Inhibitors of reactive oxygen species accumulation delay and/or reduce the lethality of several antistaphylococcal agents

Abstract

Perturbation of hydroxyl radical accumulation by subinhibitory concentrations of 2,2'-bipyridyl plus thiourea protects Escherichia coli from being killed by 3 lethal antimicrobial classes. Here, we show that 2,2'-bipyridyl plus thiourea delays and/or reduces antimicrobial killing of Staphylococcus aureus by daptomycin, moxifloxacin, and oxacillin. While the protective effect of 2,2'-bipyridyl plus thiourea varied among strains and compounds, the data support the hypothesis that hydroxyl radical enhances antimicrobial lethality.

Fig 1

Effects of inhibitors of hydroxyl radical accumulation on lethality of daptomycin, moxifloxacin, and oxacillin. Exponentially growing S. aureus strain RN450 was treated with a fixed concentration of an antimicrobial for the indicated times (A, C, E, and F) or with the indicated concentrations of an antimicrobial for a fixed time (B and D) in the absence (empty circles) or presence (filled circles) of one-half of the MIC of 2,2′-bipyridyl plus thiourea. (A) Concentration of 1.9 μg/ml (32-fold MIC) daptomycin for the indicated times. (B) Indicated concentrations of daptomycin for 1 h. (C) Concentration of 0.9 μg/ml (15-fold MIC) moxifloxacin for the indicated times. (D) Indicated concentrations of moxifloxacin for 2 h. (E) Concentration of 12.5 μg/ml (50-fold MIC) oxacillin for the indicated times. (F) A noninhibitory concentration (e.g., one-half of the MIC) of glutathione replaced the 2,2′-bipyridyl plus thiourea used for the experiment whose results are shown in panel E. All experiments were repeated 3 times with similar results; error bars indicate standard deviations.

Fig 2

Effects of fractional MIC of 2,2′-bipyridyl plus thiourea on bacterial growth and moxifloxacin lethality. (A) Effect of 2,2′-bipyridyl plus thiourea on bacterial growth. S. aureus strain RN450 was grown to exponential phase (empty circles). At the indicated time (arrow), 2,2′-bipyridyl and thiourea at one-half of the MIC were added into the growing culture (filled circles). Culture turbidity was measured as absorption (optical density [OD]) at 600 nm. (B) Effect of fractional MIC of 2,2′-bipyridyl plus thiourea on bacterial killing by moxifloxacin. Exponentially growing S. aureus strain RN450 was treated with 0.9 μg/ml (15-fold MIC) moxifloxacin for 90 min in the presence of the indicated concentrations of 2,2′-bipyridyl plus thiourea. All experiments were repeated 3 times with similar results; error bars indicate standard deviations.

Fig 3

Effect of moxifloxacin and 2,2′-bipyridyl plus thiourea on intracellular hydroxyl radical accumulation. Exponentially growing S. aureus strain RN450 was treated with 0.9 μg/ml (15-fold MIC) moxifloxacin in the presence or absence of one-half of the MIC of 2,2′-bipyridyl plus thiourea for 35 min. Hydroxyphenyl fluorescein (HPF) was added to both treated samples and an untreated control to a final concentration of 5 μM. Cells were further incubated for 10 min at 37°C in the dark before they were subjected to fluorescence-activated cell sorting (FACS) analysis. In total, 100,000 cells were analyzed for each sample. y axis indicates fluorescence-labeled-cell numbers and x axis represents fluorescence intensity of HPF-labeled cells. A peak shift to the right indicates an increase in hydroxyl radical production/accumulation. Black, untreated control sample; red, moxifloxacin-treated sample; blue, moxifloxacin- and 2,2′-bipyridyl–plus–thiourea-cotreated sample.