The type strains S. aureus ATCC 25923 (MSSA) and S. aureus ATCC 43300 (MRSA) were purchased from the American Type Culture Collection (ATCC). Clinical MRSA isolates, including strain SA-11, were obtained from the Third Xiangya Hospital of Central South University. All clinical strains were identified using the VITEK 2 Compact system (bioMérieux, France) and confirmed by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry (BD, Germany). Bacterial cultures were grown in Tryptic Soy Broth (TSB) (Solarbio, Beijing, China). Nifuratel, vancomycin (VAN), tetracycline (TCY), ciprofloxacin (CIP), and other antimicrobial agents were purchased from MedChem Express (New Jersey, USA) and dissolved in either deionized water or dimethyl sulfoxide (DMSO) as stock solutions for subsequent experiments.

The MICs and MBCs of S. aureus were tested in cation-adjusted Mueller-Hinton (MH) broth. Briefly, 50 μL of 2-fold serially diluted Nifuratel were added into a 96-well plate. Then, 50 μL of the bacterial suspensions were added to the plate to yield 5×10⁵ CFU/mL final inoculum. After incubated at 37°C for 24h, MICs were defined as the lowest concentration showing complete growth inhibition. Further, Minimum Bactericidal Concentration (MBC) is determined by subculturing broth from MIC wells showing no visible growth onto sheep blood agar plates after overnight incubation (CLSI, 2024).

S. aureus was adjusted to 0.5 McFarland (McF) standard in saline, and spread evenly onto MH agar plates by using a moist swab. Then, sterile empty disks in the presence or absence of Nifuratel were aseptically placed on the agar surface. The plates were incubated at 37°C for 16–18 h, and the inhibition zones were measured with a caliper (She et al., 2022).

The assay was conducted following the aforementioned micro-broth dilution method, with minor modifications. Briefly, Nifuratel was serially diluted in TSB or MH broth, and equal volumes of log-phased S. aureus cultures were added to a 96-well plate to the final density of ~5 × 10⁵ CFU/mL (100 µL/well). After 16h incubation at 37°C, bacterial growth was measured at 630 nm using a microplate reader.

Log-phased S. aureus was diluted and inoculated at ~1 × 10⁶ CFU/mL in TSB medium in the presence or absence of antimicrobial agents at indicated concentrations. The bacterial suspension was incubated at 37°C 180 rpm. Then, aliquots of the bacterial suspension were serially diluted in saline and plated on sheep blood agar at intervals of 0, 2, 4, 8, 12, and 24h, respectively. After incubation at 37°C for 24h, viable colonies were counted to calculate log₁₀ CFU/mL reductions (Visca et al., 2019).

Bacterial viability was assessed using dual-fluorescence staining with the LIVE/DEAD^® BacLight™ Bacterial Viability Kit (L7012, Thermo Fisher Scientific). Briefly, bacterial suspensions (∼1×10⁶ CFU/mL) or biofilms were treated with a premixed dye cocktail, including SYTO9 and PI, according to the manufacturer’s protocol. After incubated in the dark for 15 min at room temperature, the samples were washed in 1×phosphate-buffered saline (PBS) and immediately visualized under confocal laser scanning microscopy (CLSM) (LSM800, ZESS, Germany) with dual-channel detection.

Bacterial cultures were grown overnight in TSB supplemented with 0.5% glucose (TSB-G). The cultures were then diluted 1:100 in fresh medium, and aliquoted (100 μL/well) into a sterile 96-well plate. Then, equal volume of 2-fold diluted Nifuratel were added into each well. After 24 h of static incubation at 37°C, planktonic cells were removed by gentle washing with PBS. For crystal violet staining, biofilms were added with 0.15% crystal violet for 10 min, washed, and solubilized in 95% ethanol. Absorbance was measured at 570 nm to quantify total biofilm biomass (Xu et al., 2016). For the CFU counting assay, the supernatant in each well was serially diluted 10-fold with 1× PBS and spotted onto sheep blood agar. Then, the supernatant was removed, and the adherent biofilms were gently washed with 1× PBS. Subsequently, 150 µL of PBS was added to each well, and the biofilms were thoroughly dispersed and homogenized using pipette tips. After appropriate dilution, the suspensions were plated onto sheep blood agar. All the agars were incubated overnight at 37°C, after which CFUs were enumerated.

Bacterial cultures were grown overnight in TSB supplemented with 0.5% glucose (TSB-G). The cultures were then diluted 1:100 in fresh medium, and aliquoted (200 μL/well) into a sterile 96-well plate. After 24 h of static incubation at 37°C, planktonic cells were removed by gentle washing with PBS, and the remined biofilms were treated with 2-fold diluted Nifuratel. After incubation for another 24h, the biofilms were washed again and quantified by crystal violet staining (Nair et al., 2016; Xu et al., 2016) and CFU counting assay, respectively, as described above.

TSB plates containing 0.24% (wt/vol) agar were prepared uniformly in the presence or absence of Nifuratel. Then, the plates were dried for 30 min at room temperature, and equilibrated at 37°C for 1h. S. aureus overnight cultures were adjusted to OD₆₃₀ = 0.5, and 2 µL droplets were inoculated centrally onto the agar surface. Plates were incubated statically at 37°C for 24 h and the spreading zone was recorded with a camera (Wu et al., 2024).

