The Molecular Biology Research Of Integrative Conjugative Elements In The Vibrios

Vibrios areopportunistic pathogens in aquatic environments, the WHO has promulgated12speciesof the vibrio genera which could do the harm to the aquatic products even human health, three ofthe most common is: Vibrio cholerae, Vibrio parahemolyticus, Vibrio alginolyticus. In recent years,as the widely existence of antibiotic residues in the environment which push the bacterialsgenerating antibiotic resistance is becoming more and more grievous and bring the clinicaltreatment to huge difficulties.Thus, the mechanism of vibrio’s antibiotic resistance plays animportant role of prevention and treatment of the diseases caused by vibrio.Integrating Conjugative Elements (ICEs) were initially found in vibrio cholerae O139groupcarrying a variety of antibiotic resistance genesin1992. These elements primarily reside in a hostchromosome but retain the ability to excise and to transfer the resistance genes to the recepors byconjugation. Although ICEs use a range of mechanisms to promote their core functions ofintegration, excision, transfer and regulation, there are common exclusion system that avoidduplicating the same elements in one host.In this study, samples were collected from the aquatic productsin aquatic market, aquaculturewater and enviromental waters. By thiosulfate citrate bile salt of sucrose AGAR medium (TCBS),464strains were isolated.Total six groups of nine different antibiotics including trimethoprim,streptomycin, sulfamethoxazole, ampicillin, rifampicin, tetracycline, gentamicin, chloramphenicoland kanamycin were chosen to survey the antimicrobial susceptibility of the464isolates. Theoverall resistance to ampicillin is the highest, with49%of strains resistant, the resistance togentamicin is the lowest, only3%of the isolates resistant to it. Isolates from the Yangtze Riverestuary samples were the worst multi-drug resistant strains (71%) while isolates from putuoaquatic market were the most multi-drug sensitive strains, only8%strains were multi-drugresistant. Through PCR detection of ICE conservative gene among the MDR isolates,32strains ofvibrio strains were determinedcontaining ICEs. Through the PCR detection of pathogenic genes,all of the22vibrio cholerae strains carried only toxR, all of the3Vibrio parahaemolyticus strainswere detected carrying only tlh, the only1Vibrio alginolyticus was found carrying fur and valC.Serotype identification showed all the vibrio cholerae strains are non-O1none O139which wasn’sthe typical pathogenic serotype,3Vibrio parahaemolyticus were also tested to be none pathogenicserotype strainsPrimers were either designed or from the references were employed to PCR amplification andsequencing and comparison targeted at ICE’s five conservative sites, Int, traI, traC, setR, attR.theresults showed that these five sites of the ICEs this research were highly conservative as its aminoacid sequence similarity to SXT/R391above97%. Primers wers specifically to amplify ICE’s fivevariable region (VR) and hotspot (HS) to sequencing and comparison. The results showed that ICEs int this study were not found carrying resistance genes indicating the resistance gene clustersmay located in other sites of the ICE. In this study, insertion of227Aas deoxyribonuclease werefirst discovered in the region of the HS4in ICEs. Insertions in the hotspos of ICEs showeddiversity and mosaic in this study.According to references, PCR amplification of eexand traG were performed, the amino acidsequence alignment analysis after sequencing showed7S-type,5R-type and3failed to determinethe type. The exclustion rate of between R or S type and untypable type needs to be further study.After determine the MIC of32ICE containing strains, appropriate antibiotics selective markers ofdonor bacteria were chosen. E.coli MG1655without ICE, plasmid and other mobile geneticelements butresistant to chloramphenicol were chosen to be receptor for conjugative experiments.Results showed that Vp25and Vc108carrying ICE are able to transfer antibiotic resistance inICEs to the E.coli MG1655through conjuation, hence, preliminary verified the competenc of ICEshorizontal antibiotic resistance genes transferace among the bacterials.Due to the increasing outbreaks of puffer fishepidemics in early summer, we gather the sample ofthe sick fish mainly rot in body, gill and tail and the same site in the health fish as control,via16srDNA library sequencing and analyzing, the major pathogenic bacteria were found to beAeromonas. As a result to provide a theoretical basis for practical production of the farmers andcorresponding measures for preventing diseases.