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

Summary

This study examines how vitamin C modulates the kinetic behavior of a Fenton reaction under in vitro conditions. Chemical-dye probe and bacterial assays show that controlled vitamin C dosing alters the reaction's oxidative behavior and is associated with increased antibacterial activity against Helicobacter pylori in a simplified model system.

Abstract

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.

Introduction

H. pylori, a Gram-negative bacterium with a spiral shape that belongs to the genus Helicobacter, has been shown through population health studies to have a high prevalence of infection across geographical areas, sex, and age, with a global prevalence rate of approximately 50%¹. The International Agency for Research on Cancer (IARC) has classified H. pylori as a Group 1 carcinogen, underscoring its strong connection with gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma. At present, the clinical management of H. pylori infection primarily relies on pharmacological regimens. The most extensively studied a....

Protocol

Ethics statement
This study was an in vitro bacterial study and did not involve human participants, animal subjects, or patient-derived materials. Therefore, institutional review board approval and animal ethics approval were not required.

Evaluation of AO7 degradation in the vitamin C–assisted Fenton system
To assess the effect of vitamin C on the observable oxidative behavior of the Fenton reaction, Acid Orange 7 (AO7) degradation was used as an indirect chemical readout of oxidative activity. This assay does not specifically quantify individual reactive oxygen species. Prepared....

Results

Evaluation of AO7 degradation in the vitamin C–assisted Fenton system
AO7 degradation was used as a chemical probe to examine how vitamin C influences the observable oxidative behavior of the Fenton reaction under acidic conditions. In the absence of vitamin C, the conventional Fenton system resulted in partial AO7 degradation over the 10 min reaction period. When vitamin C was introduced, the extent of dye degradation increased, with the effect depending on the molar ratio between vitamin C and Fe2⁺.......

Discussion

The present study examined whether vitamin C dosing could modulate the oxidative behavior and antibacterial readouts of a Fenton-based system under defined acidic in vitro conditions. Across the chemical and microbiological assays, stepwise vitamin C supplementation altered the observable reaction behavior and was associated with greater antibacterial activity against H. pylori than the conventional Fenton reaction under the tested conditions. Importantly, this system should be interpreted as a simplifi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acid Orange 7 (AO7)	Aladdin Reagent (Shanghai) Co., Ltd.	O113230	Used as a model dye for the AO7 degradation assay.
Bovine serum albumin (BSA)	Shanghai Macklin Biochemical Technology Co., Ltd.	B885114	Used for the matrix-interference assay.
Brain Heart Infusion (BHI) broth powder	Sichuan Hapyear Bio-engineering Co., Ltd.	N/A	Used for bacterial culture and cryostorage.
Columbia blood agar plates (5% sheep blood)	Changde Bkmam Biotechnology Co., Ltd.	110701028.0	Used for bacterial culture and CFU enumeration.
Critical point dryer (EM CPD300)	Leica Microsystems	EM CPD300	Used for SEM sample preparation after graded ethanol dehydration.
Deferoxamine (DFO)	Shanghai Macklin Biochemical Technology Co., Ltd.	D873692	Used as an iron chelator in the rescue assay.
Disodium terephthalate	Shanghai Macklin Biochemical Technology Co., Ltd.	D835937	Used as the TA fluorescence probe for •OH-associated signal detection.
Ethanol, absolute	Sigma-Aldrich	E7023	Used to prepare the graded ethanol series for SEM sample dehydration.
Fluorescence microscope (DMi8)	Leica Microsystems	DMi8	Used for live/dead fluorescence imaging.
Glutaraldehyde solution	Sigma-Aldrich	G5882	Used for fixation of bacterial samples before SEM.
Glycerol	Sigma-Aldrich	G5516	Used for bacterial cryostorage in BHI broth.
GraphPad Prism 10	GraphPad Software, LLC	Prism 10	Used for statistical analysis and graphing.
Helicobacter pylori strain ATCC 26695	American Type Culture Collection (ATCC)	700392	Additional reference strain used for limited cross-strain validation.
Helicobacter pylori strain ATCC 43504	American Type Culture Collection (ATCC)	43504	Additional reference strain used for limited cross-strain validation.
Helicobacter pylori strain G27	Center of Infectious Diseases, West China Hospital, Sichuan University	N/A	Primary strain used for colony counting, rescue assay, matrix-interference assay, live/dead staining, and SEM.
Hydrogen peroxide solution	Sigma-Aldrich	H1009	Used as the peroxide substrate in Fenton reaction assays.
Iron(II) sulfate monohydrate (FeSO4·H2O)	Shanghai Macklin Biochemical Technology Co., Ltd.	F904376	Used as the ferrous ion source in the Fenton reaction.
Microplate reader (Synergy H1)	Agilent BioTek	Synergy H1	Used to measure TA-derived fluorescence (Ex/Em = 315/425 nm).
Phosphate-buffered saline (PBS), pH 7.4	Sigma-Aldrich	P4417	Used for post-treatment dilution/neutralization before plating and sample washing.
Physiological saline (0.9% sodium chloride solution)	Millipore	567442	Used for bacterial suspension and preparation of the acidic saline-urea model.
Scanning electron microscope (INSPECT F)	FEI Company	INSPECT F	Used for bacterial morphology analysis.
Sodium hydroxide	Sigma-Aldrich	S5881	Used to alkalinize TA assay aliquots and stabilize fluorescence.
Sputter coater (EM ACE200)	Leica Microsystems	EM ACE200	Used to apply the thin gold coating before SEM imaging.
SYTO 9/PI Live/Dead Bacterial Double Stain Kit	Shanghai Maokang Biotechnology Co., Ltd.	MX4234	Used for fluorescence-based live/dead bacterial staining.
Thiourea	Shanghai Macklin Biochemical Technology Co., Ltd.	T81602	Used as a radical scavenger in the rescue assay.
Urea	Shanghai Macklin Biochemical Technology Co., Ltd.	U820349	Used to prepare the acidic saline-urea model.
UV-Vis spectrophotometer (UV-2600i Plus)	Shimadzu Corporation	UV-2600i Plus	Used to measure AO7 absorbance at 483 nm.
Vitamin C (L-ascorbic acid)	Shanghai Macklin Biochemical Technology Co., Ltd.	A800295	Used as the reductant in the vitamin C-assisted Fenton system.