Thymine Sensitizes Gram-Negative Pathogens to Antibiotic Killing

Abstract

Diminished antibiotic susceptibility of bacterial pathogens is an increasingly serious threat to human and animal health. Alternative strategies are required to combat antibiotic refractory bacteria. Bacterial metabolic state has been shown to play a critical role in its susceptibility to antibiotic killing. However, the adjuvant potential of nucleotides in combination with antibiotics to kill Gram-negative pathogens remains unknown. Herein, we found that thymine potentiated ciprofloxacin killing against both sensitive and resistant-E. coli in a growth phase-independent manner. Similar promotion effects were also observed for other bactericidal antibiotics, including ampicillin and kanamycin, in the fight against four kinds of Gram-negative bacteria. The mechanisms underlying this finding were that exogenous thymine could upregulate bacterial metabolism including increased TCA cycle and respiration, which thereby promote the production of ATP and ROS. Subsequently, metabolically inactive bacteria were converted to active bacteria and restored its susceptibility to antibiotic killing. In Galleria mellonella infection model, thymine effectively improved ciprofloxacin activity against E. coli. Taken together, our results demonstrated that thymine potentiates bactericidal antibiotics activity against Gram-negative pathogens through activating bacterial metabolism, providing a universal strategy to overcome Gram-negative pathogens.

Keywords: Gram-negative bacteria; antibiotic resistance; antibiotic tolerance; metabolism; thymine.

FIGURE 1

Thymine potentiates ciprofloxacin killing against both sensitive and resistant-E. coli in a growth phase-independent manner. (A) Effect of five nucleotides on ciprofloxacin activity against exponential and stationary phase E. coli ATCC 25922. (B) Adenine and thymine drastically improved ciprofloxacin killing against exponential and stationary phase MDR E. coli B2. All data were obtained in three independent experiments and shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, determined by one-way ANOVA.

FIGURE 2

Synergistic bactericidal activity of thymine and ciprofloxacin against E. coli persisters. Percentage survival of E. coli 25922 persisters (A) and E. coli B2 persisters (B) after exposure to nucleotides (10 mM) or in combination with ciprofloxacin (20-fold MIC, 0.16 μg/mL and 320 μg/mL for E. coli 25922 and E. coli B2 persisters, respectively) for 4 h. All data were obtained in three independent experiments and shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, determined by one-way ANOVA.

FIGURE 3

Thymine sensitizes Gram-negative pathogens to antibiotic killing through upregulating bacterial metabolism. (A) Thymine decreased the ratio of NAD⁺/NADH, measured by using the water-soluble tetrazolium salt WST-8. (B,C) Thymine significantly enhanced bacterial respiration in the absence (B) or presence (C) of ciprofloxacin through monitoring reduction of iodonitrotetrazolium chloride (INT). (D) Dose-dependent increase of intracellular ATP level by thymine. ATP level was determined based on bioluminescent reaction catalyzed by firefly luciferase. (E) Combination of ciprofloxacin and thymine enhanced ROS production, determined by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, 10 μM). (F) Addition of ROS scavenger thiourea (100 mM) abolished the synergistic bactericidal activity of ciprofloxacin and thymine. All data were obtained in three independent experiments and shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, determined by unpaired t-test.

FIGURE 4

Thymine restores ciprofloxacin activity in Galleria mellonella infection model. Galleria mellonella larvae (n = 10 per group) was given a lethal inoculum of E. coli ATCC 25922 (10⁷ CFUs, A) or E. coli B2 (10⁶ CFUs, B). After 1-h post-infection, a single dose of PBS, ciprofloxacin (0.08 mg/kg for 25922 or 50 mg/kg for B2), thymine (10 mg/kg) and their combination were given. Survival rates of Galleria mellonella were monitored during 5 days. P-values were determined by the log rank (Mantel-Cox) test.