrios from the molecular level, provide theoretical guidance for reasonable use of quinolone drugs in aquaculture disease prevention and treatment of Vibrios.90 Vibrio strains were isolated, the Vibrio parahaemolyticus accounted for 27.8%(25 strains), Vibrio harveyi 23.3%(21 strains), Vibrio alginolyticus 8.9%(8 strains), Vibrio vulnificus 10.0%(9 strains), Vibrio anguillarum 4.4%(4 strains), Vibrio splendidus 4.4%(4 strains), Vibrio cholerae 7.8%(7 isolates), and other Vibrio species 13.3%(12 strains). Susceptibility results showed that the resistance rates of sulfamethoxazole isoxazole, ampicillin, neomycin and oxytetracycline to the isolate strains from different regions were higher,in which the majority of Jiangsu isolate strains to quinolones(44.7% ciprofloxacin, norfloxacin 36.8%), aminoglycosides(52.6% neomycin sulfate, gentamicin 34.2%), macrolides( erythromycin 42.1%) drugs also showed high levels of resistance. Resistance genes detection result shown that: positive strains detection rate of blaTEM-1, carB, cat2, aadA, aacA, strA, strB, sul1, sul2, qnrVC, floR, tetB, tetM and arr were 5.6%, 37.8%, 1.1%, 8.9%, 13.3%, 23.3%, 27.8%, 12.2%, 30.0%, 50.0%, 6.7%, 14.4%, 8.9%, 5.6%, respectively, while the macrolide resistance genes(eg ermA and ermB) is not detected. Conclusion: the status of Vibrio resistance in aquaculture in our country is worrying, multiple drug resistant strains are in the majority, we recommend to strengthen the supervision of the use of fishing drugs, timely guide the production of rational drug use.Integron plays an important role in the wide distribution of antimicrobial resistance genes and especially in the transfer of multiple drug resistance in Gram negative bacteria. In order to establish a multi-PCR detection method for the simultaneous detection of class 1 integron(Int1), class 4 integron(SXT) and insertion sequence common region 1(ISCR1), three pairs of specific primers were designed using PrimerPlex 2.61 based on the gene sequence of int1, intSXT and ISCR1 in GenBank. The multiplex PCR system was established and optimized by changing the primer volume and annealing temperature. The spectivity and sentivity of the optimized multiplex PCR were also evaluated, and then the multiplex PCR was applied to the detection of three kinds of integron-related elements in 90 Vibrio strains of mariculture source. The results showed that the above primers could amplify three bands of 569 bp, 430 bp and 651 bp specifically. The optimized reaction conditions were as follows: the volume of primers 0.3μL for int1, 0.6μL for intSXT, and 0.6μL, and anneal temperature 50℃. Under the conditions, the detection minimum limit was 5ng/μL of genome DNA. The results of detction for the above three integron-related elements in 90 Vibrio strains, using the optimized multiplex PCR, were the same as that of the control assays. All together, this study provide a effective multiplex PCR method for the simultaneous detction of int1, intSXT and ISCR1, which could be applied to studies on integron-associated antimicribal resistance.In this chapter, 45 strains of qnrVC positive strains were detected in 90 strains of aquaculture Vibrios. Susceptibility results shown that, MIC values of the quinolones distributed 0.25-2 μg / m L in majority of Vibrio, resistance rate will remain at around 25%, the maximum number of Vibrio nalidixic acid resistance, resistance rate of 37.8%(34 / 90). There were 6 qnrVC positive strains performance out of all these five kinds of quinolones resistant, and 19 positive strains showed a entirely sensitive to the detection of five quinolones, and the resistance is not serious. The conjugation tests selected from two positive strains were successful, and the MIC value of the transconjugants were all improved. Conclusion: qnrVC positive for Vibrio parahaemolyticus and Vibrio harveyi were PFGE typed, the results showed that: between the two strains of Vibrios just several strains have homology, but there was no epidemiological correlation, and shown sporadic fashion trends. QnrVC gene was popular in Vibrios, but not necessarily present on the drug-resistant strains, a lot of sensitive strains also carry it; the qnrVC gene was metastatic, and can spread horizontally; Vibrio parahaemolyticus and Vibrio harvey that carried with qnrVC was sporadic epidemic trend. |
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