Study On The Acid Tolerance Of Vibrio Parahaemolyticus From The Biological Characteristics 、Transcriptome And Comparative Proteom Analysis

Vibrio parahaemolyticus is an important food borne pathogen, exists widely in sea products, it can cause gastrointestinal discomfort, diarrhea, vomiting and other symptoms if consumers eat the seafood which is not processing or incomplete. The global warming caused the marine factors changing and the emergencing of all kinds of extreme climate phenomena, promoting pathogenic bacteria evolution and improving their survival system to resist external environment that can survival and attack host. The most important factors are temperature and the p H whatever in environment and eafood processing or transportation process. This study is to compare the difference between the original strain and acid tolerance strains from the biological characteristics, movement, biofilm formation ability, resistance and cytotoxicity combined with the results of transcriptome and comparative proteomic, and analyze the mechanism of the survival and tolerance of Vibrio parahaemolyticus in acid condition then to provide basic data and theoretical basis for the risk assessment of Vibrio parahaemolyticus.1. Obtaining of the acid tolerant strains.The preserved strains were recovered and cultured for twice, purified for one time and cultured until to the middle of the log(OD600=0.6, as working strain) before use. The working strain was treated in different p H TSB-3% Na Cl for 2 h, and colony counting by the plate coating, and the p H which the number of the strain was less than 105CFU/m L is the challenge p H. And the results indicated that the p H=4 was the challenge p H, and then cultured in the challenge until the number of the strain was more than the 105CFU/m L.2. The differences in biological characteristics, motility, biofilm formation ability, drugresistance and toxicity changes between the original strain and acid tolerant strain.This section aim to know the adaptation mechanism of the acid tolerant strain by VI comparing the biological characteristics, movement, biofilm forming ability, and drugresistance between the original strains and acid tolerant strain. The results showed that the colony morphology of the acid tolerance strains have the radiate from the center to the periphery and the edge is not smooth compared with the original strain through visual inspection. By observation of the scanning electron microscope, there was no significant difference in shape and size, the external shape of the strain were all found to be short rods. Cell motility experiment, which uses the sterilization bamboo replace a single colony to 3 kinds of active plates, incubated at 37℃ education 20-24 h, observing and measuring the size of turbid areas, which bacteria from the point of inoculation migration turbid areas, the results show that there was a significant difference in swimming but not in swarming and the twitching. Comparing the biofilm-forming ability through the traditional crystal violet staining method at the 72 h between the original strains and acid tolerance strains. The results showed that the ability of biofilmforming of acid tolerant strain was significantly reduced compared with the starting strain in the 72 h. According to the CLSI standard(the clinical and Laboratory Standards Organization) the susceptibility test of the original strain and acid tolerant strain to 18 kinds of drugs. The results indicated that the original strain was resistance to Ampicillin, and in the intermediary level to streptomycin, kanamycin, cefazolin and piperacillin, and was sensitive to other drug. While the acid tolerance strains appeared sensitive to ampicillin and others, indicated that the acid tolerant strain drug-resistance was reduced.Toxicity changes were compared by cytotoxicity assays. The Caco-2 cell and bacteria at the ratio of 1:1 were infected for 4 h, using two kinds of dyes to stain live cells and dead cells, and then to detect cell survival number by Merck flow cytometry. It was found that the number of living cells in about 11% for infected of the original strains for 4 h, and in contrast, by the acid tolerant strain for 4h of infected cells, cell survival number rate at around 60%, the survival rate significantly increased that the cell infected under the acid tolerant strain, means that toxicity is greatly reduced.3. Transcription sequencing analysis, selection of the referenc and confirmation of thetranscriptional data.There were total 4603 transcripts were obtained after filtering, splicing and assembling the sequencing data, in which 4447 transcripts were annotated as known function protein. The expression of 871 genes were the differentially expressed genes, all of these, 414 genes were upregulated and 457 genes were downregulated. Biological function and signaling pathway enrichment analysis showed that, the DEGs mainly enrich to the metabolic pathway, secondary metabolite biosynthesis, different environmental microbial metabolism and amino acid biosynthesis related signal pathway. Significantly DEGs, about 33 was the membrane protein related genes, 21 was the flagellar genes, 6 genes was about the resistance, 22 genes associated with toxicity, 51 genes was the transcription regulation related genes, and about metal metabolism and heat stress related genes respectively were 16 and 4 genes. Then evaluated the stability of 6 reference genes(16S r RNA, rec A, rpo S, pvs A, pvu A, and gapdh) in 5 V. parahaemolyticus strains(O3:K6-clinical strain-tdh+, ATCC33846-tdh+, ATCC33847-tdh+, ATCC17802-trh+, and F13-environmental strain-tdh+) cultured at 4 different temperatures. Stability values were calculated using Ge Norm, Norm Finder, Best Keeper, and Delta CT algorithms. The results indicated that rec A was the most stably expressed gene in the V. parahaemolyticus strains cultured at different temperatures. This study examined multiple V. parahaemolyticus strains and growth temperatures, hence the finding provided stronger evidence that rec A can be used as a reference gene for gene expression studies in V. parahaemolyticus. The rec A and 16 S r RNA were used as the reference genes in the q RTPCR to detect the results of the RNA-seq with 10 genes, in which 4 were up-regulat, 4 down-regulat, and 2 were not the DEGs. It was found that the q RT-PCR results were consistent with the sequencing results. The results showed that the transcription group sequencing results were reliable.4. Comparative proteomic analysisQuantitative analysis of protein by non labeling quantitative method, and then the differential expression analysis was carried out between O3:K6-C and O3:K6-E. 1231 DEPs were obtained, 571 proteins were expressed in the two kind of samples, and 305 proteins only detected in the O3:K6-C and 355 in O3:K6-E. Combined the transcriptome data together, there were a total of 546 results are consistent in sequence annotations. Three DEPs: ATS、Dps and Omp T were significantly high expression in O3:K6-E, and this perhaps was related to the acid tolerance mechanism. In the O3:K6-E sample, the T3 SS was not detected while the T2 SS up high expression, this results may be explain the result of the cytotoxicity test. And also the expression of MSHA may be explain the decreased the ability of biofilm formation and adhesion.