Horizontal Gene Transfer And Expression Characteristics Of Vibrio Parahaemolyticus In Shrimp Culture System

Vibrio parahaemolyticus is a kind of halophilic bacteria,which mainly exists in sea water and fish,shellfish and other seafood near the coast,posing a huge threat to our country’s aquaculture and human health.Among them,acute hepatopancreas necrosis(AHPND)caused by Vibrio parahaemolyticus is a bacterial disease that causes devastating deaths in shrimp farming(up to 100% mortality).This disease has caused significant damage to the global shrimp farming industry.Economic losses.The main research content of this study is to monitor the shrimp farms in 5 cities in northern China(Dalian,Yingkou,Tianjin,Tangshan,Huanghua)for many years,through drug susceptibility experiments,multi-site serial typing analysis,and comparative genomics Analysis,transcriptome analysis,preliminary exploration of the evolution of Vibrio parahaemolyticus.The research results obtained are as follows:1.The use of antibiotics in shrimp farms drives the evolution of drug resistance and virulence of Vibrio parahaemolyticus.In this study,we first monitored the real-time evolution of Vibrio parahaemolyticus in response to successive antibiotic treatment in three shrimp farms(Tianjin,Tangshan,Huanghua)in North China from 2011 to 2018 by multilocus sequence typing(MLST)and whole-genome sequencing.The results showed that the gradual emergence of drug resistance of florfenicol and enrofloxacin was related to the use of antibiotics.Genomic analysis of ST122 strains isolated from 2014 to 2016 showed that the acquisition of drug-resistant mobile gene elements with insertion sequence(ISVal1)was closely related to the antibiotics used in shrimp farms since2014.In addition,we also found that ISVal1 transposon formed 50 insertion sites in30 genomes,which caused partial gene inactivation and promoted the horizontal gene transfer of pathogenic Island(PAI)and adaptive island.In addition,comparative genomic analysis of strains since 2016 showed that the virulence genomic island of strain ST1 trh-nik-ure was transferred from chromosome to plasmid in Tianjin farm,and then transferred to strain ST422 through gene horizontal transfer,which improved the virulence of strain ST422.These results show that the use of antibiotics not only promotes the emergence of drug-resistant gene island,but also drives the virulence evolution of Vibrio parahaemolyticus.2.Comparison of the horizontal gene transfer of Vibrio parahaemolyticus under different culture modes.We monitored Vibrio parahaemolyticus in shrimp pond culture system(Yingkou)and circulating water culture system(Dalian)for three years.No antibiotics were used in these farms to exclude the potential impacts of antibiotics on the emergence of antibacterial resistance.Multilocus sequence typing(MLST)was utilized to characterize the dynamics of V.parahaemolyticus populations.Whole-genome sequencing(WGS)was conducted to determine the representative sequence types(STs)at each farm.Results revealed that the population structure of V.parahaemolyticus remained stable over time in both RAS farms,during the three-year monitoring process,4,8 and 3 ST types were found.On the contrary,12,16 and 9 ST types were found in pond culture,and the population of Vibrio parahaemolyticus changed every year.Based on the phylogenetic analysis of the whole genome sequence,50 isolated strains were divided into five clusters.Among them,the strain with ST917 A contained a genomic island inserted into the rec A gene.Pair-wised genomic comparison of isolates from the same STs showed that they were genetically related but belonged to different clones associated with geographical distribution.These results suggested that RAS presented a specific ecological niche by minimizing the water exchanges with the external environment.3.After acquisition of the plasmid with T6 SS,the adaptability of Vibrio parahaemolyticus to the environment was improved.We isolated and identified two strains of Vibrio parahaemolyticus with ST114 in Yingkou pond farm in 2020 and 2019,respectively,strain VP14-2020(new type VI secretion system carried in plasmid)and strain YK114(wild type).In this chapter,we conducted genomic comparison for two isolates and compared the phenotype difference between two strains.Results showed that VP14-2020 harbored a 103 kb plasmid which consist of two fragments: 73 kb fragment with 97.4% DNA identity with plasmid p TJ114-1 and 36 kb T6SS1 operon.Microbial blastn found that this T6SS1 operon was also presented in two strains with a similar plasmid and chromosome I of strain VP368.The transcriptome results showed that after VP14-2020 obtained T6 SS through horizontal gene transfer,its growth ability,virulence and environmental adaptability were enhanced relative to that of strain YK114.Co-culture assay suggested that VP14-2020 had significant higher antibacterial activity against Vibrio cholerae relative to isolate YK114.In conclusion,t6 ss promotes the acquisition of genetic traits to improve the adaptability and virulence of Vibrio parahaemolyticus.Therefore,the newly obtained T6 SS may be crucial for the survival of Vibrio parahaemolyticus because it reduces the selection pressure of adaptive evolution of Vibrio parahaemolyticus.This study is the first to propose the evolutionary trajectory of bacterial genes,which helps us understand the mechanism of the emergence of new genotypes.