甲氧西林耐药金黄色葡萄球菌（MRSA）是引起医院内感染的重要多重耐药细菌之一，世界卫生组织对多重耐药细菌进行分类，将MRSA列为严重威胁行列。前期的分子流行病学数据提示，中国大部分城市从1994年到现在一直流行MRSA ST239克隆。本研究以MRSA ST239为研究对象，深入分析MRSA ST239起源、传播和宿主内进化。 本研究选取了1994-2016年中国多个城市医院内分离的132株MRSA ST239菌株，对其进行全基因组测序（WGS），同时下载了全球多个国家已测序的153个MRSA ST239基因组序列。本研究对这285株MRSA ST239基因组进行了深入的研究。Bayesian系统发育分析推测，全球MRSA ST239于1957年（1951 – 1961, 95% 最高概率密度，Highest probability density, HPD）起源于澳大利亚，随后分化成两大分化枝（clade）：Asia clade和non-Asia clade，进化速率为2.0 ?10-6 (1.9?10-6 - 2.1?10-6, 95% HPD)。MRSA ST239在世界多个国家流行，多个国家之间存在传播事件。中国MRSA ST239分成3大世系（Lineage），定义为Lineage A、B、C，其中Lineage A和B来源于新加坡，Lineage C来源于土耳其。MRSA ST239在中国首先在北京流行，然后传播到其他城市，并且多个城市之间也存在传播事件。Lineage B和C的遗传多样性大于Lineage A，Lineage A已经在中国消失，但Lineage B和C依然在中国流行。从土耳其来源的Lineage C的附属基因组与Lineage A和B的附属基因组大不相同，并且每个Lineage有其各自不同的可移动遗传元件（Mobile genetic elements, MGEs）谱。Lineage B的耐药基因的数量高于Lineage A和C，并且随着时间的推移，MRSA ST239的耐药基因的数量在降低，而其毒力基因的数量在升高。 进一步，本研究对MRSA ST239小菌落变异体（SCVs）的宿主内进化进行了研究。本研究选取了分离自亚急性细菌性感染性心内膜炎患者的1株MRSA ST239野生菌株IE1和1株MRSA ST239 SCVs，对其进行了表型实验、比较基因组、转录组和蛋白质组分析。结果显示，与MRSA ST239野生菌株IE1相比，MRSA ST239小菌落变异体SCVs IE2菌落明显变小、生长速率和毒力显著降低。比较基因组结果显示SCVs IE2基因组上有9个基因15个位点发生突变，包括丝氨酸/苏氨酸蛋白激酶PrkC (prkC)、甘油-3-磷酸盐酰基转移酶 (plsY)、2-脱氧核糖-5-磷酸盐醛缩酶 (deoC)、细胞外粘附蛋白Eap/Map (eap)、铁化合物ABC摄取转运体底物结合蛋白 (sstD)、RecU霍利迪连接体解离酶 (recU)、核酸外切酶ABC亚基B (uvrB)、Ⅰ型限制修饰系统M亚基 (hsdM)和平滑肌钙调素结合蛋白。其中prkC、plsY、deoC、sstD、hsdM和平滑肌钙调素结合蛋白发生氨基酸改变，eap、recU、uvrB未发生氨基酸改变。比较转录组结果显示，在SCVs IE2转录组中，321个基因的表达上调，582个基因的表达下调。大多数差异表达的基因参与代谢。上述9个基因中6个基因plsY、deoC、eap、sstD、uvrB和hsdM在DNA和RNA水平上均发生了变化。基因plsY、deoC、eap、sstD所在通路上的一些基因的表达水平也发生了变化，这些通路与金黄色葡萄球菌的代谢和毒力有关。比较蛋白质组结果显示，在SCVs IE2蛋白质组中，共有773个差异蛋白，其中表达上调的有439个，表达下调的有334个；Pathway富集结果与GO富集结果显示，差异蛋白主要富集在代谢相关通路上。蛋白质组与转录组关联分析结果显示，就差异蛋白和差异基因而言，有286个蛋白和基因被关联到，其中216个蛋白和基因表达方向相同；关联聚类分析结果显示SCVs IE2表达下调的基因数目比表达上调的多；关联富集分析结果显示，7个有显著性差异的通路依次为不同环境中的微生物代谢、抗菌药物生物合成、代谢通路、精氨酸生物合成、丙氨酸、天冬氨酸和谷氨酸生物合成、核糖体和次级代谢生物合成。MRSA ST239 SCVs在宿主内进化中基因组、转录组和蛋白质均发生了变化，SCVs菌落变小和生长速率降低的原因与代谢相关基因的突变和代谢途径大量基因表达发生变化相关，SCVs毒力降低与毒力相关基因突变和表达发生变化相关。 综上，MRSA ST239在进化过程中发生了一系列的适应性变化，并且在中国多个城市之间存在传播事件，其未来的进化趋势值得本研究继续追踪和研究。

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important multi-drug resistant bacteria causing nosocomial infections. MRSA is considerred as a serious threat by world health organization. Previous molecular epidemiological data suggested that MRSA ST239 clone has been popular in most cities in China since 1994. In this study, the origin, spread and within-host evolution of MRSA ST239 were analyzed using whole-genome sequencing (WGS). One hundred and thirty two MRSA ST239 strains were isolated from hospitals in several cities in China from 1994 to 2016 to perform WGS. At the same time, 153 MRSA ST239 genome sequences were downloadded from several countries around the world. The Bayesian phylogenetic analysis speculated that the global MRSA ST239 originated in Australia in 1957 (1951 – 1961, 95% highest probability density, HPD) and subsequently differentiated into two major clades: Asia clade and non-Asia clade, with an evolution rate of 2.0×10-6 (1.9×10-6 - 2.1×10-6, 95% HPD). MRSA ST239 spread in many countries around the world, and there were several transmission events between many countries. Chinese MRSA ST239 were divided into three major lineages, defined as lineage A, B, C. Lineage A and B were from Singapore, while lineage C was from Turkey. MRSA ST239 was first prevalent in Beijing, then spread to other cities, and there were some transmission events between several cities. Lineage B and C had greater genetic diversity than lineage A. Lineage A has disappeared in China over time, but lineage B and C were still popular in China. The accessory genome of lineage C from Turkey was quite different from the