Delineating molecular mechanisms on the acquisition of in vitro-adapted colistin-resistant Klebsiella pneumoniae by transcriptomic analysis.

The worldwide dissemination of colistin-resistant pathogens, despite global efforts, is a serious public health concern, considering that colistin is regarded as a last-resort antimicrobial agent for the clinical treatment of multidrug-resistant (MDR) Gram-negative bacterial infections. Since the development of novel strategies and technologies, such as limiting bacterial adaptation, is essential for our society, adaptive laboratory evolution (ALE) was conducted under colistin pressure to uncover poorly understood mechanisms of colistin adaptation and provide a fundamental basis for future technologies. In this study, two isolates of Klebsiella pneumoniae were subjected to ALE under colistin pressure, with each isolate serving as the ancestor of three mutant strains resulting from independently conducted ALE experiments. Consequently, all strains surpassed the resistance threshold, and the resistant ratio of the bacterial population exceeded 60% by at least the seventh day of colistin pressure. Whole-genome resequencing revealed multiple gene variants potentially associated with colistin adaptation, warranting further assessment. The RNA-seq results from each stage of pressure revealed that in the early stage, pathways related to quorum sensing are involved, while in the later stage, pathways related to colistin target modification mechanisms were activated. This suggests that different reactions and pathways contribute to colistin survival at the early and later stages. The findings presented in this study will contribute to a deeper understanding of colistin adaptation in K. pneumoniae and provide valuable insights for further studies aimed at establishing new strategies to prevent the further emergence of colistin-resistant K. pneumoniae.

Importance: Nowadays, only a few treatment options remain for widespread multidrug-resistant (MDR) Gram-negative bacterial infections, including the old antibiotic colistin. However, colistin-resistant clinical pathogens are spreading globally, further limiting treatment options. The future does not look promising due to the increasing global use of antibiotics and the uncontrolled spread of MDR pathogens. Moreover, the development of novel antibiotics has been limited in the industrial sector due to the rapid adaptation of the clinical pathogens. Therefore, the hope relies solely on the development of novel strategies and technologies to manage bacterial infections or limit bacterial adaptations. In this study, a new strategy has been proposed based on findings from in vitro colistin pressure to limit bacterial adaptation to colistin. Given the growing future concerns, the novel perspectives proposed in this study could provide a fundamental basis for developing novel materials or measures to limit antimicrobial adaptation in clinical environments.