Bacterial Single-cell RNA-seq and Multi-omics Reveal Effects of Proline in Arresting the Evolution of Antibiotic Resistance

Antibiotic pollution over a range of environmental reservoirs imposed selection pressure and contributed to the evolution of antimicrobial resistance, posing a major world health crisis. Although recent work has revealed several ways to control the evolution and transmission of antibiotic-resistance genes in environmental settings, evolution-slowing strategies have been systemic underexplored. Previous laboratory evolution experiments have noticed that altering the metabolic state could change the bacterial evolution of antibiotic resistance. Here, we identify that exogenous proline can arrest the resistance evolution to environmental-level ciprofloxacin exposure of Escherichia coli. Through transcriptome and metabolomic analysis, we show that exogenous proline greatly suppressed the gene expressions and relative metabolite abundances of the TCA cycle. The mechanism underlying this phenomenon is that exogenous proline demotes the TCA cycle metabolic flux, which in turn decreases production of NADH and proton motive force and reduces uptake of antibiotic. Furthermore, bacterial single-cell RNA-seq and deep resequencing demonstrate that exogenous proline reduced the proportion of resistant subpopulations and delayed the appearance of resistant mutants. Overall, our results demonstrate how exogenous proline can help arrest the evolution of antibiotic resistance against pathogenetic E. coli, which may suggest prospective metabolism reprogramming applications for mitigation of antibiotic resistance progression in antibiotic-polluted environments.

各类环境储库中的抗生素污染可施加选择压力，推动抗菌耐药性的演化，进而构成重大全球公共卫生危机。尽管近期研究已揭示多种可在环境场景中管控抗生素耐药基因演化与传播的手段，但减缓耐药性演化的策略仍未得到系统性深入探索。既往实验室演化实验已发现，改变代谢状态可调控细菌的抗生素耐药性演化进程。本研究发现，外源脯氨酸（exogenous proline）可阻断大肠杆菌（Escherichia coli）在环境浓度环丙沙星（ciprofloxacin）暴露下的耐药性演化。通过转录组（transcriptome）与代谢组学（metabolomics）分析，本研究证实外源脯氨酸可显著抑制三羧酸循环（tricarboxylic acid cycle, TCA）的基因表达与代谢物相对丰度。该现象的核心机制为：外源脯氨酸会降低TCA循环的代谢通量，进而减少烟酰胺腺嘌呤二核苷酸（NADH）的生成与质子动力势（proton motive force），并降低细菌对抗生素的摄取量。此外，细菌单细胞RNA测序（single-cell RNA-seq）与深度重测序结果显示，外源脯氨酸可降低耐药亚群的比例，并延缓耐药突变体的出现。综上，本研究结果阐明了外源脯氨酸如何助力阻断致病性大肠杆菌（Escherichia coli）的抗生素耐药性演化，这一发现可为在抗生素污染环境中通过代谢重编程（metabolism reprogramming）缓解耐药性演化提供潜在的应用思路。