枯草芽孢杆菌对嗜水气单胞菌毒力的影响以及群体感应基因luxS的表达

发布时间：2018-06-09 16:08

本文选题：枯草芽孢杆菌 + 嗜水气单胞菌　；参考：《华中农业大学》2017年硕士论文

【摘要】：在鱼类养殖中,细菌性疾病造成的经济损失严重。为了应对细菌引起的鱼病,人们在过去多年使用各类抗生素来抑制细菌的生长和繁殖,由于用药不当,从而导致细菌耐药性的产生和逐渐增强,在鱼体内形成药物残留,破坏水体生态系统。为了解决这一难题,大家将目光转向了能改善水体环境,无药物残留,提高鱼类生长性能和免疫力的益生菌。枯草芽孢杆菌是在水产动物中广泛使用的益生菌之一,本实验室在草鱼的肠道内筛选出一株对嗜水气单胞菌等水产病原菌有拮抗作用的枯草芽孢杆菌(Ch9菌株)。本文研究了该枯草芽孢杆菌对嗜水气单胞菌的影响。分析比较了不同接种量的枯草芽孢杆菌对嗜水气单胞菌产生生长抑制的适宜接种量。在共培养中,当枯草芽孢杆菌和嗜水气单胞菌的初始接种量为1×10~8 CFU/ml和1×106 CFU/ml时,枯草芽孢杆菌和嗜水气单胞菌在共培养条件下与单培养相比,生长均变缓,但均未出现十分明显的生长抑制。而当枯草芽孢杆菌和嗜水气单胞菌的初始接种量为1×10~9 CFU/ml和1×10~8 CFU/ml时,枯草芽孢杆菌在共培养和单培养下的生长不受影响,而嗜水气单胞菌在共培养4h之后,细菌数量显著减少,与单培养相比,生长受到显著的抑制。因此,枯草芽孢杆菌和嗜水气单胞菌的初始接种量为1×10~9 CFU/ml和1×10~8 CFU/ml是适宜接种量。将初始接种量为1×10~9 CFU/ml枯草芽孢杆菌与1×10~8 CFU/ml的嗜水气单胞菌和共培养,对嗜水气单胞菌和枯草芽孢杆菌在共培养和单培养条件下,嗜水气单胞菌毒力基因转录水平,溶血活性以及对细胞的毒性进行了分析。结果表明,当嗜水气单胞菌和枯草芽孢杆菌共培养,通过分析荧光定量的结果发现,嗜水气单胞菌的毒力基因包括aer,ahy B,emp,hcp基因的转录水平都出现下降。在培养6h和8h时,嗜水气单胞菌aer基因在共培养条件下表达量与单培养相比分别下降了18.01倍和3.05倍。在培养6h、8h和10h时,ahy B基因在共培养条件下表达量与单培养相比分别下降了2.07倍、1.43倍和3.07倍。在培养4h、6h、8h、10h和12h,嗜水气单胞菌hcp基因在共培养条件下的表达量与单培养相比分别下降了10.99倍、218.48倍、830.28倍、565.48倍和481.34倍。在培养6h和8h,嗜水气单胞菌emp基因在共培养条件下的表达量与单培养相比分别下降了1.76倍和1.46倍。当嗜水气单胞菌和枯草芽孢杆菌共培养,共培养上清液对血细胞没有出现溶血现象,而嗜水气单胞菌单培养上清液随着培养时间的增加,对血细胞的溶血活性也在增强。这说明,当嗜水气单胞菌和枯草芽孢杆菌共培养,嗜水气单胞菌溶血活性减弱。当通过测定细胞乳酸脱氢酶的释放量,发现在嗜水气单胞菌和枯草芽孢杆菌共培养情况下,共培养上清液对CIK细胞系的细胞毒性显著低于嗜水气单胞菌单培养上清液对CIK细胞的毒性,说明,嗜水气单胞菌与枯草芽孢杆菌共培养,嗜水气单胞菌的细胞毒性减弱。利用RT-PCR方法检测了嗜水气单胞菌和枯草芽孢杆菌共培养,两者的lux S基因的转录水平。结果表明,当嗜水气单胞菌和枯草芽孢杆菌共培养,嗜水气单胞菌的lux S基因表达量显著下调,而枯草芽孢杆菌的lux S基因表达量显著上调。表明,枯草芽孢杆菌对嗜水气单胞菌生长毒力抑制的益生作用可能与群体感应相关。
[Abstract]:In fish culture, the economic loss caused by bacterial disease is serious. In order to cope with the disease caused by bacteria, people have used various kinds of antibiotics to inhibit the growth and reproduction of bacteria in the past years. Because of improper use of drugs, the drug resistance of bacteria is produced and gradually increased, and the drug residues in the fish are formed and the water ecosystem is destroyed. In order to solve this problem, people turn to the probiotics that can improve the water environment, have no drug residues, improve the growth performance and immunity of fish. Bacillus subtilis is one of the probiotics widely used in aquatic animals. In this laboratory, a strain of Aquatic Pathogenic bacteria such as Aeromonas hydrophila, such as Aeromonas hydrophila, is screened in grass carp's intestines. The effect of Bacillus subtilis (Ch9). The effect of Bacillus subtilis on Aeromonas hydrophila was studied in this paper. The suitable inoculation amount of Bacillus subtilis on the growth inhibition of Aeromonas hydrophila was analyzed and compared. In co culture, the initial inoculation of Bacillus subtilis and Aeromonas hydrophila was 1 Under co culture conditions, the growth of Bacillus subtilis and Aeromonas hydrophila was slow, but no obvious growth inhibition was found in the co culture conditions of Bacillus subtilis and Aeromonas hydrophila under the co culture conditions, while the Bacillus subtilis and Aeromonas hydrophila were cultured under the co culture condition, while the