探索肠球菌对利奈唑胺低水平耐药的分子机制

Enterococci have been known for their role as a common cause of nosocomial infection. Linezolid is one of most effective agents for treating multidrug-resistant Enterococci infection in clinics. Recently, the emergence of resistance to linezolid in Enterococci has been documented increasingly and become a severe challenge for clinical therapy. The most common mechanism of resistance found in enterococci involves mutations in genes encoding domain V of the 23S rRNA or mediated by the acquired gene cfr which encodes a methyltransferase that modifies 23S rRNA. However, resistant mechanisms mentioned above were not existed in clinical isolates of Enterococcus faecalis in our previous study. The common characters of these Enterococcus faecalis strains were exhibiting low level resistance to Linezolid(MIC=4～16mg/L). Exploring the new resistant mechanism in this type Enterococci is great significant for infection control and development of new antimicrobial agents.. Traditional technique for resistant mechanism research focus on single or several related genes and fails to provide a key insight of critical metabolic changes at the global level in bacteria which under antibiotic pressure. In this research, traditional technique of shotgun cloning combined with transcriptome sequencing based on RNA-sequencing and iTRAQ proteomic sequencing based on Tandem Mass Spectrometry will be applied to analyze high-throughput the products of gene transcription and translation in Linezoid low-resistant Enterococcus faecalis strains. Applying this Proteogenomics methodology, by comparing the expression difference in gene and protein level between Linezoid low-resistant and sensitive strains, allows for discovery of mutation and metabolism regulation without expectations that can bias experimental methods away from capturing potentially important information. The global understand the changes associated with drug resistance in Enterococci will be highly possible to screen out new drug resistant targets and elucidate new resistant mechanism.

利奈唑胺是治疗医院感染耐药肠球菌的最有效药物。但近年来出现利奈唑胺耐药肠球菌给临床治疗带来严峻挑战。其耐药机制为核糖体23SrRNA V区突变和cfr基因介导的甲基化酶修饰。前期研究中发现相当部分对利奈唑胺呈低水平耐药（MIC=4～16mg/L）的粪肠球菌不存在上述机制，探索该类肠球菌新的耐药机制对于感染防控和新药开发具有重要意义。. 传统的耐药研究方法多采用单个或少数几个关联基因的研究方式，不能整体了解抗生素压力环境下细菌关键代谢的变化，本研究采用传统鸟枪克隆技术并结合RNA-seq 为核心的转录组测序技术和以串联质谱为核心的iTRAQ蛋白质组测序技术，高通量地对基因的转录和翻译产物进行精确测定，此种“蛋白质基因组学”的研究手段可以通过比较利奈唑胺敏感和耐药肠球菌基因和蛋白表达的差异，全面了解细菌在抗生素压力下突变和代谢调节的全貌，极有可能筛选出新的耐药靶标，阐明新的耐药机制。

利奈唑胺为治疗耐药肠球菌的首选药物，但目前临床分离的低水平耐药菌株的耐药机制不明，为探索该类肠球菌的耐药机制，我们采用抗生素体外诱导、外排泵抑制、RNA-Seq、定量蛋白质组技术、基因定位、全基因和质粒测序、结合试验及流行病学与危险因素分析等研究利奈唑胺低水平肠球菌的耐药及传播机制。发现低水平利奈唑胺耐药粪肠球菌(P10748 MIC:8 mg/L)可以在抗生素诱导下发生高水平耐药，耐药菌细胞壁增厚导致利奈唑胺摄入量减少，Illumina HiSeq 4000高通量转录组测序技术筛选出与耐药相关的基因及通路，发现生物膜形成和外排泵相关基因esp、optrA、fexA在耐药菌中显著上调，TMT技术对膜蛋白进行定量分析得到差异表达蛋白,结合RNA-Seq结果，发现显著上调蛋白Sea1、RepA、TraB、Esp、OptrA等与利奈唑胺耐药密切相关。对44株临床分离的利奈唑胺低水平耐药粪肠球菌进行S1-PFGE和Southern-blot分析，有26株菌（59%）optrA位于质粒，18株菌（41%）optrA则位于染色体上， 23株（88.5％，23/26）位于质粒上的optrA都能进行水平转移，而位于染色体的optrA不能进行转移。质粒测序分析显示质粒中存在耐药基因（optrA和fexA）和多个性信息素反应基因，提示携带optrA基因的质粒具备信息素质粒特征，其水平转移过程可能由Sea1、TraB、RepA及XRE家族转录调控蛋白参与调节。44株菌未发现23S rRNA V区突变,也未检出cfr基因，所有菌株均检测出optrA基因，表明optrA是利奈唑胺低水平耐药粪肠球菌的重要耐药标记基因。MLST结果显示感染病例呈现散发性而不是暴发性。质粒分型表明，rep9型质粒（pCF10原型，性信息素应答质粒）占主导地位（18/23，78％）。在携带optrA的接合菌株中存在三个关键的性信息素应答基因（prgA，prgB和prgC），凝集诱导试验细菌呈现不同程度的聚集，表明optrA介导的利奈唑胺抗性可能通过性信息素质粒转移而广泛传播。optrA基因发挥耐药的机制（外排或核糖体保护）有待进一步证实。