Hydrogen sulfide demonstrated potent antimicrobial activity against fungi and bacteria that cause nail infections through a novel mechanism involving cytochrome C oxidase inhibition, reactive oxygen species generation, and protein S-sulfhydration, according to research published in Scientific Reports.
Using sodium hydrogen sulfide (NaHS) as an H₂S donor, researchers found the gaseous molecule exhibited strong activity against dermatophytes, with minimum inhibitory concentration values of 0.082 to 0.16 µg/mL. These concentrations are 50- to 2,000-fold more potent than aqueous forms depending on pH, lead author Fritz Ka-Ho Ho, PhD, of the Department of Life Sciences, University of Bath, reported with colleagues. The study demonstrated that hydrogen sulfide (H₂S) has potent activity against causative agents of nail infections, including fungi and bacteria. The most active form appeared to be H₂S rather than the anion HS⁻, though the researchers noted that this was most likely related to faster cellular uptake of H₂S.
Mechanism of Action
The investigators showed that H₂S inhibits cytochrome C oxidase (COX), a key respiratory enzyme, increases reactive oxygen species and induces protein S-sulfhydration. In crude mitochondrial extracts from T rubrum, H₂S inhibited COX activity to 42% at 3.4 µg/mL and 15% at 17 µg/mL.
Transcriptomic analysis revealed a stress response: 96 genes were upregulated and 117 were downregulated, which indicated efforts to reduce oxidative stress. The researchers identified differential expression in genes encoding membrane transport proteins, ribosomal components, and oxidative stress regulators. Notably, a putative Cu/Zn superoxide dismutase gene showed threefold upregulation, while catalase genes showed no statistically significant expression changes. The researchers noted that "SODs are required for resistance to oxygen radicals, but a recent study showed that a Cu/Zn SOD (denoted SOD1) also functions as an H₂S oxidase, protecting against H₂S toxicity."
The authors concluded that "COX inhibition likely causes electron leakage, generating ROS and oxidising cysteine residues, which then react with H₂S to form S-sulfhydrated proteins." Using 5'-iodoacetamide fluorescein labeling, the investigators observed a 70% reduction in free thiol groups following H₂S treatment, which confirmed extensive protein modification.
Spectrum of Activity
The researchers tested H₂S against multiple pathogens that cause onychomycosis. Dermatophytes (Trichophyton and Microsporum spp) appeared more sensitive to H₂S than most nondermatophytes, with minimum inhibitory concentration values of 0.082 to 0.16 µg/mL vs 0.32 to 1.6 µg/mL, respectively. Activity extended to terbinafine-resistant T indotineae isolates.
Among bacterial pathogens, 2 S aureus strains, including multidrug-resistant MRSA252, were sensitive to H₂S with minimum inhibitory concentration values of 0.16 µg/mL, while P aeruginosa was not inhibited at the highest concentration tested (greater than 8.2 µg/mL). E. coli strain BW25113 was highly resistant, but the recA- strain DH5α showed partial sensitivity with a minimum inhibitory concentration value of 3.2 µg/mL, the researchers wrote.
pH and Formulation Dependencies
At pH 5, T rubrum showed 20-fold lower aqueous minimum inhibitory concentration than at pH 7 or 8, suggesting that H₂S has higher antifungal activity than HS⁻. The equilibrium between H₂S and HS⁻ determines the predominant species at different pH values: approximately 100:1 at pH 5, 1:1 at pH 7, and 1:10 at pH 8.
Gas release studies showed that at 0.1 to 1 mM NaHS concentrations, 16% to 18% of total available sulfide was released as H₂S within 3 hours, compared with only 2% with 10 mM NaHS. The researchers found that 3 to 6 hours of H₂S exposure was sufficient to reach the minimum inhibitory concentration value.
Developmental Stage Sensitivity
Germination of T rubrum spores proved more sensitive to H₂S because the minimum inhibitory concentration increased when applied during hyphal formation. Preincubation of conidia for 9 to 12 hours before H₂S exposure increased the minimum inhibitory concentration from 0.082 to 0.32 µg/mL and the minimum fungicidal concentration from 0.082 to 1.6 µg/mL.
Confocal microscopy and flow cytometry demonstrated that sublethal H₂S concentrations inhibited mycelium formation and caused conidial clumping with membrane damage. Treatment with reactive oxygen species scavengers N-acetylcysteine or glutathione increased the minimum inhibitory concentration eightfold and confirmed oxidative stress involvement.
Clinical Implications
The study positioned H₂S as promising for treating onychomycosis due to good nail penetration and antimicrobial properties. Current topical treatments achieve complete cure rates of only 15% to 18%, with mycological cure rates of 53% to 55% for efinaconazole, one of the most effective agents.
"This innovative mechanism of action," the authors wrote, "combined with the ability of H₂S to penetrate the nail plate, suggest that topically delivered H₂S—via donors such as NaHS—is a promising and innovative therapeutic approach." They noted that H₂S, with molecular weight 34 g/mol penetrates nails more efficiently than topical antifungals such as amorolfine or ciclopirox.
Regarding safety, the researchers noted, occupational exposure limits in the UK and the EU are 5 ppm for 8-hour time-weighted average and 10 ppm for 15-minute periods. They calculated that applying 50 mg of 10% H₂S donor gel would generate 0.35 ppm in a small unventilated 10 m³ room, which is well below safety thresholds.
The study was led by Fritz Ka-Ho Ho, PhD, of the Department of Life Sciences, University of Bath, and colleagues. The research received funding from the Engineering and Physical Sciences Research Council.through grants EP/V009621/1 and EP/V009567/1. The authors declared no competing interests.
Source: Scientific Reports
