Ethylenediaminetetraacetic Acid Enhances Vancomycin and Reactive Oxygen Species-Mediated Killing of Vancomycin-Intermediate Staphylococcus aureus

Abstract

Background: The emergence of antibiotic-tolerant staphylococcal strains, such as vancomycin-intermediate Staphylococcus aureus (VISA), poses a significant healthcare challenge and complicates treatment regimens. VISA often exhibits mutations in Krebs cycle enzymes, promoting anaerobic metabolism under physiological conditions and reducing susceptibility to antibiotics and innate immune defenses-factors not typically captured in standard susceptibility testing. Building on these findings, we investigated ethylenediaminetetraacetic acid (EDTA), a chelator that enhances reactive oxygen species (ROS) production, as an adjunct to vancomycin for combating VISA infections.

Methods: RNA sequencing analysis compared gene expression of a well-characterized VISA strain (D712) under physiological versus nutrient-rich conditions. Hydrogen peroxide (H₂O₂) killing assays were conducted with and without the hydroxyl radical quencher thiourea, while neutrophil killing assays used ROS scavenger butylated hydroxyanisole (BHA). A murine bacteremia model assessed the effects of vancomycin, EDTA, or their combination on VISA.

Results: VISA exhibited downregulation of Krebs cycle enzymes and other genes associated with resistance to iron and ROS-mediated killing under physiological conditions. EDTA, alone or with vancomycin, improved H₂O₂-mediated killing compared to vancomycin alone, a response counteracted by thiourea. The combination of EDTA and vancomycin enhanced neutrophil killing of VISA more effectively than either treatment alone, an effect negated by BHA. In vivo, EDTA enhanced vancomycin activity against VISA.

Conclusions: EDTA potentiates vancomycin efficacy against VISA in vivo and enhances susceptibility to H₂O₂ and neutrophil killing. Reduced expression of Krebs cycle enzymes in VISA suggests that EDTA promotes ROS-mediated bacterial killing, targeting a key mechanism by which persistent staphylococci evade host defenses.

Keywords: EDTA; ROS; VISA; antibiotic tolerance; vancomycin.

Figure 1.

Physiological conditions induce gene expression changes in vancomycin-intermediate Staphylococcus aureus (VISA) D712. RNA sequencing analysis was conducted on VISA strain D712 after 3 hours of growth in either standard bacteriological (cation-adjusted Mueller-Hinton broth) or physiological (Roswell Park Memorial Institute medium supplemented with 10% Luria-Bertani broth [RPMI + 10% LB]) conditions. Under physiological conditions, VISA D712 showed a downregulation of genes involved in electron transport, including key Krebs cycle enzymes such as citrate synthase, isocitrate dehydrogenase, and succinate dehydrogenase, which may inhibit energy-dependent processes. Additionally, siderophore genes (eg, staphyloferrin B), responsible for iron uptake, were downregulated, while genes involved in protection against iron-mediated oxidative stress were upregulated. These included PerR, a regulator of intracellular iron and hydrogen peroxide levels, and DNA starvation protection proteins that help prevent hydroxyl radical production via the Fenton reaction.

Figure 2.

Effect of adjunctive ethylenediaminetetraacetic acid (EDTA) on hydrogen peroxide (H₂O₂) and human neutrophil killing of vancomycin-intermediate Staphylococcus aureus (VISA) D712. A, Adjunctive EDTA (150 μg/mL) or monotherapy increases VISA D712's susceptibility to oxidative stress (H₂O₂ killing) compared to vancomycin (VAN, 1 μg/mL) alone at 45 minutes. This effect was reduced by the hydroxyl radical quenching agent thiourea (150 mM), leading to bacterial growth similar to that of bacteria grown without H₂O₂ (n = 3). B, Adjunctive EDTA increases VISA D712's sensitivity to neutrophil killing (at 45 minutes) in the presence of VAN (1 μg/mL), compared to VAN alone (n = 4). This effect was diminished by the reactive oxygen species scavenger butylated hydroxyanisole (BHA, 10 µM). *P < .05 and **P ≤ .01, 2-tailed Mann-Whitney test.

Figure 3.

Efficacy of adjunctive ethylenediaminetetraacetic acid (EDTA) with vancomycin (VAN) against vancomycin-intermediate Staphylococcus aureus (VISA D712) in a murine bacteremia model. Bacterial counts from kidneys (colony-forming units [CFU]/g) after 24 hours of treatment with VAN (100 mg/kg/day) alone, EDTA (200 mg/kg/day) alone, VAN + EDTA combination, or no treatment (control) are shown. The VAN + EDTA combination significantly reduced recoverable VISA D712 from kidneys compared to VAN alone, EDTA alone, and the control (n = 16). Horizontal bars represent the mean. ****P ≤ .0001, 2-tailed Student t test.

Figure 4.

The Fenton reaction, ethylenediaminetetraacetic acid (EDTA), and bacterial adaptation to oxidative stress: a proposed mechanism for enhancing antibiotic efficacy. The Fenton reaction involves the oxidation of ferrous iron (Fe²⁺) by hydrogen peroxide (H₂O₂), producing reactive oxygen species (ROS), such as hydroxyl radicals (•OH). This reaction is pH-dependent, with optimal rates at acidic pH (around pH 3). At physiological pH, ferric iron (Fe³⁺) becomes less soluble, potentially slowing the reaction. EDTA may enhance ROS production by increasing the solubility of Fe³⁺, as it has a higher binding affinity for Fe³⁺ compared to Fe²⁺, thus facilitating the Fenton reaction over a broader range of physiological pH conditions. Staphylococcal persisters, which exhibit low adenosine triphosphate (ATP) levels and anaerobic metabolism, often harbor mutations in tricarboxylic acid (TCA) or Krebs cycle enzymes [10, 11]. These enzymes play a key role in generating Fenton-mediated radicals. Overstimulation of the bacterial electron transport chain can release iron from iron-sulfur clusters, potentially activating the Fenton reaction and causing damage to bacterial DNA, proteins, and lipids. EDTA may catalyze this process in the presence of immune factors that generate ROS and antibiotics like vancomycin, potentially overcoming bacterial adaptations to oxidative stress and improving antibiotic efficacy against antibiotic-tolerant and/or -resistant strains.