多重耐药肺炎克雷伯菌的临床流行状况及镓离子对其生长曲线的影响机制

Multidrug-resistant Klebsiella pneumoniae infection can often lead to intractable threat to human health, with the increase of multi-drug resistant strains, which pose serious challenges to public health and safety. How to overcome multidrug resistance of K. pneumoniae is a very difficult problem that requires urgent action. Our preliminary work indicates that gallium ion has bactericidal activity against K. pneumoniae and can increase Reactive Oxygen Species (ROS) production. So we speculate that the molecular mechanisms of gallium's bactericidal effect have connection with ROS pathway. In order to know its infection characteristics, we are going to carry out case-control study to understand the epidemiological characteristics, such as the high risk of infection factors, resistant phenotype, and genotype of epidemic strains. Simultaneously we will systemically evaluate multiple gallium-antimicrobial combinations for synergistic antimicrobial effect. The combinations that show maximal synergistic effect will be further evaluated for pharmacokinetics, pharmacodynamics, and therapeutic efficacy in a rabbit tissue cage model of infection. In another arm of studies, we will be to first select gallium-resistant and gallium-tolerant mutants. Comparison of whole genome sequences between wild-type and the resistant/tolerant strains, transcriptom analysis in the presence/absence of gallium and between wild-type and mutant strains, and evaluation of the effect of deficiency in ROS detoxification pathway genes on gallium susceptibility will be used to explore and elucidate molecular mechanisms underlying gallium antimicrobial effect. The outcome of the present work will form knowledge base for developing gallium-containing antimicrobial regimens that help overcome the growing problem of multidrug resistant bacterial infection.

多重耐药肺炎克雷伯菌常可导致难治性感染,威胁着人类健康,随着多重耐药菌株的增加,其给公共卫生安全带来严峻的挑战。如何克服肺炎克雷伯菌的多重耐药性是人类亟需解决的公共卫生难题。本课题组预实验表明:镓离子对肺炎克雷伯菌有杀菌活性;且镓离子能够上调细菌内活性氧的产生,由此我们推测镓杀菌的分子机理与活性氧通路有关。为了掌握多重耐药肺炎克雷伯菌医院感染的特征,本课题拟进行病例对照研究,了解其感染的高危因素、耐药表型及流行株的基因型等流行病学特征;同时拟系统探索镓-抗菌药物组合的协同杀菌效应,并对最佳组合在兔组织笼模型中进行药代药效动力学及疗效验证;然后筛选肺炎克雷伯菌镓耐药、耐受突变体,对比野生型进行全基因组测序,以及在有镓无镓条件下全转录组对比测序,检测活性氧解毒通路基因敲除对镓杀菌敏感性的影响,阐明镓抗菌杀菌的分子机理,为开发高效、新型含镓抗菌药物,克服日趋严重的细菌耐药问题提供理论依据。

多重耐药肺炎克雷伯菌（MDR-KP）常可导致难治性感染,威胁着人类健康,随着多重耐药菌株的增加,其给公共卫生安全带来严峻的挑战。本课题组多中心收集临床MDR-KP及相应临床资料，通过病例对照及分子流行病学研究发现临床流行MDR-KP主要为碳青霉烯耐药肺炎克雷伯菌（CRKP），其耐药机制为产KPC及NDM型碳青霉烯酶，对除替加环素外的大多数抗菌药物耐药，ST11型为优势克隆，存在区域流行播撒，临床床合理经验用药及住院总时间与CRKP感染预后（30天死亡率）有独立相关性，该型可作为感染预后的独立危险因素。本地区业已存在碳青霉烯耐药高毒力肺炎克雷伯菌（CR-hvKP）流行趋势，其碳青酶烯耐药基因均由分子量大小28.878.2kb质粒携带。硝酸镓（GaN）对CRKP最低抑菌浓度分布范围为0.0625~2μmol/mL，MIC50、MIC90分别为0.25和0.5μmol/mL。1/2MIC的GaN可显著抑制CRKP的生物膜，以庆大霉素作为对比，GaN抑制CRKP生物膜能力优于庆大霉素。阐明了GaN介导杀菌机制与增加的细胞内ROS积累相关，该积累被可耐受相关基因Evgs的突变消除。二甲亚砜可降低GaN介导的杀伤作用，耐受株evgs的突变上调了编码ROS解毒和三羧酸乙醛酸分流酶的基因循环。综上表明，GaN通过提高ROS杀死细菌，evgS的功能获得突变通过组成性地激活EvgA-YdeO/GadE的酸抗性通路级联，进而通过上调ROS解毒和转移三羧酸循环碳通量产生耐药/耐受。鉴于GaN较强的杀菌活性及相应耐药机制表明，临床使用该药物后产生耐药株的周期较长。