Rapid evolution of colistin resistance in a bioreactor model of infection of Klebsiella pneumoniae

Objectives. We seek to understand the dynamics of de novo resistant mutations arising during colistin treatment of an antibiotic-naïve population of K. pneumoniae. Methods. We used a bioreactor model of infection and colistin treatment against the colistin susceptible K. pneumoniae Ecl8. Whole-genome sequencing and MIC profiling was used to characterize genetic and phenotypic state of the bacterial culture at three time points: before treatment, immediately after regrowth following challenge, and at the end point of the experiment. A mathematical model based on the birth-death process was used to gain further insights on the population dynamics of emerging resistant variants. Results. We find that, after an initial decline, the population recovers within 24h due to the evolution of highly resistant clones which exhibit MICs >100-fold higher than the parental strain. Recovery is caused by a small number of “founder cells” which have single point mutations mainly in the regulatory genes encoding crrB and pmrB. The mutants arise through spontaneous mutations prior to colistin treatment. Conclusions. Development of colistin resistance during treatment of K. pneumoniae infections is readily achieved if colistin is used as a monotherapy.

研究目的：本研究旨在阐明未接触过抗生素的肺炎克雷伯菌（K. pneumoniae）菌群在接受多粘菌素E（colistin，又名粘菌素）治疗过程中，新发耐药突变的动态变化规律。研究方法：本研究采用感染生物反应器模型，针对多粘菌素敏感型肺炎克雷伯菌Ecl8菌株开展多粘菌素处理实验。研究团队分别于三个时间点采集细菌培养物，通过全基因组测序（whole-genome sequencing）与最低抑菌浓度（minimum inhibitory concentration，MIC）谱分析，表征其遗传与表型状态：治疗前、给药后即刻恢复生长时，以及实验终点。此外，基于生死过程（birth-death process）构建数学模型，以进一步解析新发耐药变异株的种群动态特征。研究结果：本研究发现，菌群在初始下降后可在24小时内完成恢复，这一过程由高度耐药克隆的演化驱动，该类克隆的最低抑菌浓度较亲本菌株高出100倍以上。菌群恢复源于少量携带单点突变的‘创始细胞（founder cells）’，这些突变主要发生于编码crrB与pmrB的调控基因中。上述突变实则在多粘菌素治疗前便已通过自发突变产生。研究结论：若将多粘菌素作为单一疗法（monotherapy）使用，在肺炎克雷伯菌感染治疗过程中极易诱导产生多粘菌素耐药性。