Repurposed darifenacin and betamethasone potentiate streptomycin against resistant Staphylococcus aureus in vitro

A laboratory study in multidrug-resistant Staphylococcus aureus found that a formulation containing repurposed darifenacin and betamethasone increased the activity of streptomycin without showing a marked antibacterial effect on its own. The combination reduced the fitted streptomycin half-maximal inhibitory concentration by 2.2-fold in the selected CS9 isolate and was associated with increased oxidative stress and reduced RecA-associated ATPase activity. These findings concern in vitro bacterial assays and do not establish clinical efficacy in people.

The researchers used an integrated strategy combining virtual screening, formulation modeling, biochemical assays and in vitro antibacterial testing. They docked 1,500 FDA-approved compounds against an AlphaFold-predicted model of the S. aureus RecA protein using AutoDock Vina. Twelve compounds were shortlisted for inspection, and darifenacin, digoxin and betamethasone were selected for experimental testing as potential antibiotic adjuvants.

The bacterial panel included four S. aureus isolates from food products—CS4, CS8, CS9 and ZS—and the antibiotic-susceptible quality-control strain ATCC 6538. Streptomycin susceptibility was assessed with broth microdilution and an MTT viability assay rather than a conventional MIC endpoint. Four-parameter logistic models were fitted to characterize each strain’s response. CS9 was selected for subsequent experiments because it did not reach 90% inhibition at the highest tested streptomycin concentration and showed a shallow dose-response curve.

When tested individually at concentrations of 0.125, 0.0625 and 0.03125 mg/mL, betamethasone, darifenacin hydrobromide and digoxin did not show a clear concentration-dependent antibacterial effect against CS9. The researchers then used an augmented simplex-centroid mixture design to model combinations of the three compounds. Response-surface optimization generated five formulations for experimental validation. F1 was excluded after a large mismatch between predicted and observed viability, while F2, F3, F4 and F5 were retained for combination assays.

F5, containing betamethasone and darifenacin hydrobromide without digoxin, was identified as the strongest adjuvant candidate. At an antibiotic concentration of 1.5 times the CS9 IC50, F5 plus streptomycin produced significantly greater inhibition than streptomycin alone, with an adjusted p value of about 0.001. F5 alone did not differ significantly from the untreated control, supporting adjuvant activity rather than substantial direct antibacterial activity. In dose-response testing, the combination performed better than streptomycin alone at most tested doses; the exception was four times the IC50, where streptomycin was already near maximal inhibition.

Four-parameter logistic regression estimated an IC50 of approximately 25.04 for streptomycin alone and 11.38 for streptomycin combined with F5, a 2.2-fold reduction. The source text does not state the concentration unit for these fitted IC50 values. Growth monitoring over 18 hours showed stronger intermediate suppression with the combination than with antibiotic alone, although relative viability increased later in all groups, consistent with partial regrowth rather than complete inhibition.

Colony enumeration supported the optical-density findings. At six hours, the combination produced 4.65 log10 CFU/mL, compared with 5.06 log10 CFU/mL for streptomycin alone, a statistically significant reduction of 0.41 log10 in viable bacterial counts. Plates from untreated and formulation-only groups were classified as too numerous to count, so the quantitative CFU comparison was limited to the antibiotic and combination groups.

Mechanistic assays found the strongest oxidative-stress response under combination treatment. Hydrogen peroxide levels differed across conditions, with p = 0.0017 and the highest levels in the combination group. Activities of superoxide dismutase, catalase, thioredoxin reductase and isocitrate dehydrogenase were also highest with the combination. These measurements were associated with a stronger oxidative and metabolic stress response than streptomycin alone. In an indirect NADH-coupled assay of single-stranded-DNA-dependent ATPase activity in crude extracts, the formulation produced a near-zero or negative ssDNA-dependent signal, while the combination yielded 0.209 ± 0.076 ΔA340·min−1·mg−1, compared with 0.865 ± 0.104 for the untreated control and 0.358 ± 0.044 for antibiotic alone.

The study did not directly isolate RecA-specific inhibition: crude extracts may contain other ssDNA-responsive ATPases, and compound-dependent interference can affect coupled assays. Computational results also remained predictions. Docking scores were −8.1 kcal/mol for darifenacin and −7.3 kcal/mol for betamethasone, while 100-nanosecond molecular dynamics simulations and MM/PBSA calculations estimated favorable binding for both complexes, with total binding energies of approximately −15 kcal/mol for darifenacin and −10 kcal/mol for betamethasone. The work was conducted entirely in vitro; host-cell compatibility and human treatment effects were not evaluated, and the results therefore support further investigation of the formulation as a stress-response-targeted antibiotic adjuvant rather than a demonstrated clinical therapy.

Subject of Research: In vitro testing of repurposed darifenacin and betamethasone as antibiotic adjuvants against multidrug-resistant Staphylococcus aureus

Article Title: Drug repurposing enhances antibiotic activity in multidrug-resistant staphylococcus aureus through modulation of oxidative stress homeostasis

Article References: Chagra, Z., Ouedrhiri, W., Loukman, S., El Asri, S., El Fatimy, R., & Ben Mrid, R. (2026). Drug repurposing enhances antibiotic activity in multidrug-resistant staphylococcus aureus through modulation of oxidative stress homeostasis. Frontiers in Pharmacology, 17, Article 1873160. https://doi.org/10.3389/fphar.2026.1873160

Image Credits: AI Generated

DOI: 10.3389/fphar.2026.1873160

Keywords: Staphylococcus aureus, antibiotic resistance, drug repurposing, darifenacin, betamethasone, streptomycin, RecA, oxidative stress, antibiotic adjuvants, Drug, repurposing, enhances