【作者】 王佳；

【导师】 贾英民；

【作者基本信息】 河北农业大学 ， 微生物学， 2014， 硕士

【摘要】 金黄色葡萄球菌(Staphylococcus aureus，简称SA)是一种重要的病原菌，由它分泌产生的肠毒素(SEs)更是诱发食物中毒的主要因素，世界各地关于金葡菌引起的食物中毒事件有很多。而且因SA导致的肺炎及伤口感染，占医院感染病例的10％；抗生素的广泛应用，使MRSA大量出现，让它一举成为与艾滋和乙型肝炎并列的三大恶性传染性病菌。本实验从实验室现有菌株出发，分析研究了sec、sed和MRSA中mecA基因在不同来源SA中的分布及其规律性，并结合之前的研究成果，在转录水平上对SA中肠毒素基因在不同培养基和不同培养阶段表达的影响，以及调控因子的调控作用进行研究。在保藏的436株金黄色葡萄球菌（来自理发用具、人体化脓组织、刀具、空气、果蔬、豆制品、肉制品、速冻食品、生牛乳和生鲜肉）中sec、sed和mecA的总检出率分别为24.1%、8.3%和8.7%，sec检出率明显高于其他两种毒素基因。sec在各种来源中均有分布，速冻食品所占比例最大。sed在空气、刀具、理发用具、水产品及果蔬中都未检出，生鲜肉中的检出率最高。mecA除理发工具、刀具和豆制品，在其余来源中都有分布，果蔬和水产品中的检出率较高。本试验选择金黄色葡萄球菌SA30、SA75、SA83这三株菌，分析了它们在不同培养基中各毒素基因的转录情况。结果发现：菌株SA83中hlb进入对数期时表达量最大；SEU的表达量一直不多；hlg没有检测到。菌株SA75中hlb和SEU在进入对数期时表达量最大；hlg随着菌株生长时间的增长，表达的也越来越多。菌株SA30中hlb在进入稳定期后表达量最大；hlg在进入稳定期时表达量最大；SEU在全过程中都有表达，在10h时表达量最多。金黄色葡萄球菌对不同糖的分解利用能力不同，对乳糖及可溶性淀粉的分解利用能力较低，对葡萄糖和蔗糖的分解利用能力较高。蔗糖最有利于SEA的表达；可溶性淀粉对SEI表达的增强作用最明显；葡萄糖的添加对hld在对数末期的表达有减弱作用；hla仅在葡萄糖、蔗糖中有表达，而且都是在6h，进入对数期时表达；hlb只在含有乳糖的TSB培养基中有极微量的转录，8h时表达量最多，即进入对数期时；蔗糖对hlg的表达可能有抑制作用。RNAⅢ作为调控因子，虽然在每个时段都有检测到，但是它对毒素产生的调控作用并未有明显的体现。许多研究表明金黄色葡萄球菌所生长的环境状况对于不同类型肠毒素的形成有重要影响。鉴于目前的状况，进一步研究金黄色葡萄球菌毒素产生的具体培养条件就显得极为重要。这些科研成果将为环境及食品受金葡菌的污染情形和该菌的流行状态作出快速判断，并为监控由金黄色葡萄球菌所引起的疾病的爆发提供判断的科学根据。更多还原

【Abstract】 Staphylococcus aureus is an important pathogen that enterotoxin (SEs)generated by its secretion is an important factor in inducing food poisoning. Thereare many food poisoning caused by Staphylococcus aureus around the world. Andpneumonia and wound infections caused by SA accounted for10%in hospitalinfections; the antibiotics extensive use lead methicillin-resistant aureus (MRSA)appeared in large numbers. It has become one of the world’s three infection germsand others are HIV and hepatitis B tied.In this experiments combined with the results of previous studies, we studiedthe transcriptional level in the SA enterotoxin gene impact in different cultures and atdifferent stages of the medium of expression, as well as regulatory factors in theregulation.The strains we used are all from the laboratory, analyzes distribution andregularity of sec, sed and mecA gene in SAs in different sources.The total detection rates of sec, sed, and mecA in436Staphylococcus aureuspreservation of (from hair tools, human purulent tissue, knives, air, fruits andvegetables, soy products, meat, frozen food, raw milk and raw meat etc.) were24.1%,8.3%and8.7%, the sec’s rate was significantly higher than the other twotoxin genes.Sec is distributed in a variety of sources, the largest proportion of frozen food.Sed neither detected in the air, knives, hair tools, aquatic products and fruits andvegetables; the highest detection rate is raw meat. MecA except hair tools, knivesand soy products, the rest are distributed sources, including fruits and vegetablesand aquatic products in high detection rate.In this study we select the strains SA30, SA75, SA83which three strainsanalyzed the situation of each toxin genes are transcribed in a different medium. Theresults showed that: the expression of hlb is the maximum when strain SA83grewinto log phase; has little expression of the SEU; hlg not detected. Strain SA75hlband SEU in the largest expression is in the logarithmic phase; hlg were expressedmore and more, with the growth of time. The hlb in strain SA30expression of themaximum after entering the stable; hlg when entering the expression of themaximum stable; SEU are expressed in the whole process, expressed maximumamount at the10h.Staphylococcus aureus on the ability to use and catabolism different sugars aredifferent, less ability to lactose utilization and soluble starch, higher ability toglucose and sucrose. Sucrose most conducive to the expression of the SEA; solublestarch enhancement of expression of SEI is obvious; glucose played the weakenedrole when hld in the late logarithmic expression; hla only expressed with glucoseand sucrose, both are at6h, when access to a number of expression; hlb in TSB medium containing lactose just transcripted a very small amount, up to theexpression amount that enter the logarithmic phase at8h; expression of sucrose onhlg may have inhibited.RNA Ⅲ as a regulatory factor, although in each period has detected, but itsrole in the regulation of toxin production has not been clearly reflected.Many researches have shown that the growth environmental conditions ofStaphylococcus aureus has important implications for to form different types ofenterotoxins. Given the current situation, the specific culture conditions ofStaphylococcus aureus toxin produced becomes extremely important in further study.These academic achievements will be state to make quick judgments and providesjudged according to monitoring and tracking outbreaks and pollution disease whenenvironmental and food contamination cased by Staphylococcus aureus and thisprevalence.更多还原