L-leucine increases the sensitivity of drug-resistant Salmonella to sarafloxacin by stimulating central carbon metabolism and increasing intracellular reactive oxygen species level (LC-MS positive mode)

The overuse of antibiotics has made public health and safety face a serious crisis. It is urgent to develop new clinical treatment methods to combat drug-resistant bacteria to alleviate the health crisis. The efficiency of antibiotics is closely related to the metabolic state of bacteria. However, studies on fluoroquinolone-resistant Salmonella are relatively rare. In this study, non-targeted metabolomics was used to analyze the metabolic difference between sarafloxacin-susceptible Salmonella Typhimurium (SAR-S) and sarafloxacin-resistant Salmonella Typhimurium (SAR-R), and found that the central carbon metabolism was weakened due to drug resistance. We also confirmed that exogenous L-leucine increased the killing effect of sarafloxacin on SAR-R and other clinically resistant Salmonella serotypes. Exogenous L-leucine stimulated the metabolic state of bacteria, especially the TCA cycle, which increased the working efficiency of the electron transfer chain and increased the intracellular NADH, ATP concentrationa nd reactive oxygen species level. Furthermore, we used real-time quantitative PCR to confirm the changes in various metabolic characteristics. Our results suggest that when the metabolism of drug-resistant bacteria is reprogrammed, the bactericidal effect of antibiotics improves. This study further enhances research in the anti-drug resistance field at the metabolic level and provides theoretical support for solving the current problem of sarafloxacin drug resistance, a unique fluoroquinolone drug for animals. To sum up, our research results showed that exogenous L-leucine promotes the bactericidal effect of antibiotics through metabolic modification of drug-resistant bacteria, which has the potential as a new adjuvant of antibiotics.

LC-MS positive mode is reported in the current study MTBLS7711.
LC-MS negative mode is reported in MTBLS7713.

抗生素过度滥用已使公共卫生安全面临严峻危机，亟需开发新型临床治疗手段以对抗耐药菌，缓解当前的健康危机。抗生素的抗菌效率与细菌的代谢状态紧密相关，但目前针对耐氟喹诺酮类（fluoroquinolone）沙门氏菌的研究相对匮乏。本研究采用非靶向代谢组学（non-targeted metabolomics）技术，分析了沙拉沙星（sarafloxacin）敏感型鼠伤寒沙门氏菌（Salmonella Typhimurium，SAR-S）与沙拉沙星耐药型鼠伤寒沙门氏菌（Salmonella Typhimurium，SAR-R）的代谢差异，发现耐药性会导致细菌的中央碳代谢通路减弱。本研究同时证实，外源性L-亮氨酸可增强沙拉沙星对SAR-R及其他临床分离的耐药沙门氏菌血清型的杀伤作用。外源性L-亮氨酸能够激活细菌的代谢状态，尤其是三羧酸循环（tricarboxylic acid cycle, TCA cycle），进而提升电子传递链的运作效率，提高细胞内NADH、ATP浓度与活性氧（reactive oxygen species, ROS）水平。此外，本研究通过实时定量PCR（real-time quantitative PCR）验证了多项代谢特征的变化。研究结果表明，当耐药细菌的代谢发生重编程后，抗生素的杀菌效果可得到显著提升。本研究进一步推进了代谢层面的抗耐药领域研究，为解决当前沙拉沙星——一种专属动物用的氟喹诺酮类药物——的耐药问题提供了理论支撑。综上，本研究结果显示外源性L-亮氨酸可通过重编程耐药细菌的代谢增强抗生素的杀菌效果，具备开发为新型抗生素佐剂的潜力。

本研究的正离子模式液相色谱-质谱联用（Liquid Chromatography-Mass Spectrometry, LC-MS）数据已上传至MTBLS7711。
本研究的负离子模式LC-MS数据已上传至MTBLS7713。