【作者】 陈燕丽；

【导师】 朱兆荣；

【作者基本信息】 西南大学 ， 基础兽医学， 2007， 硕士

【摘要】 金黄色葡萄球菌(Staphylococcus aureus，简称SA)是临床上引起化脓性感染和医院内感染以及引起食品污染和细菌性食物中毒的常见的病原菌。兽医临床上常因养殖场饲养密度过大、通风不良、卫生条件差，地面有碎玻璃、铁丝等尖锐杂物刺破动物皮肤而感染金黄色葡萄球菌，如鸭的蹼或趾被划伤而感染。此病易传染，不易治愈，给养殖户造成极大的经济损失。为了解决这一问题，不少学者开展了防治畜禽感染金黄色葡萄球菌的研究工作。目前，国内外治疗SA的药物众多，主要是抗生素治疗。由于SA对抗生素极易产生耐药性，而且近年来其耐药周期越来越短，因此金黄色葡萄球菌病已成为临床治疗棘手的难题。茵栀黄注射液是由茵陈、栀子、金银花中药复方提取物及黄芩甙组成的制剂，具有清热、解毒、利湿、退黄等药理作用，临床上常用于治疗肝胆湿热、恶心呕吐、小便黄赤、急慢性肝炎等病症。国内外研究表明，茵栀黄注射液中的有效成分，如茵陈挥发油、栀子甙、氯原酸、黄芩甙等均有较好的抗SA的作用。本课题在总结国内外有关中药防治金黄色葡萄球菌引起疾病方面的研究资料的基础上，以茵栀黄注射液为研究对象，研究药物抗SA的作用机理，以便为寻找天然抑菌剂提供理论依据。第一，利用杯管法测定不同浓度的受试药物对SA的抑菌圈大小，采用二倍稀释法测定最低抑菌浓度值(MIC)，求出MPC／MIC值。结果：茵栀黄注射液对SA的抑菌圈达20.50mm，接近阳性对照组(2.5％庆大霉素对SA的抑菌圈为23.40mm)，药液稀释2000倍仍能抑制细菌生长。就MPC／MIC值而言，茵栀黄注射液对SA的MPC／MIC值最低，比值为4，远低于青霉素钾、氟苯尼考注射液等其他抗菌药物。第二，40只鸭随机分组、攻毒，复制鸭金黄色葡萄球菌病理模型，用茵栀黄注射液及氟苯尼考注射液对照治疗，观察药物对鸭金黄色葡萄球菌病的治疗效果。结果：攻毒组的凝血时间与其他三个组相比差异极显著(P＜0.01)。茵栀黄组与生理盐水组相比差异不显著(P＞0.05)。攻毒组及氟苯尼考组的病死率与生理盐水组相比差异极显著(P＜0.01)，而茵栀黄组与生理盐水组相比差异不显著(P＞0.05)。从相对增重率来看，各组差异不显著(P＞0.05)，但茵栀黄组15d增重最接近生理盐水组。两个用药组之间及分别与生理盐水组相比脾系数差异均不显著(P＞0.05)，各组的法氏囊系数差异不显著(P＞0.05)。第三，采用玻片法和试管法检测药物对血浆凝固酶活性的影响，甲苯胺蓝核酸琼脂法测定药物对耐热核酸酶(TNase)活性的抑制作用。根据已公布的金黄色葡萄球菌耐热核酸酶nuc基因的序列，设计并合成一对特异性的引物，以茵栀黄注射液作用的细菌基因组DNA为模板，利用PCR技术对其耐热核酸酶nuc基因片段进行扩增，获得了预计大小的片段。琼脂糖凝胶电泳分析，与标准菌株的nuc基因对比分析。结果：茵栀黄注射液在1：1～1：128浓度范围内对SA的结合凝固酶和游离凝固酶活性无影响。茵栀黄注射液1：1～1：8浓度范围内可完全抑制SA的耐热核酸酶活性，即使稀释512倍仍对耐热核酸酶活性有抑制作用，其各浓度抑制率与空白组相比均有极显著差异(P＜0.01)。琼脂糖凝胶电泳显示：利用合成的引物成功地扩增出三条长达867bp的特异性条带，其大小与预计的相符。把测序结果与标准mu50菌株序列相对比，二者完全一致。结论：(1)茵栀黄注射液在杀灭SA的同时还具有较强的抑制细菌耐药性产生的能力。(2)茵栀黄注射液体内抗金黄色葡萄球菌的疗效显著。(3)药物没有改变nuc基因的碱基序列，即未对nuc基因造成突变影响。更多还原

