Purpose: Bacterial metabolism regulators offer a novel productive strategy in the eradication of antibiotic refractory bacteria, particularly bacterial persisters. However, the potential of amino acids in the fight against Gram-negative bacterial persisters has not been fully explored. The aim of this study is to investigate the potentiation of amino acids to antibiotics in combating Gram-negative bacterial persisters and to reveal the underlying mechanisms of action.
Methods: Bactericidal activity of antibiotics in the absence or presence of amino acids was evaluated through detecting the reduction of bacterial CFUs. The ratio of NAD⁺/NADH in E. coli  B2 persisters was determined using assay kit with WST-8. Bacterial respiration and ROS production were measured by the reduction of iodonitrotetrazolium chloride and fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate, respectively.
Results: In this study, we found that cysteine possesses excellent synergistic bactericidal activity with ciprofloxacin against multiple Gram-negative bacterial persisters. Furthermore, the potentiation of cysteine was evaluated in exponential and stationary-phase E. coli  ATCC 25922 and E. coli  B2. Interestingly, cysteine significantly improves three bactericidal antibiotics killing against stationary-phase bacteria, but not exponential-phase bacteria, implying that the effect of cysteine correlates with the metabolic state of bacteria. Mechanistic studies revealed that cysteine accelerates the bacterial TCA cycle and promotes bacterial respiration and ROS production. These metabolic regulation effects of cysteine re-sensitive bacterial persisters to antibiotic killing.
Conclusion: Collectively, our study highlights the synergistic bactericidal activity of bacterial metabolism regulators such as cysteine with commonly used antibiotics against Gram-negative bacterial persisters.
Keywords: amino acids, bacterial persisters, bactericidal antibiotics, cysteine, gram-negative bacteria