泛耐药肺炎克雷伯氏菌耐药基因分布、多态性及新型耐药基因挖掘研究

Klebsiella pneumoniae is an important opportunistic pathogen. The irrational application of antibiotics lead emergency pandrug-resistant klebsiella pneumoniae in recent years. Research of antibiotics resistance related genes of the pandrug-resistant klebsiella pneumonia is one of research trend. But the distribution, polymorphisms of antibiotic resistance gene and novel antibiotic resistance gene in pandrug-resistant klebsiella pneumonia are not clear. Aim to this scientific questions, Eighty two pandrug-resistant klebsiella pneumonia strains were screened in our previous programe and hundred kinds of antibiotic genes such KPC-2 were found in one draft genome sequence of the representative strain. On the basis of previous research works, this research will done as follows: 1) Klebsiella pneumoniae genome will being squenced by the high throughput sequence platform, based on the genome sequence datas of the pandrug-resistant klebsiella pneumonia determine the antibiotic gene distribution and SNP alleles. The PCR be used to verify them. Accordingly, The results can be illuminates the distribution and polymorphisms of antibiotic resistance genes. 2) RNA-seq method be used to sequence the transcriptome of the pandrug-resistance klebsiella pneumonia. Bioinformatics methods be used to analyz and predict novel transcripts.Novel transcripts will be cloned and expressed. The positive bacteria strain will be test antibiotic sensitive to ascertain novel antibiotic resistance gene. This project's development has critical scientific significance and application prospect to research new antibiotic resistence gene , resistant mechanisms, target drugs and clinical medication.

肺炎克雷伯氏菌为重要条件致病菌，抗菌素广泛不合理使用导致泛耐药肺炎克雷伯氏菌不断出现，其耐药相关基因的研究为国内外研究热点。但其基因组耐药相关基因分布、多态性如何？是否存在新型耐药相关基因？尚不明确。针对该科学问题，本项目组在前期所分离鉴定82株泛耐药肺炎克雷伯氏菌，测定并获得其中1株全基因组草图，分析发现含有KPC-2等100多种耐药相关基因基础上，拟开展以下研究：1）测定泛耐药肺炎克雷伯氏菌基因组，分析其耐药相关基因分布、挖掘耐药基因SNP位点，PCR方法进行验证，阐明耐药相关基因分布及多态性。2）测定泛耐药肺炎克雷伯氏菌转录组，分析转录本结构，挖掘新转录本，对新转录本进行克隆、表达，测定表达菌株抗菌素敏感性，挖掘出泛耐药肺炎克雷伯氏菌新型耐药基因与耐药基因新亚型。本项目的开展为泛耐药肺炎克雷伯氏菌耐药基因及其机理，靶向药物的研究，为临床抗感染药物的选择具有重要科学价值。

抗菌药物的广泛使用导致细菌对抗生素耐药性日益增加，已成为全球的公共卫生问题。本研究在自然基金的资助下开展了如下工作：（1）从四川省、上海、北京、湖南，福建等多个省市多家医院分离到不同耐药水平的肺炎克雷伯菌2000余株，肺炎链球菌100余株，沙门氏菌78株，大肠杆菌189株，不动杆菌67株。（2）筛选出218株耐碳青霉烯肺炎克雷伯菌，对其6大类耐药基因进行了检测和序列分析，阐明了耐碳青霉烯肺炎克雷伯菌耐药基因的分布和多态性，并首次从肺炎克雷伯菌中发现携带产金属酶新亚型基因blaNDM-19。（3）测定高毒力粘液性肺炎克雷伯菌SWU01菌株全基因组，测定同时产NDM,KPC大肠杆菌。（4）对肺炎克雷伯菌临床菌株耐受性进行了初步研究，并挖掘出多个可能与细菌耐受有关的基因。（5）开展肺炎链球菌中新型抗菌肽的研究。（6）对肺炎克雷伯菌及本文的研究发现肺炎克雷伯菌能产生高水平的耐受，并在各种高浓度抗生素的杀伤作用下存活。本研究的开展为细菌耐药性的防控具有重要意义。