体外诱导耐甲氧西林金黄色葡萄球菌利奈唑胺耐药相关新分子及机制研究

Linezolid is an effective antibiotic against most gram-positive bacteria including drug-resistant strains such as methicillin-resistant Staphylococcus aureus. Recently, resistance to LZD in MRSA isolates was often detected from patients. Mounting evidences have indicated linezolid resistance is usually due to point mutations in domain V of the 23SrRNA and the level of resistance generally correlates with the number of mutated copies of 23SrRNA in the Enterococcus and Staphylococcus respectively. In addition to mutations in the 23SrRNA, deletion or point mutations in the ribosomal protein L3 and L4 have been reported in many clinical bacterial isolates with linezolid resistance. Moreover, an rRNA methyltransferase translated by the cfr (chloramphenicol-florfenicol resistance) gene was reported to confer resistance to linezolid in Enterococcus and Staphylococcus. Recently, novel molecules including RNA methyltransferase and ABC tansporter proteins in Streptococcus have been identified to be involved in linezolid resistance. However, whether genetic mutations or abnormal expression of novel molecules contribute to linezolid resistance in Staphylococcus is still elusive. In our previous study, we selected five linezoid-susceptive strains of MRSA, which subsequently selected with linezoid in vitro. By this way, four groups of linezolid-resistant strains with different MIC values were identified. The nucleotide mutations in domain V of the 23SrRNA in these strains were verified and subsequently, we applied the whole-genome sequencing to compare the nucleotide mutations in the resistant strain with the sensitive in one group, suggesting additional genes possibly associated with linezolid resistance in S.aureus. Our results indicated many potential novel molecules contributing to linezolid resistance in S.aureus. In this study, we plan to further apply high-throughput techniques, including whole-genome sequencing, microarray and proteomic techniques, to screen and identify linezolid-resistant associated novel molecules of MRSA and subsequently elucidate their mechanism involved in bacterial resistance.

利奈唑胺(LZD)是临床治疗耐甲氧西林金黄色葡萄球菌(MRSA)感染的主要药物，耐药性问题日益突出。研究发现23SrRNA V区基因突变是金葡菌LZD耐药的主要机制，但革兰阳性菌LZD耐药机制复杂，LZD耐药相关新分子及机制仍有待揭示。本课题通过体外诱导LZD敏感MRSA获得4组系列耐药菌株，选取2株母株与相应耐药株，高通量测序技术比较全基因序列差异，发现多个基因产生突变。本课题拟在这一工作基础上筛选MRSA LZD耐药相关新分子并阐明其机制，具体内容包括：①高通量测序技术分析MRSA敏感株和耐药株全基因组差异并进行功能注释，初筛耐药相关新突变基因；②采用扩大样本检测和基因重组方法鉴定候选基因与LZD耐药发生发展之间的关系；③阐明鉴定的LZD耐药相关新基因突变参与耐药的相关机制。本课题旨在从新的角度认识MRSA LZD耐药发生机制，为LZD耐药的防控、治疗乃至药物开发提供理论基础。

利奈唑胺(LZD)是临床治疗耐甲氧西林金黄色葡萄球菌(MRSA)感染的主要药物，耐药性问题日益突出。本项目分析本地区金黄色葡萄球菌抗菌药物敏感性及 ST 分型特点，揭示金黄色葡萄球菌在本地区的临床规律。采用 Illumina Miseq 结合第三代测序技术和生物信息学分析手段完成一株LZD敏感MRSA菌株（MS4）的全基因组完成图绘制，申请获得 Genebank 号 CP009828。采用不同浓度梯度的LZD对遗传背景清晰的MSRA-MS4母株进行诱导，获得不同MIC值的一系列LZD耐药菌株， 通过全基因组测序研究LZD敏感株和诱导耐药MRSA之间基因变异位点差异，PCR扩增MRSA-MS4-LZD100 23sRNA V区及核糖体蛋白L3/L4基因，测序后与野生株比较，获得相应的突变位点；采用Illumina Hiseq 2000测序技术对样品DNA进行paired-end(PE) 测序，构建Illumina PE文库，利用生物信息学完成该菌株的全基因组测序。外显子的SNP占16个，导致氨基酸序列改变的蛋白质包括IstB ATP 结合区域包含蛋白、凝集因子A及转座子IS1272等，而发生于外显子的Small indel 3个，导致氨基酸序列改变的蛋白质包括假设蛋白、30S核糖体蛋白S1、凝集因子A。测序分析提示该菌株存在除23S rRNA V区及L3蛋白基因以外的突变位点，介导LZD耐药。以红霉素（ERY）为代表大环内酯类抗生素具有的和利奈唑胺具有相同的药物作用靶位，且易和利奈唑胺形成交叉耐药，使用利奈唑胺-红霉素连续体外培养诱导四株甲氧西林敏感金黄色葡萄球菌S2、S3、S5和S7株，成功地建立了利奈唑胺-红霉素耐药菌株系。在S2-、S5-和S7衍生的耐药菌株中，L22蛋白首先发生突变，导致与酮内酯类抗生素产生较低水平的交叉耐药性 (≤4μg/mL)。L4蛋白突变依赖于L22蛋白，导致与酮内酯类抗生素产生高度交叉耐药（≥8μg/mL）。在S3衍生的菌株中，在23S rRNA结构域II/V区和L22蛋白中同时发生突变的情况下出现与酮内酯类抗生素产生高水平的交叉耐药的现象，因此抗生素药物结合位点的突变可增加对酮内酯类抗生素的最小抑菌浓度（MIC），包括泰利霉素和新型化合物青霉素，但对林可酰胺、氯霉素或恶唑烷酮类抗生素的最小抑菌浓度（MIC）没有增加。