肺炎支原体耐药相关基因点突变的作用及新耐药机制的研究

Mycoplasma pneumoniae ( Mycoplasma pneumoniae, MP ) is one of the important pathogens of respiratory tract infection. The drug resistance of this pathogen is serious in recent years, which make the clinical treatment is very difficult. The serious drug resistance caused great harm for the adolescent health. .Previous analysis on the macrolide antibiotics target gene sequences indicated that the point mutations of 23S rRNA V region gene are highly correlated with the drug resistance, which had not been experimentally confirmed. In addition, our previous studies found that another kind of drug resistant strains have no 23S rRNA gene mutations, but some of their genes and proteins' expression levels are significantly different from those of the drug sensitive strains. This may indicated that there are some new resistance mechanisms in the gene encoding or expression level which had not been revealed yet. Terefore, this research plan focuses on sequencing the whole genomes and transcriptomes of the representative strains of these two types of MP resistant isolates,and compare the new genomes and transcriptomes with those of the type strains to identify the resistance-related genes; We will further knockout the resistance-related genes, especially 23S rRNA V region genes, and elucidate the relationship between drug resistance and resistance-related genes according to the MP mutant phenotype. This study will systematically elucidate the MP macrolide antibiotic resistance mechanisms, find new resistance mechanisms, and lay the foundation for future clinical typing and the development of new drugs.

肺炎支原体（Mycoplasma pneumoniae, MP）是社区获得性肺炎的重要病原体，近年耐药严重，治疗困难，尤其危害青少年健康。以往对大环内酯类抗生素靶基因进行序列分析的研究表明，23S rRNA Ⅴ区编码基因点突变与MP耐药性高度相关，但未有实验证明；另外，我们前期研究发现另一类耐药株虽不具有23S rRNA基因突变，但其部分基因和蛋白的表达水平与敏感株显著不同，说明可能在基因编码或者表达调控层面存在尚未揭示的新耐药机制。本研究拟对上述两种类型的MP耐药株代表株进行全基因组和转录组测序，通过与标准株进行比较分析，初步鉴定耐药相关基因；进一步对耐药相关基因尤其是23S rRNAⅤ区编码基因进行基因敲除操作，根据MP突变体表型研究阐明耐药相关基因与耐药性的关系。本研究不仅从基因组水平对MP大环内酯类抗生素耐药机制进行全面系统分析，明确其机制；还对控制耐药和耐药诊断具有重要意义。

肺炎支原体（Mycoplasma pneumoniae，MP）是社区获得性肺炎的重要致病菌，近年来MP耐药率不断上升，是导致难治性或重症肺炎增多的重要原因。本研究通过分离培养肺炎支原体流行株，选择其中的代表株分为耐药组和敏感组进行比较基因组学和转录组学分析，预测了MP中可能与耐药相关的24个SNP位点，系统研究了MP可能存在的耐药机制；从分离培养的同源克隆中选择耐药相关位点基因型不同的高度同源单克隆菌株，经测序验证后得到23SrRNA耐药相关位点的突变株和野生株，并对其进行了全基因组测序分析和表型研究，验证了23SrRNA 2063位点与耐药性的关系；通过从临床样本中分离培养MP单克隆的方法，发现MP在感染者体内是以混合菌群的状态存在，菌群的组成及各类菌株的比例可能随着菌株的变异而不断变化，这在国内外研究中尚数首次发现，该发现对MP耐药的产生及发展规律的研究具有重要意义；针对研究过程中的新发现，我们建立了用以检测肺炎支原体耐药突变基因（23S rRNA V区A2063G和A2064G）的等位基因特异性扩增实时定量PCR（Allele-specific real-time PCR, ASPCR）方法，该方法不仅能够检测耐药相关基因的基因型，同时还能检测各种基因型所占比例。对该方法的准确性、灵敏度、特异性进行了验证，并采用该方法检测了182份临床样本。本研究结果对系统阐明MP 对大环内酯类抗生素耐药机制具有重要意义，对研究MP耐药的发生发展机制、MP在感染人体后的进化变异规律具有重要作用，ASPCR方法的建立和应用能够研究体内MP菌群的动态变化过程，是研究耐药发生发展变化规律的有力手段，对MP感染的临床用药也具有重要的指导意义。