Survey Of Antimicrobial Resistanceand Detection Of Integron Gene Cassettes Of Vibrio Isolates From Mariculture

In recent years, with the rapid development of aquaculture, large-scale intensivefarming leads to farming environmental deterioration and diseases occurrence. Inorder to combat bacterial diseases, a large number of antibiotics have been used inaquaculture of various stages. Any use of antibiotics can increase selective pressureso that the types and quantities of the drug-resistant bacteria increase significantly inaquaculture waters and resistant pathogens diseases occur more and more frequentlyin aquaculture. Prevalence of antibiotic resistance in aquaculture water increases thedifficulty of prevention and control for aquatic animal diseases, which will causeenormous losses to the aquaculture industry. And what is more, antibiotic resistancecan infect human and transfer to human pathogens through the food chain withpotential hazards to food safety and human health. Therefore, to promote thedevelopment of bacterial resistance risk assessment, to protect aquaculturedevelopment and food safety and to safeguard human health, it is an urgent demandto monitor the bacterial resistance in aquaculture.At present, antibiotic resistance testing of aquatic bacteria is mostlyconcentrated in the resistant phenotypes and mainly uses drug sensitive slips andMIC determination method. With the development of molecular biology techniques,it plays a very important role in the detection of resistance genes, as well as newresistance mechanisms.In this study, the samples of Vibrio strains were collected from marineaquaculture regions in Chongming (Shanghai), Qidong (Jiangsu) and Qionghai(Hainan). The Vibrios were identified to species according to Vibrionaceaeidentification standards. Disk diffusion methodmethod (K-B testing) was used to testsusceptibility of15antimicrobial agents. The results were interpreted according toNational Committee for Clinical Laboratory Standards(NCCLS) and the antimicrobial resistance of these Vibrio isolates form different regions was evaluatedand analyzed. Based on susceptibility testing results, multiple drug-resistant strainswere chosen to detect Class I integron gene using PCR-sequencing method and toanalyze the antimicrobial resistance gene cassettes. This study was classified intotwo parts as follows.1. Survey of antimicrobial resistance among Vibrio isolates from differentmarine aquaculture regionsIn2009-2011,224isolates of Vibrio strains were collected from marineaquaculture regions in Chongming (Shanghai) Qidong (Jiangsu) and Qionghai(Hainan). Among these,69strains were from Chongming,84strains from Qidongand71strains from Qionghai. The Vibrios were identified to species according toVibrionaceae identification standards. Disk diffusion test (K-B testing) was used totest susceptibility of15antimicrobial agents. It was revealed that224Vibrio strainsshowed different antimicrobial resistance to15antimicrobial agents and theresistance to most antibiotics in different regions was basically similar. But to asmall number of antibiotics, there was regional difference. The strains from differentregions has a higher resistance rates of aminoglycosides, sulfonamides. Theresistance rates for tobramycin, cotrimoxazole, novobiocin, rifampicin exceeded60%. But the vibrio strains was more sensitive to neomycin and the resistance ratesremained at a lower level. The resistance rate of spectinomycin in three regionsremained at about40%. Vibrio strains in three regions had higher drug-resistancemedium rates to kanamycin, neomycin and spectinomycin, which indicated thatantimicrobial resistance would tend to rise up.Multiple drug-resistance analysis showed that multidrug-resistance rates ofVibrio strains from Chongming, Qidong and Qionghai were100%,76.2%and77.5%, respectively. It showed that multiple drug resistance were serious.Comparing the multiple drug-resistance of three regions, multiple drug resistancewas mainly concentrated in the resistance between3-7drugs.2. Detection of integron-gene cassettes among Vibrio spp strains from differentmarine aquaculture regionClass I integron gene, variable segment and3’-conserved segment of genomicDNA and plasmid DNA from188multidrug-resistance Vibrio isolates wereamplified using PCR technique. The PCR results indicated that84genomic DNAand56plasmid DNA contained intI1integrons and the detection rate were44.7% and29.8%, respectively. Amplification for variable segment of Int I1-positive strainsshowed that44strains were found to carry antimicrobial resistance gene cassettes ingenomic DNA, and34strains were found in plasmid DNA.Restriction enzyme of PCR amplification products of variable regions by Hinf1Endonuclease revealed that the same restriction map were gotten and the variableregion of the different strains carried the same resistance gene cassette. Sequencingresults of resistance gene cassette showed a single type of gene cassette arrangement(dfrA12+orf+aadA2), which encoded resistance to sulfonamides andaminoglycosides.According to the above results, the relationship between Class I integron andmultidrug resistance showed that for single vibrio strain there was no completecorrespondence between resistance gene and phenotype, but integrons wererelatively concentrated in resistance strains. In Chongming, of53multi-resistantstrains of4-7kinds of drugs,22strains were found to carry int I integron in genomeDNA and15strains in plasmid DNA. In Qidong, of58multi-resistant strains of3-7kinds of drugs,6strains were found to carry int I integron in genome DNA and9strains in plasmid DNA. In Qionghai, of48multi-resistant strains of3-7kinds ofdrugs,16strains were found to carry int I integron in genome DNA and10strains inplasmid DNA. In this study, int I integrin-positive strains showed multi-drugresistance, which there was a close relationship between int I integrin and multi-drugresistance of vibrio strains.