發(fā)布時間：2018-06-09 16:08

  本文選題：枯草芽孢桿菌 + 嗜水氣單胞菌　； 參考：《華中農(nóng)業(yè)大學》2017年碩士論文

【摘要】：在魚類養(yǎng)殖中,細菌性疾病造成的經(jīng)濟損失嚴重。為了應對細菌引起的魚病,人們在過去多年使用各類抗生素來抑制細菌的生長和繁殖,由于用藥不當,從而導致細菌耐藥性的產(chǎn)生和逐漸增強,在魚體內(nèi)形成藥物殘留,破壞水體生態(tài)系統(tǒng)。為了解決這一難題,大家將目光轉(zhuǎn)向了能改善水體環(huán)境,無藥物殘留,提高魚類生長性能和免疫力的益生菌�？莶菅挎邨U菌是在水產(chǎn)動物中廣泛使用的益生菌之一,本實驗室在草魚的腸道內(nèi)篩選出一株對嗜水氣單胞菌等水產(chǎn)病原菌有拮抗作用的枯草芽孢桿菌(Ch9菌株)。本文研究了該枯草芽孢桿菌對嗜水氣單胞菌的影響。分析比較了不同接種量的枯草芽孢桿菌對嗜水氣單胞菌產(chǎn)生生長抑制的適宜接種量。在共培養(yǎng)中,當枯草芽孢桿菌和嗜水氣單胞菌的初始接種量為1×10~8 CFU/ml和1×106 CFU/ml時,枯草芽孢桿菌和嗜水氣單胞菌在共培養(yǎng)條件下與單培養(yǎng)相比,生長均變緩,但均未出現(xiàn)十分明顯的生長抑制。而當枯草芽孢桿菌和嗜水氣單胞菌的初始接種量為1×10~9 CFU/ml和1×10~8 CFU/ml時,枯草芽孢桿菌在共培養(yǎng)和單培養(yǎng)下的生長不受影響,而嗜水氣單胞菌在共培養(yǎng)4h之后,細菌數(shù)量顯著減少,與單培養(yǎng)相比,生長受到顯著的抑制。因此,枯草芽孢桿菌和嗜水氣單胞菌的初始接種量為1×10~9 CFU/ml和1×10~8 CFU/ml是適宜接種量。將初始接種量為1×10~9 CFU/ml枯草芽孢桿菌與1×10~8 CFU/ml的嗜水氣單胞菌和共培養(yǎng),對嗜水氣單胞菌和枯草芽孢桿菌在共培養(yǎng)和單培養(yǎng)條件下,嗜水氣單胞菌毒力基因轉(zhuǎn)錄水平,溶血活性以及對細胞的毒性進行了分析。結果表明,當嗜水氣單胞菌和枯草芽孢桿菌共培養(yǎng),通過分析熒光定量的結果發(fā)現(xiàn),嗜水氣單胞菌的毒力基因包括aer,ahy B,emp,hcp基因的轉(zhuǎn)錄水平都出現(xiàn)下降。在培養(yǎng)6h和8h時,嗜水氣單胞菌aer基因在共培養(yǎng)條件下表達量與單培養(yǎng)相比分別下降了18.01倍和3.05倍。在培養(yǎng)6h、8h和10h時,ahy B基因在共培養(yǎng)條件下表達量與單培養(yǎng)相比分別下降了2.07倍、1.43倍和3.07倍。在培養(yǎng)4h、6h、8h、10h和12h,嗜水氣單胞菌hcp基因在共培養(yǎng)條件下的表達量與單培養(yǎng)相比分別下降了10.99倍、218.48倍、830.28倍、565.48倍和481.34倍。在培養(yǎng)6h和8h,嗜水氣單胞菌emp基因在共培養(yǎng)條件下的表達量與單培養(yǎng)相比分別下降了1.76倍和1.46倍。當嗜水氣單胞菌和枯草芽孢桿菌共培養(yǎng),共培養(yǎng)上清液對血細胞沒有出現(xiàn)溶血現(xiàn)象,而嗜水氣單胞菌單培養(yǎng)上清液隨著培養(yǎng)時間的增加,對血細胞的溶血活性也在增強。這說明,當嗜水氣單胞菌和枯草芽孢桿菌共培養(yǎng),嗜水氣單胞菌溶血活性減弱。當通過測定細胞乳酸脫氫酶的釋放量,發(fā)現(xiàn)在嗜水氣單胞菌和枯草芽孢桿菌共培養(yǎng)情況下,共培養(yǎng)上清液對CIK細胞系的細胞毒性顯著低于嗜水氣單胞菌單培養(yǎng)上清液對CIK細胞的毒性,說明,嗜水氣單胞菌與枯草芽孢桿菌共培養(yǎng),嗜水氣單胞菌的細胞毒性減弱。利用RT-PCR方法檢測了嗜水氣單胞菌和枯草芽孢桿菌共培養(yǎng),兩者的lux S基因的轉(zhuǎn)錄水平。結果表明,當嗜水氣單胞菌和枯草芽孢桿菌共培養(yǎng),嗜水氣單胞菌的lux S基因表達量顯著下調(diào),而枯草芽孢桿菌的lux S基因表達量顯著上調(diào)。表明,枯草芽孢桿菌對嗜水氣單胞菌生長毒力抑制的益生作用可能與群體感應相關。
[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.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S942.3

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本文編號：2000337