Variation Of Vibrio Parahaemolyticus Under Stresses And Comparison Of The Growth Rate Of Different Strains

In this study, we used Vibrio parahaemolyticus as the research material. The research comprises two aspects:Part I Variation in Morphology and some genes expression of Vibrio parahaemolyticus under cold and starvation stresses:Vibrio parahaemolyticus as an important foodborne pathogen, the function of this gene is not completely clear. In different conditions, change of sone genes expression of Vibrio parahaemolyticus is not clear too. Therefore, in this study, we check the change of morphological and some genes expression of Vibrio parahaemolyticus in cold and starvation stresses.The changes of surface morphology of three different genotypes of Vibrio parahaemolyticus (8G, ATCC33847, ATCC17802 )are similar under the condition of the at 4℃. The center part of the cell became saged inwardly, and the cell wall rolled inwardly subsequently. However, this depression is different from the common phenomenon of air deflated. The outside cell rolled inwardly in center but the remaining parts were relatively full, similar to the shape of donut. With the processing time increasely, cell wall became more smoothly and folding. Finally, the cell wall became loose and ulceration, and amoticed as mucosa. In the early period of cold treatment, The expression of cytoskeletal genes(MreB, FtsZ and MinE) of three kinds of Vibrio parahaemolyticus are Up-regulated. These changes may lead to the change of bacterial cell morphology. In addition, the cellular antioxidant gene AHPC which associated with resistance also slightly raised, indicating cold treatment could cause ROS levels change in cell. Cell metabolism genes (EF-TU and GAPD have no significant change in the early cold treatment, indicating low-temperature could not affect cell metabolism in a short period.Three kinds of bacteria showed different morphological changes after complete starvation (complete absence of carbon and nitrogen resources). The starvation stress resistance of three kinds of bacteria is different. 8G is Superior, 33847 followed by, and 17802 is the worst. 8G did not occur significant changes after 72 hours treatment. However, 33847 was totally different from 8G. After 72 hours treatment , the sticky mucous-like substance was observed in liquid medium, indicating that the fold-like substance outside the cell wall had loose and ulcerated. This changes had occured in 17082 just in 12 hours after starvation streatment. Degradation of membranous material maybe as carbon and nitrogen sources can be consumed in order to maintain the survival of bacterial cells. Cytoskeletal genes and antioxidant gene AHPC expression levels are down in three kinds of Vibrio parahaemolyticus in the early stage of starvation, indicated that cell morphology, cell division activity and intracellular reactive oxygen were affected in the starvation conditions. Cell metabolism genes’changes were not obvious in early stage of starvation, indicating that the metabolic genes are not the primary class of genes which were affected in a short period of starvation.In addition, we found that three virulent genes TDH , TRH , TLH were down-regulated in two kinds of stress conditions, indicating that stresses would less the level of poison of Vibrio parahaemolyticus. This results makes a certain sence in the research of pathogenicity of Vibrio parahaemolyticus.Part II Comparison of the growth rate of pathogenic strains and non-pathogenic strains.In the composition of bacterial food poisoning, Vibrio parahaemolyticus food poisoning has accounted to the first, and Vibrio parahaemolyticus has become the primary food-borne pathogens. Pathogenic Vibrio parahaemolyticus can produce the thermostable direct hemolysin (TDH). Some strain could also produce a small amount of the TDH-related heat-labile toxin TRH, which has hemolytic properties either. Most strains isolated of clinical can produce TDH, and about 10% strains can produce TRH. However, most strains isolated from environmental are presented in hemolysis negative, and the TDH hemolysis test positive rate of strains which separated from the marine was 10% -30%, the strains containing Trh gene is almost zero.In this study, acorrding to this phenomenon, we tested the growth rate of pathogenic strains and non-pathogenic strains, and initially confirmed that either pure culture or mixed culture, the growth rate of non-pathogenic Vibrio parahaemolyticus were better than pathogenic Vibrio parahaemolyticus.We tested the growth rate of 11 strains of three genotypes of Vibrio parahaemolyticus in pure culture conditions, and the change of proportion of pathogenic strains in the mixed culture conditions and the influence of the different concentration gradient of the ratio of pathogenic bacteria.The result indicates that there are main three reasons that could lead to the low ratio of pathogenic strains which separated from the environment condition.First, the growth rate of non-pathogenic bacteria is generally greater than the pathogenic bacteria. In the same condition, the non-pathogenic strains will grow in a single colony priority than pathogenic strains in plate.Second, Non-pathogenic strains could inhibit the growth of pathogenic strains. Therefore, after a period of mixed culture, the proportion of pathogenic strains would be much lower.Third, compare to low concentration, the percentage of pathogenic strains are small. Therefore, if we make use of higher concentration of bacteria which isolated from the environment to plate coating, would lead to single plate colony growth too density, and as a result, the detection rate of pathogenic strains is reduce.Third, non-pathogenic strains may have more vitality than pathogenic strains. Therefore, under stress conditions, compared to pathogenic bacteria, non-pathogenic bacteria has more advantages .