Vitamin C-Assisted Fenton Oxidation Improves Oxidative Killing of Helicobacter pylori In Vitro

Helicobacter pylori infection remains a major global health burden, and the increasing prevalence of antibiotic resistance underscores the need for alternative antibacterial strategies. This study examined whether vitamin C dosing could modulate Fenton oxidation and enhance oxidative antibacterial activity against H. pylori under defined acidic in vitro conditions. Oxidative activity was assessed using Acid Orange 7 decolorization as an indirect chemical readout and a terephthalic acid fluorescence assay as a relative indicator of hydroxyl radical-associated product formation in iron-peroxide reactions supplemented with vitamin C. Antibacterial efficacy was evaluated by exposing standardized H. pylori suspensions in urea-containing saline at pH 3 to hydrogen peroxide and ferrous iron with or without stepwise vitamin C supplementation, followed by colony-forming unit enumeration. Vitamin C supplementation altered oxidative readouts and was associated with greater reductions in viable bacteria compared with conventional Fenton chemistry. Rescue experiments using deferoxamine and thiourea attenuated antibacterial activity, supporting the hypothesis that antibacterial activity is associated with iron-mediated and radical-related oxidative processes. Live/dead fluorescence staining and scanning electron microscopy provided qualitative visual observations consistent with the colony-forming unit (CFU) enumeration results. Collectively, these findings indicate that controlled vitamin C dosing can modulate Fenton-based oxidative reactions and enhance antibacterial activity against H. pylori in a simplified, acidic in vitro model.