| Vibrio belongs to Gram-negative bacteria and is widely distributed.Some conditional pathogenic bacteria belong to food-borne pathogenic bacteria,which seriously threaten the safety of aquatic products and human health.At present,antibiotics are still the main method to treat Vibrio diseases.However,with the irrational use or large scale use of antibiotics,the Vibrio bacteria produce different degrees of antibiotics resistance.Therefore,it is necessary to monitor the resistance of Vibrio from aquaculture sources to provide a theoretical basis for effective control of the resistance.In order to research the epidemic characteristics and antimicrobial resistance status of Vibrio culture from the four areas of Shanghai,Jiangsu,Fujian and Hainan,the Vibrios were isolated and identificated;The antimicrobial resistance and genotyping were investigated on Vibrio parahaemolyticus isolates from cultured shrimp to understand the prevalence that in four regions,and the carrying rate of antimicrobial-resistant genes,the correlation between antimicrobial-resistant phenotypes and genotypes,which to provide basic data for the disease control of V.parahaemolyticus and the study of resistance mechanism in Vibrio parahaemolyticus;Subsequently,the qnr VC gene in 38 strains of Vibrio were detected,gene localization,transferability evaluation and gene environment analysis,to understand the prevalence and analyze the genetic background of qnr VC gene in Vibrio,and its transfer mechanism,to provide basic data support for the prevention and control and the spread of antimicrobial-resistant Vibrio from aquaculture sources.1.The prevalence of Vibrio from aquacultureIn this experiment,70 strains of Vibrio were isolated from four maricultral regions,and identified by biochemical and molecular biological identification methods.The results showed that the most isolated strain was Vibrio parahaemolyticus 51.4%(36 strains),followed by Vibrio harveyi 27%(19 strains).The number of Vibrio isolates varies in different regions,which may be related to the isolated areas and the number of samples.The investigation of the prevalence of Vibrio from aquaculture sources provides guidance for the prevention and treatment of Vibrio diseases and antimicrobial use.2.Detection of drug resistance and resistance genes of Vibrio parahaemolyticus from cultured shrimpVibrio parahaemolyticus isolates(n=36)were recovered from cultured diseased shrimps.The agar dilution method was used to determine the antimicrobial resistance of the strains to 16 antimicrobials.The presence of genes involved in antimicrobial resistance to quinolones(qnr A,qnr B,qnr S,qnr VC),phenicols(cat,optr A,flo R,cfr),tetracyclines [tet(A),tet(B),tet(M)],sulfonamides(sul1,sul2,sul3),aminoglycosides(str A,str B,aad A,aac A),rifampicin(arr),?-lactams(car B)and macrolides(erm)were detected by using PCR amplication and DNA sequencing.The results indicated that the isolates exhibited high resistance to ampicillin(88.9%),sulfamethoxazole(SMZ)(66.7%),and all the isolates were susceptible to neomycin sulfate,gentamicin,ceftriaxone and meropenem.In general,multi-drug resistance(MDR)was highly prevalent(61.1%),and 1 isolates were resistant to six antimicrobials.Furthermore,72.2% and 58.3% of the isolates were primarily mediated by qnr VC and str B,respectively;and macrolides and rifamycin resistant gene were not detected in all the V.parahaemolyticus isolates.There are obvious mismatch between the antimicrobial resistance phenotypes and genotypes.Prompt the complexity of resistance to V.parahaemolyticus.3.Molecular characterization of qnr VC in Vibrio spp.The section of the research is to understand the mechanisms of plasmid-mediated quinolone resistance in Vibrio spp.The 28 strains of qnr VC positive strains(26 V.parahaemolyticus and two strains of V.vulnificus,V.anguillarum)were detected in 38 strains of aquaculture Vibrio isolates.MIC values of two strains of V.vulnificus and V.anguillarum against five quinolones were measured,The results showed that V.vulnificus was resistant to ciprofloxacin and norfloxacin,V.anguillarum was resistant to nalidixic acid,and two strains of V.vulnificus were less resistant to quinolones;The conjugation tests selected from two positive strains(V.vulnificus,V.anguillarum)were successful,and the MIC value of the transconjugants were all improved: the MIC values of J53-Vv strain against five quinolones,ciprofloxacin,norfloxacin,enrofloxacin,ofloxacin and nalidixic acid,were increased by 8 times,4 times,4 times,16 times and 1 times,respectively.Primer walking was used to study the gene environment.The results showed that the qnr VC genes carried by V.vulnificus and V.anguillarum were located on plasmids of about 7000-8000 bp.After the comparison of plasmid sequencing results by blast analysis,it was shown that the qnr VC genes were linked to the drug resistance gene cassette dfr6 carried by class 1 integrons,and the spread of qnr VC genes might be associated with class 1 integrons.Finally,the qnr VC9 gene on V.anguillarum was knocked out,and the strain was constructed using the suicide plasmid p CVD442 to provide a basis for the functional study of the qnr VC9 gene.This suggests that plasmidmediated quinolone-resistant strains have the risk of transmitting resistance genes through plasmids,which is of guiding significance for the prevention and treatment of Vibrio species from breeding sources and the rational use of quinolones. |
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