accessory genomes of lineage A and B, and each lineage had its own different mobile genetic elements (MGEs). The number of resistant genes in lineage B was higher than lineage A and C. The number of resistant genes in MRSA ST239 was decreasing over time, while the number of virulence genes was increasing. Furthermore, we investigated the within-host evolution of MRSA ST239 small colony variants (SCVs) in this study. A wild strain of MRSA ST239 IE1 and a SCVs strain of MRSA ST239 isolated from subacute bacterial infective endocarditis patients were selected to perform phenotypic experiments, comparative genomic, transcriptomic, and proteomic analyses. Compared with MRSA ST239 wild strain IE1, the colony of MRSA ST239 SCVs IE2 was significantly smaller, growth rate significantly decreased, and virulence also significantly reduced. The results of comparative genomic showed that there were nine mutations in 15 genes in the SCVs IE2 genome. The 15 genes included the serine/threonine protein kinase PrkC (prkC), glycerol-3-phosphate acyltransferase (plsY), 2-deoxyribose-5-phosphate aldolase (deoC), extracellular adhesion protein Eap/Map (eap), iron compound ABC uptake transporter substrate binding protein (sstD), RecU holliday junction resolvase (recU), exonuclease ABC subunit B (uvrB), type I restriction modification system M subunit (hsdM) and smooth muscle calmodulin binding protein. Among them, amino acids changed in prkC, plsY, deoC, sstD, hsdM and smooth muscle calmodulin binding protein, and amino acid changes were not observed in eap, recU and uvrB. Comparative transcriptome results showed that in the SCVs IE2 transcriptome, 321 genes were up-regulated and 582 genes were down-regulated. Most differentially expressed genes were involved in metabolism. Six of the above nine genes, plsY, deoC, eap, sstD, uvrB, and hsdM, changed at the DNA and RNA levels. The expression levels of some genes in the pathways of the genes plsY, deoC, eap, and sstD have also changed, and these pathways were involved in the metabolism and virulence of S. aureus. The comparative proteome results showed that there were 773 differential proteins in the SCVs IE2 proteome, of which 439 were up-regulated and 334 were down-regulated. Pathway enrichment results and GO enrichment results showed that the differential proteins mainly related to metabolic pathways. The results of association analysis between proteome and transcriptome showed that 286 proteins and genes were associated with differential proteins and differential genes, of which 216 proteins and genes expressed in the same direction. Correlation clustering analyses showed that the number of down-regulated genes was much higher than that of up-regulated genes in SCVs IE2. The results of association enrichment analysis showed that seven pathways with significant differences were microbial metabolism in different environments, antibiotic biosynthesis, metabolic pathway, arginine biosynthesis, alanine, aspartate and glutamic acid biosynthesis, ribosome and secondary metabolic biosynthesis. The genome, transcriptome and protein of MRSA ST239 SCVs have changed in the host. The reason for the small colony phenotype and the decrease of growth rate of SCVs related to the mutation of metabolic related genes and the gene expression of metabolic pathways. The decreased virulence of SCVs was associated with virulence-related gene mutations and changes in expression. In conclusion, MRSA ST239 has undergone a series of adaptive changes in the evolution process, and there were transmission events between many cities in China, and its future evolutionary trend is worthy of continued tracking and research.