太空环境诱导肺炎克雷伯菌对碳青霉烯类药物耐药性逆转的机制

Klebsiella pneumoniae is an important pathogen of hospital and community acquired pneumonia, and carbapenems is the last resort drug to treat it. However, at present, the carbapenems resistance in Klebsiella pneumoniae is serious. It is of great therapeutic significance to find the reverse of carbapenems resistance and its mechanism in Klebsiella pneumoniae, and there is no relevant report at present. The unique and complex factors in space environment could increase microbial variation rate and variation types. In previous study, we screened a strain of Klebsiella pneumoniae which was stably sensitive to carbapenems changing from its resistance via the space platform. Based on this, we put forward that the reverse of carbapenems resistance in Klebsiella pneumoniae was due to the stable changes of the related genes induced by the environmental factors. In this project, we will use the mutant strain as the object of research, recruiting omics analysis to explore the changes of genome, transcriptome and proteome. Then we would use bioinformatics and molecular biology technique to analyze changes of the inactivated enzyme activity, drug target, bacterial outer membrane permeability, drug efflux system and biofilm in the mutation strains; to carry out regulation mechanism of resistance pathway; and to reveal the molecular mechanism of carbapenem resistance reverse in Klebsiella pneumonia induced by space environment. Completion of this project will provide a theoretical basis for the treatment of carbapenem resistant Klebsiella pneumoniae.

肺炎克雷伯菌是医院及社区获得性肺炎的重要病原菌，碳青霉烯类抗生素是治疗该菌感染最后的屏障药物。目前肺炎克雷伯菌碳青霉烯类耐药形势严峻。发现其耐药性逆转及机制具有重要的治疗意义，目前尚无相关报道。空间环境因素独特且复杂，可使细菌变异速率和变异类型加大。我们前期通过太空搭载筛选出一株对碳青霉烯类耐药逆转为敏感的肺炎克雷伯菌，且能稳定遗传。据此我们提出该菌碳青霉烯类耐药性逆转是由于空间环境因素诱导而发生了相关基因的稳定性改变。本项目以这株耐药逆转菌为研究对象，通过组学分析揭示其基因组、转录组和蛋白组层面的分子改变，通过生物信息学及分子生物学等技术手段，分析突变株的灭活酶、药物作用靶位、细菌外膜通透性、药物主动流出系统及细菌生物被膜形成等变化，开展耐药相关通路的调控机制研究，揭示空间环境诱变后肺炎克雷伯菌对碳青霉烯类耐药性逆转的分子机制。本项目的完成将为耐碳青霉烯肺炎克雷伯菌的治疗提供理论基础。

变异速率和变异类型加大。我们前期通过太空搭载筛选出一株对碳青霉烯类耐药逆转为敏感的肺炎克雷伯菌，且能稳定遗传。本项目以这株耐药逆转菌为研究对象，通过基因组、转录组、蛋白组分析及分子生物学实验等技术手段，发现突变菌株具有多组学性改变，关键的改变是相对于耐药菌株丢失了1个质粒，该质粒共携带139个基因，其中含3个耐药基因：kpc-2，Tem1，OXA-9。3个基因中存在2个碳青霉烯酶，kpc-2和OXA-9，其中kpc-2酶为原始菌株的特点基因。OXA-9对碳青霉烯类抗生素往往产生低水平耐药；发挥作用的关键基因是kpc-2。本项目最终揭示了肺炎克雷伯菌碳青酶烯类耐药性逆转的分子机制：经过太空飞行后，肺炎克雷伯菌(ATCC BAA-1705)出现携带kpc-2等碳青霉烯酶的质粒丢失，从而导致该菌对碳青霉烯类抗菌素由耐药逆转为敏感。本项目的完成说明：1.太空飞行可影响细菌质粒改变或消失。由于太空密闭舱内的特殊环境因素（包括微重力、弱磁场和粒子辐射等）作用于微生物具有双向性，也可能出现不利于人类的改变，如导致细菌对环境的适应能力更强，更加容易生长和繁殖，某些微生物的致病性、耐药性、传播效能会增强，这对空间站的防控洗消、对太空微生物防护安全提供了理论支撑。2.该结果可对临床治疗有一定的启示作用，现阶段针对质粒消除常用的措施效果欠佳，甚至不适合于临床应用；若能立足于本项目的现有结果，进一步明确该菌质粒丢失的启动蛋白，就可以针对性设计药物，达到协同抗生素治疗的效果。从而解决碳青霉烯类抗菌素耐药肺炎克雷伯菌的治疗难题。