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Isolation And Preliminary Application Of Bacteriophages Hosted Vibrio Parahaemolyticus

Posted on:2020-10-08Degree:MasterType:Thesis
Country:ChinaCandidate:Y J YinFull Text:PDF
GTID:2370330620460531Subject:Food Science and Engineering
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Vibrio parahaemolyticus is an important foodborne bacterial pathogen frequently transferred through contaminated seafood,which causes acute gastroenteritis.The bacterium can easily form biofilms on food and food contact surfaces during food processing,which makes it difficult to control by the traditional methods.In addition,antibiotics resistant of V.parahaemolyticus and environmental pollution were more popular owing to abusing of antibiotics in the aquaculture industry.Therefore,it is necessary to develop new strategies to control pathogenic bacterial transmission.Lytic bacteriophages are promising candidates,which are viruses against the specific bacterial hosts.The virus can lethally infect the pathogenic bacterial host,reduce the bacterial concentration and be less contamination to the environment.Therefore,for application in biological control,it is necessary to isolate,identify and characterize the bacteriophages.In this study,to address the need,the investigation on the bacteriophages of pathogenic V.parahaemolyticus was carried out.The detail information was as follows:Firstly,isolation and identification of V.parahaemolyticus bacteriophages.The surface water samples?N=130?were randomly collected from Yangtze river delta region during September 2016 to October 2017,including estuaries,open ocean,puddles along the shore,shellfish harvesting areas,rivers influenced by tidal influx,and so on.Thirty-four bacteriophages against V.parahaemolyticus?ATCC 17802?were isolated and purified by successive single-plaque isolation??V01-?V34?.All bacteriophages suspension(>109 PFU mL-1)was concentrated and stored at4 oC.The morphologies of the 34 isolated bacteriophages were analyzed by transmission electron microscopy.According to the morphology,all the bacteriophages belonged to the order Caudovirales,including 28bacteriophages belonged to the family Podoviridae?82.35%?,4 belonged to the family Siphoviridae?11.76%?,and 2 belonged to the family Myoviridae?5.88%?.Secondly,investigation of host ranges,physiological and biochemical characteristics of three selected bacteriophages.The diversity of the 25 strains of V.parahaemolyticus was studied by ERIC-PCR,which demonstrated multiple antimicrobial resistances to at least one kind of antibiotics.The results showed that the 25 V.parahaemolyticus strains were divided into 17 groups at a similarity of 85%.The host ranges of the 34 isolated bacteriophages were analyzed using the above described strains.The representative bacteriophage was selected from the three families according to the lysis spectrum,which had highest lysis abilities and used for further characterization.The digested patterns of the three genomes further confirmed that they were different between the nucleotide sequences of three phenotypic bacteriophages.The optimum thermotolerance,pH,and salinity concentration were 25-50?,2.0-12.0,and1.5-5.0%,respectively.The results of one-step growth curve demonstrated that three bacteriophages presented high burst sizes?458593?.Thirdly,application of V.parahaemolyticus bacteriophages.The ability to prevent biofilm formation and to destroy established biofilms were evaluated using the selected lytic bacteriophages and their bacteriophage cocktails.The data demonstrated that all bacteriophages and its mixture were promising in preventing the development of V.parahaemolyticus biofilms.However,they could not destroy the established biofilms.In addition,three selected bacteriophages and their bacteriophage cocktails were used for the depuration of oysters,which were artificially contaminated by V.parahaemolyticus?ATCC 17802?.Results indicated that selected lytic bacteriophage was effective to reduce the concentration of V.parahaemolyticus.The concentration of V.parahaemolyticus in the artificial seawater was reduced from 1.0106 CFU mL-1 to 3.6103 CFU mL-1 after48 h incubation?post-infection?.The concentration of V.parahaemolyticus of oyster tissues was 5.7102 CFU mL-1 after bacteriophage treatment.On the other hand,the concentration of the pathogenic bacterium was 7.9104CFU mL-1 without the treatment of bacteriophages,which was much higher than the treated group.In summary,34 strains of bacteriophage were isolated and identified in this study.Three of representative bacteriophages isolated in this study were stable under different conditions of temperature,pH,and salinity.In addition,the bacteriophages could efficiently inhibit the development of bacterial biofilms.However,they could not destroy the formed biofilms.Fortunately,the results showed the isolated bacteriophages are promising candidates for the depuration of pathogenic V.parahaemolyticus in oyster tissues.The results of this study provides a strategy on prevention and control of pathogenic V.parahaemolyticus by using of bacteriophages.
Keywords/Search Tags:Vibrio parahaemolyticus, bacteriophage, biofilm, aquaculture, oyster
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