【作者】 王庆伟；

【导师】 朴香淑； 谯仕彦；

【作者基本信息】 中国农业大学 ， 动物营养与饲料科学， 2014， 博士

【摘要】 金黄色葡萄球菌是化脓感染中最常见的病原菌,所产肠毒素引起的食物中毒是世界性卫生难题。枯草芽孢杆菌产生的细菌素Sublancin,对金黄色葡萄球菌等革兰氏阳性菌有良好的抑菌活性。本研究以金黄色葡萄球菌甲氧西林敏感菌株CVCC1882和耐甲氧西林菌株ATCC43300为研究对象,研究了Sublancin对金黄色葡萄球菌CVCC1882和ATCC43300的体内体外抑菌效果及作用机制。(1) Sublancin对这两种菌均有良好的抑菌活性,最小抑菌浓度分别为3.75和60mg/L。(2)以耐甲氧西林金葡菌ATCC43300为研究对象,通过电镜等形态学方法研究Sublancin对菌体结构的影响,发现其主要是通过影响细菌菌体分裂进而抑制细菌生长。利用蛋白质组学的方法,通过Sublancin处理组与未处理组差异蛋白点的比较,得出Sublancin作用于细菌的靶位点主要涉及有关能量代谢、氨基酸代谢和氧化应激的蛋白。(3) Sublancin对金黄色葡萄球菌感染小鼠的体内抑菌效果。试验分成两部分,分别以CVCC1882和ATCC43300为试验菌株。其中,CVCC1882不产肠毒素且对甲氧西林敏感,而ATCC43300产肠毒素并且对甲氧西林耐药。两个试验设计相同：将160只体重为18-22g的SPF级昆明小鼠分成8组,每组20只。对照组腹腔注射0.5mL生理盐水,其他7组腹腔注射0.5mL1.0×1010CFU金黄色葡萄球菌。6h后,对照组腹腔注射05mL生理盐水,其他七组腹腔分别注射含有0、0.5、1.0、2.0和4.0mg/kg体重(body weight, BW)的Sublancin以及1.0和2.0mg/kg BW的氨苄西林。两个试验的结果都表明,Sublancin处理组显著降低了小鼠的死亡率,保护了小鼠肠道肠绒毛的完整性,促进了肠细胞增殖,降低了肠道炎症因子含量,抑制了NF-kB和iNOS的表达,减少了脾脏中凋亡细胞的数目,增加了脾脏中CD8细胞的数目。而本研究中,小鼠感染ATCC43300菌株后,由于金葡菌ATCC43300分泌的肠毒素具有超抗原特性,能强烈的刺激淋巴细胞(尤其是CD4细胞)的增殖,发现小鼠脾脏白髓比例升高,脾脏中CD4细胞数目显著升高,呈现增殖性炎症特征。Sublancin治疗后,小鼠脾脏白髓比例和CD4细胞数目降低。综上所述,Sublancin对金黄色葡萄球菌有良好的体外抑菌活性,其抑菌机制可能是通过抑制细菌的能量代谢而抑制了细菌的分裂。Sublancin对金黄色葡萄球菌同样有良好的体内活性,能有效的保护由于金葡菌感染所导致的肠道和脾脏的损伤。更多还原

【Abstract】 Staphylococcus aureus is one of the most common pathogen in suppulative infection, and food poisoning caused by its enterotoxin is a globle health problem. Sublancin, a bacteriocin of the Bacillus subtilis, exerts powerful antibacterial activity against a broad spectrum of gram-positive bacteria including S. aureus. We have developed a highly efficient expression system using Bacillus subtilis800in order to obtain large amounts. To investigate the effect of sublancin on S. aureus and possible antibacterial mechanism, methicillin susceptible S. aureus CVCC1882and methicillin resistant S. aureus ATCC43300were used.(1) The minimal inhibitory concentrations of sublancin for the two stains are3.75and60mg/L, and exert powerful antibacterial activity.(2) In order to identify the intracellular target site. ATCC43300cells were incubated with sublancin and electron micrographs and proteomic analysis were done. Electron micrographs results confirmed that S. aureus cells division was interrupted. The differentially expressed S. aureus proteins involve in energy metabolism, amino acid metabolism and response to stress.(3) The effect of sublancin on S. aureus in vivo. The experiments were done using the two stains, respectively. A total of160, four week old mice were randomly assigned to1of8treatments. Mice in the control group were injected intraperitoneally with0.5mL of0.9%saline. Mice in the other seven groups were given an intraperitoneal injection of0.5mL saline containing1.0×1010CFU/mL S. aureus. Six hours after inoculation, mice in the control group were again injected with0.5mL of0.9%saline. Mice in the other seven groups were injected intraperitoneally with0.5mL of0.9%saline containing0,0.5,1.0,2.0or4.0mg/kg BW sublancin or1.0or2.0mg/kg BW ampicillin. The results showed that sublancin significantly reduced mice mortality. The height and the number of proliferated cells from the intestinal villi in the sublancin treated mice were higher than in the control. Sublancin also downregulated inflammatory cytokines, the NF-κB and iNOS in the intestine of mice. Sublancin also reduced the number of apoptosis cells and increased number of the CD8cells in spleen. ATCC43300express enterotoxins while CVCC1882doesn’t, and enterotoxins sharply stimulate lymphocyte (especially CD4cells). Thus, the ratio of white pulp and the number of CD4cells in the spleen of mice increased significantly. After sublancin treatment, the ratio of white pulp and the number of CD4cells in the spleen of ATCC43300infected mice significantly decreased. In conclusion, sublancin powerfully inhibited S. aureus growth through inhibiting the energy metabolism and thus inhibiting the cell division. Sublancin also showed antibacterial effects in vivo and protect the intestine and spleen injury.更多还原