【摘要】 家畜相关耐甲氧西林金黄色葡萄球菌(livestock-associated methicillin-resistant Staphylococcus aureus,LA-MRSA)在畜牧业和动物性食品中多次被检测到，可以向人群传播，造成严重的公共卫生问题，因此研究其耐药性、毒力因子的表达与调控对LA-MRSA的防治及后续药物的开发具有重要意义。本研究以宁夏规模化奶牛养殖场乳腺炎患牛牛乳中分离出的9株LA-MRSA为试验对象，通过抗菌药物敏感性、生物被膜形成能力检测和细菌体外侵袭力试验筛选出具有高耐药、高毒力特征的LA-MRSA菌株，结合本实验室前期LA-MRSA分子分型结果，鉴定出1株具有高毒力特征的代表菌株，然后将代表菌株和标准菌株ATCC 33591进行转录组测序(RNA-seq)分析，最后通过RT-qPCR验证RNA-seq结果准确性。高毒力LA-MRSA鉴定结果显示，9株LA-MRSA中编号为D10的菌株对13种抗菌药物耐药，且生物被膜形成能力、体外侵袭力较强，具有高耐药、高毒力特征。RNA-seq分析结果显示，与标准菌株相比，D10株存在大量差异表达基因(differential expression genes, DEGs),其中多种生物被膜形成相关基因、侵袭性毒素基因、与宿主免疫逃逸相关基因及编码甲氧西林抗性的mecA基因等过度表达。KEGG分析结果显示，DEGs共参与了84种不同代谢途径，主要在新陈代谢、次生代谢产物的生物合成、碳代谢、逆环境下的微生物代谢等通路大量聚集，但整体差异不显著。RT-qPCR结果显示，mecA、hld、thyA等重要基因的表达趋势与RNA-seq基本一致，表明RNA-seq结果良好，数据真实可靠。以上结果表明，本研究筛选的宁夏地区LA-MRSA流行株具有较高的毒力水平及较强的感染能力。更多还原

【Abstract】 Livestock-associated Methicillin-resistant Staphylococcus aureus(LA-MRSA) has been detected many times in animal husbandry and animal food, and can spread to the population, causing serious public health problems.It is of great significance to study the expression and regulation of its virulence factors and drug resistance for the prevention and treatment of LA-MRSA and the subsequent drug development.In this study, 9 strains of LA-MRSA were isolated from the milk of cows suffering from mastitis in a large-scale dairy farm in Ningxia.The LA-MRSA strains with drug-resistant and high virulence characteristics, combined with the previous LA-MRSA molecular typing results of our laboratory, identified a representative strain with high virulence characteristics, and then transcribed the representative strain and the standard strain ATCC 33591 Group sequencing(RNA-seq) analysis, and finally verifies the accuracy of RNA-seq results by RT-qPCR.The identification results of high virulence LA-MRSA showed that among the 9 strains of LA-MRSA,the strain numbered D10 was resistant to 13 kinds of antibacterial drugs,and had strong biofilm formation ability and in vitro invasiveness,and had the characteristics of high drug resistance and high virulence.RNA-seq analysis results showed that compared with standard strains,D10 strain has a large number of Differential Expression Genes(DEGs),including a variety of biofilm formation related genes,invasive toxin genes,and host immune escape related genes and codes mecA gene for methicillin resistance is overexpressed.KEGG analysis results show that DEGs participate in 84 different metabolic pathways,mainly in metabolism,biosynthesis of secondary metabolites,carbon metabolism,and microbial metabolism under adverse environments,but the overall difference is not significant.RT-qPCR results showed that the expression trends of important genes such as mecA,hld,and thyA were basically the same as RNA-seq,indicating that the RNA-seq results are good and the data is true and reliable.The above results indicate that the epidemic strain of LA-MRSA in Ningxia selected in this study has a high level of virulence and a strong infection ability.更多还原