基于特定菌种的氨基糖苷类药物协同敏感机制研究

In recent years, researchers found that bacteria evolved resistant to antimicrobials under selective pressure, will give rise to collateral sensitivity, which exhibite increasing susceptibility to one or more antimicrobials. However, the mechanism of collateral sensitivity has not been clarified yet. And there is no related research in domestic. In our previous studies, we found that both Klebsiella pneumoniae and Enterococcus faecalis exhibited significant increase in susceptibility to aminoglycosides when evolved resistant to other antimicrobials. Thus, we speculated that through elucidating the mechanism and regularity of collateral sensitivity, it will help predicting resistance evolution in clinic. Alternating therapy based on collateral sensitivity may mitigate the evolution of resistance. In this study, clinical isolated and standard strains of Klebsiella pneumoniae and Enterococcus faecalis will be evolved resistant to various antimicrobials through adaptive laboratory evolution. The cross-susceptibility will be analyzed. Aminoglycosides and corresponding drugs which show collateral sensitivity are selected. Bacteria will be challenged repeatedly by two antibiotics. Subsequently, transcriptome analysis, whole genome sequencing and gene recombination are applied to clarify the mechanism of collateral sensitivity. Finally, a prediction model is going to be established by combining collateral sensitivity phenotype and genotype. This research will help provide scientific basis to carry out more reasonable and effective anti-infection treatment strategies in clinic.

近年来，人们发现细菌在抗菌药物选择性压力下耐药的同时，会发生协同敏感（collateral sensitivity），即对其他药物敏感性升高，但机制尚未明确，并且国内尚无相关研究。我们在前期研究中发现肺炎克雷伯菌和粪肠球菌在抗菌药物诱导耐药后，均表现出对氨基糖苷类药物敏感性显著升高。我们推测通过对协同敏感现象及其机制的阐明，能够帮助临床预测治疗中耐药性的变化，交替使用协同敏感药物可能延缓耐药性的发生发展。本研究中我们利用各类抗菌药物对肺炎克雷伯菌及粪肠球菌临床及标准菌株对进行耐药诱导，分析抗菌药物间的交叉敏感性；选取氨基糖苷类药物及其协同敏感的药物进行交替用药诱导，比较单药及多药诱导后细菌耐药性变化；随后对诱导前后菌株进行转录组、全基因组测序和基因重组，阐释协同敏感的发生机制；最后，结合药敏表型及基因型建立协同敏感预测模型。本项目将为临床制订合理有效的用药策略提供科学依据。

多重耐药病原菌感染对人类健康造成严重威胁。近年来，人们发现细菌在抗菌药物选择性压力下发生耐药的同时，对其他抗菌药物发生协同敏感（collateral sensitivity），即对其他药物敏感性升高。阐明这一现象的发生发展规律及机制，可能为多重耐药病原菌的治疗提供新的思路和策略。本课题研究中，我们对临床常见的革兰阴性杆菌（肺炎克雷伯菌）和革兰阳性球菌（肠球菌属细菌）进行协同敏感现象及机制的研究。我们选取分离自温州医科大学附属第一医院不同标本来源、不同耐药背景的菌株进行研究，包括耐碳青霉烯肺炎克雷伯菌、粪肠球菌和耐万古霉素屎肠球菌。对上述菌株采用多种抗菌药物进行体外诱导耐药实验（experimental evolution）。研究结果显示诱导耐药突变株表现出广泛的交叉耐药和协同敏感现象。我们对体外诱导获得的耐药突变株进行生长特性、竞争能力和毒力实验等适合度代价（fitness cost）分析，研究显示细菌由不同种类抗菌药物诱导耐药后适合度代价不同，主要表现为生长能力减弱、竞争能力下降及毒力减低等。为深入研究协同敏感的发生机制，课题组对发生协同敏感现象的诱导耐药突变株及其亲本菌株进行全基因组测序及生物信息学分析，研究发现协同敏感现象的发生是多因素导致的，其中耐药质粒的丢失、插入序列破坏耐药基因结构进而影响其功能等是引起协同敏感的主要机制。通过研究我们发现诱导耐药突变株抗菌药物敏感谱的改变具有一定的规律性，且协同敏感现象的发生具有一定的规律性，我们可以通过分析临床抗菌药物的用药种类、用药浓度水平、用药时长等特点结合特定基因型改变的检测，预测协同敏感现象的发生，并指导临床合理用药。