initial inoculation of Bacillus subtilis and Aeromonas hydrophila was 1 * 10~9 CFU/ ml and 1 x 10~8 CFU/ml, the Bacillus subtilis was cocultured with 10~8 CFU/ml and 1 x 106. The growth of the cultured and single culture was not affected, and the number of bacteria decreased significantly after co culture of 4H. Compared with the single culture, the growth was significantly inhibited. Therefore, the initial inoculation amount of Bacillus subtilis and Aeromonas hydrophila was 1 * 10~9 CFU/ml and 1 x 10~8 CFU/ml. The initial inoculation amount was 1 * 10~9 CFU/ml Bacillus subtilis and 1 x 10~8 CFU/ml were Aeromonas hydrophila and co culture. The transcriptional level, hemolytic activity and toxicity of Aeromonas hydrophila to Aeromonas hydrophila and Bacillus subtilis were analyzed under co culture and single culture conditions. The results of fluorescence quantitative analysis showed that the virulence genes of Aeromonas hydrophila, including aer, ahy B, EMP and HCP, were all decreased. The expression of Aeromonas hydrophila aer gene decreased by 18.01 times and 3.05 times compared with that of single culture in culture of 6h and 8h. The expression of ahy B gene decreased 2.07 times, 1.43 times and 3.07 times as compared with that of single culture. In culture 4h, 6h, 8h, 10h and 12h, the expression of HCP gene of Aeromonas hydrophila decreased 10.99 times, 218.48 times, 830.28 times, 565.48 times and 481.34 times, respectively. The expression of EMP gene of monomonas aeruginosa decreased 1.76 times and 1.46 times than that of single culture. When Aeromonas hydrophila and Bacillus subtilis were co cultured, the co culture supernatant did not appear hemolytic, while the single culture supernatant of Aeromonas hydrophila increased with the time of culture and hemolysis to blood cells. The activity was also enhanced. This indicated that the hemolytic activity of Aeromonas hydrophila was weakened when Aeromonas hydrophila and Bacillus subtilis were co cultured. The cytotoxicity of co cultured supernatant to CIK cell lines was significantly lower than that of Aeromonas hydrophila and Bacillus subtilis by measuring the release of lactate dehydrogenase. The toxicity of single culture supernatant to CIK cells showed that Aeromonas hydrophila and Bacillus subtilis were co cultured, and the cytotoxicity of Aeromonas hydrophila was weakened. The co culture of Aeromonas hydrophila and Bacillus subtilis was detected by RT-PCR method. The transcription level of lux S gene of both Aeromonas hydrophila and Bacillus subtilis showed that it was an Aeromonas hydrophila. With Bacillus subtilis co culture, the expression of lux S gene of Aeromonas hydrophila was significantly down, while the lux S gene expression of Bacillus subtilis was significantly up-regulated, indicating that the probiotic effect of Bacillus subtilis on the inhibition of virulence of Aeromonas hydrophila may be related to the quorum induction.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S942.3

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