【Abstract】 Staphylococcus aureus （SA to abbreviate） causes pyogenic infection, nosocomial infection, food contamination, bacteritic food poison is a common pathogenic bacteria. In the nursery, breed density is large, ventilation and hygienic condition is bad. There is some sundry goods for example glass fragments, iron wire on the groud. Duck infects SA when it’s web or the toe scratched by glass fragments or iron wire. The incidence rate and the mortality rate of the disease are very high. The cultivation household loss is serious. At present internal and overseas experts mainly use antibiotic to treat this disease. SA is extremely resistant to the antibiotic; the disease is difficult to healing well.Yinzhihuang injection is a compound preparation made up with Herba Artemisiae Scopariae, Fructus Gardeniae, Flos Lonicerae and baicalin. The drug has pharmacological action including relieving fever、detoxication and damp elimination. It has been used in traditional Chinese medicine to treat a wide range of infectious diseases such as hepatochlic hygropyrexia、nausea and vomiting、yellowish or reddish urine、acute or chronic hepatitis. We don’t found any researchers to study the antimicrobial mechanisms of Yinzhihuang. So we designed a series of experiments to detect of SA affected by Yinzhihuang.First, we use cup tube method to detect the capability of different density drug inhibiting SA. The antimicrobial activity of Yinzhihuang against eight bacteria strains examined was assessed by the presence or absence of inhibition zones and Minimum Inhibition Concentration （MIC） values. Results: Yinzhihuang have significant antibacterial activities to both gram positive and negative bacteria. The inhibition zones of Yinzhihuang to SA is 20.50mm, the physic liquor to dilute 2000 folds still inhibits the growth of bacteria. The MPC/MIC ratio of Yinzhihuang to SA is the lowest of the antimicrobial drugs.Second, Forty ducklings were randomly assigned to four groups. Each duck of experimental group was vaccinated broth of SA 0.35ml of 1×10¹⁰cfu/ml and we investigated the feasibility of Yinzhihuang to protect ducks against infectious disease of SA Results: All ducks in Yinzhihuang experimental group were protected against infectious disease of SA and remained alive. The death rate was significant difference between 1 group and 4goup （P<0.01）, the same difference existed between 2 group and 4 goup. The difference between 3 group and 4 goup was not found to be statistically significant （P>0.05）. The difference of weight gain ratio was not statistically significant between the four groups （P>0.05）. We found the difference of bursa of Fabricius quotient was not statistically significant between the four groups （P>0.05）. The spleen quotient showed that there was significant difference between 1 group and 2, 3,4 goup （P<0.01）.Third, Use tube coagulase test （TCT） to detect SA plasma coagulase effected by Yinzhihuang and detect thermonuclease （TNase） activity by Toluidine blue DNA agar. On the basis of classic thermonuclease nuc gene sequence, we designed a couple of primers. We take the genome DNA affected by drug fluid as template, utilize PCR technology to amplify nuc gene, use agarose gel electrophoresis to analyze the results. Yinzhihuang injection has no effect on activity of plasma coagulase. The addition of Yinzhihuang at certain dilution 1:1～1:16 can inhibit the activity of TNase. We amplified three specificity strap of 867 bp. We contrasted the sequencing result and classic thermonuclease nuc gene sequence and found that the two sequences are completely the same.From the experiments, we conclude:（1） It suggests that Yinzhihuang can strongly inhibit the resistance of SA（2） The efficacy of Yinzhihuang in the treatment of disease caused by SA appears to be higher under the treatment conditions studied.（3） It is suggested that Yinzhihuang injection hasn’t mutational influence to nuc gene.更多还原