| Vibrio parahaemolyticus(VP)is a Gram-negative bacterium belonging to the genus Vibrio,Vibrionacea.It is halophilic and widely found in the marine environments.This is a worldwide food-borne pathogenic bacterium.Human can get acute gastroenteritis mainly due to consumption of contaminated raw or undercooked seafood.In this thesis,starting from the investigation,risk identification of contamination in RTE food and aquatic products.We studied the phenotypes and genetic type characteristics of the isolated strains,such as serotypes,antimicrobial susceptibility,virulence gene and molecular typing.Based on this,we obtained the complete genetic information of main representative strains by genome sequencing.Finally,the overall regulation processes of V.parahaemolyticus under cold stress were obtained by combined analysis of transcriptome and proteome.The specific contents are as follows: 1.The prevalence of V.parahaemolyticus in 2531 samples from 43 cities had been investigated.The contamination rate and level reached 24.26% and the average MPN was 11.19 MPN/g.The contamination rate in summer was 30.66%,which significantly higher than that in winter(17.66%).In the Southern China,especially in the coastal areas,the contamination rate and level are higher than those in the Northern China(30.07% and 13.99% respectively).The contamination rate of aquatic products was higher which reach 33.53%;5.79% of RTE foods which have high risk to human were also contaminated,and the average MPN was 1.62 MPN/g.Meanwhile,the contamination of V.parahaemolyticus was more serious in bazaars(30.17%),but the prevalence rate in supermarkets also reached 17.82% which is regulated more stringently.Therefore,it is necessary to carry out the risk monitoring of V.parahaemolyticus in different food types and environments,then carry out risk assessment and management for high-risk contamination nodes.2.Complete the phenotypic and genetic diversity analysis of 39 V.parahaemolyticus strains isolated from RTE food and 150 strains from South China aquatic products.The susceptibility tests of RTE food isolates showed that these strains were highly antimicrobial resistant,and that 53.85% strains were resistant to more than two antibiotics.The serotypes O2 were the most common,and the O3 type has same pandemic strains.The clustering of ERIC-PCR and MLST were obvious,especially in the MLST typing.That has many new loci and ST types.The contamination rate of V.parahaemolyticus strains in aquatic products from southern China were 43.75%.The positive rate of trh was 40.67% and 40% of the isolates were O2 serotype.The ERIC-PCR typing revealed the relevance between different strains,clinical and food strains.Antibiotic resistance to streptomycin,cefazolin and ampicillin were obvious.The multi-resistant strains also had a significant proportion.3.Nineteen V.parahaemolyticus strains were sequenced.Comparative analysis showed that the genomic size and GC content of these strains were similar,but the rate of core genome in clinical strains(68.97%)was higher than food strains(48.05%).The core genome is mainly involved in RNA modification,defense mechanism,energy production and conversion.No unique core gene cluster was found in clinical isolates,but food strains carried less than 50% gene clusters among the five gene clusters in the clinical isolates.Analysis of virulence-associated and antibiotic resistance genes showed the risk of the food strains.Phage appeared in all strains.Although there were differences in fragment size,GC content and number between food strains and clinical strains,the functions were mainly related to transcription regulators,membrane proteins and DNA modified proteins.The membrane-associated proteins were same,which was inconsistent with the result by scanning electron microscopy(SEM).4.Transcriptome sequencing revealed an average of 4216 genes at low temperature.In food strain V82,118 genes has significantly expression changed,including 42 up-regulated and 76 down-regulated genes.These genes were involved in glutamine,arginine metabolic process and oxidoreductase activity.There were 513 different genes in clinical strain VL8,of which 182 genes were up-regulated,331 genes were down.These genes mainly participate in catabolism,phosphate ion transport and oxidoreductase activity.These genes were enriched in carbon metabolism,arginine biosynthesis,pyruvate metabolism,and butyrate metabolism.The difference was that the ABC transporter changed most obviously in VL8,while in V82 was amino acid biosynthesis pathway.An average of 1539 proteins were obtained by iTRAQ.There were 20 up-regulated and 25 down-regulated proteins in VL8,which were mainly involved in the organic nitrogen compounds metabolism,cytoplasm and the molecular function is binding;While in V82,17 up-regulated and 129 down-regulated proteins were found,which were mainly involved in the biosynthesis process,cell and the molecular function is binding.These difference proteins were mainly associated with carbon metabolism and pentose phosphate pathway.The ribosome and nucleotide excision repair protein clusters also changed.5.Comprehensive analysis of two V.parahaemolyticus strains by transcriptome and proteome revealed 22 common pathways changes under low-temperature,such as carbon metabolism,amino acid biosynthesis and fatty acid metabolism.Among them,pyruvate metabolism and ABC transporter pathway had obvious variation in the number of genes and proteins.When added different concentrations of exogenous pyruvate at low temperature,fatty acids content in V.parahaemolyticus was decreased.Meanwhile,bacterium growth was inhibited and difficult to recover.The changes of key genes related to pathways were also consistent.The result proved that pyruvate can reduce the hypothermia adaptability of V.parahaemolyticus.In summary,the thesis was first time to comprehensively analyze on the prevalence of V.parahaemolyticus in RTE food and aquatic products in our country.Then,the genetic diversity had studied.The result provided us a better foundation for further understanding of the molecular evolution,epidemiology trend and risk identification of this bacterium.Genomic sequencing provided detailed genetic information on V.parahaemolyticus strains.Finally,we clarified the regulation mechanism of V.parahaemolyticus under cold stress.Demonstrated that exogenous addition of pyruvate can reduce the cold adaptation,and provided a theoretical basis for controlling V.parahaemolyticus under cold storage and transportation. |
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