Overnight cultured S. aureus was collected by centrifugation at 16,000 × g for 2 min, and re-suspended in 3 mL 1× PBS, in the presence or absence of Nifuratel. The suspension was incubated statically at 37°C for 24h and the turbidity was monitored by measuring the OD630nm (Yu et al., 2021).

Log-phased S. aureus cultures were diluted and the initial MIC was determined by broth microdilution assay as described above. After incubation at 37°C for 24 h, the MIC was recorded, and the bacterial suspension in the 1/2× MIC wells were 1:1000 diluted with fresh MH broth subsequent passages. The MIC was detected daily for a consecutive 24 passages. CIP was used as a positive control (Song et al., 2021).

Prepare agar (MH broth, 17 g/L agarose) with indicated concentrations of Nifuratel or CIP (positive control). Then, 100 μL of S. aureus suspension with 10⁸ CFU/mL of bacterial cells was inoculated on the surface of the agar. After incubated at 37°C for 48 h, the CFUs on the plates were counted (Smith et al., 2018).

Specific pathogen-free outbred female ICR mice, aged six to seven weeks, were anesthetized using 1% sodium pentobarbital at a dosage of 50 mg/kg. Their back hair was removed by shaving. Fifty microliters S. aureus ATCC 43300 bacterial suspension containing 1× 10⁸ CFU/mL was administered by subcutaneous injection to establish infection. The mice were randomly assigned to two groups with six animals each: one group received a dose of DMSO as vehicle control while the other was treated with 30 mg/kg Nifuratel. Both groups received their respective subcutaneous treatments 1h after infection. Twenty-four hours post-infection, the mice were humanely euthanized and the resulting abscesses were surgically excised. The collected abscesses were then either homogenized for quantifying bacterial load or fixed in 4% paraformaldehyde solution for subsequent histological and immunohistochemical analysis (Pletzer et al., 2018).

Female ICR mice aged 6–8 weeks were anesthetized intraperitoneally with 1% sodium pentobarbital, after which the dorsal skin was shaved and disinfected with 75% ethanol. A full-thickness excisional wound 6 mm in diameter was created, penetrating both the epidermis and dermis. The wound was then inoculated 50 μL of MRSA ATCC 43300 at the concentration of 1×108 CFU/mL. After 1h post infection, topical treatment was administered by applying 2% (wt/vol) of the test compound directly to the wound site once every 24 h for a total of 7 days. At the experimental endpoint, wound tissues were excised for analysis: bacterial burden was quantified by homogenizing tissue in PBS and performing serial dilution plating for CFU counts. Histopathological evaluation was conducted on 4% paraformaldehyde-fixed, paraffin-embedded sections stained with H&E staining (Rehberg et al., 2020).

Mice were randomly assigned to two groups (n=6 per group) and received an intraperitoneal injection of either the vehicle control (5% Cremophor EL combined with 5% ethanol) or 30 mg/kg Nifuratel. At 24h post-injection, blood was collected for the quantification of hematological parameters and organic biomarkers. Concurrently, major organs, including the heart, liver, spleen, lungs, and kidneys, were harvested and fixed in 4% paraformaldehyde solution for subsequent histological examination by H&E staining (Wu et al., 2023).

All experiments were performed with three independent replicates. Statistical analyses were carried out using GraphPad Prism 9.0 software, employing the Student’s t-test for comparisons between two groups and one-way ANOVA for comparisons among multiple groups. A P-value of less than 0.05 was considered statistically significant.

Additional details regarding the materials and methods are described in the Supplementary Information.

Nifuratel, 5-[(Methylthio)methyl]-3-[[(5-nitro-2-furyl)methylene]amino]-2-oxazolidinone (Figure 1A), exhibited effective bactericidal activity against S. aureus. The MIC and MBC values against MSSA and MRSA type strains and clinical isolates were consistently 2-8 μg/mL and 8–16 μg/mL, respectively (Figures 1B, C). Notably, Nifuratel also demonstrated moderate efficacy against E. faecalis ATCC 29212 with MIC of 16 μg/mL. In K-B disk diffusion assay, Nifuratel showed concentration-dependent zones of inhibition against MRSA ATCC 43300 (Figures 1D, E) as well as other type strain and clinical isolate (Supplementary Figures S1A, 1B). And enhanced antimicrobial susceptibility of Nifuratel at sub-MICs was observed in MH broth when compared with TSB (Figure 1F), which suggested cation composition in broth may influences the antimicrobial efficacy by Nifuratel. By time-killing assay, Nifuratel exhibited concentration- and time-dependent bactericidal activity against S. aureus by both type strains and clinical isolate (Figure 1G and Supplementary Figure S2). For example, Nifuratel treatment led to CFU reduction at the concentration of 1–2× MIC against ATCC 43300 with no detectable colonies at the time point of 24h (Figure 1G). In consistence, SYTO9/PI viability staining visualized bacterial damage with enhanced PI uptake (red fluorescence) after 1h exposure to 1× MIC of Nifuratel (Figure 1H).

Nifuratel effectively inhibited S. aureus biofilm formation and virulence factor production at sub-MICs. Crystal violet staining demonstrated that 4 μg/ml of Nifuratel significantly inhibited biofilm formation (Figure 2A). In accordance, Nifuratel reduced the number of viable cells in both the supernatant and the adherent biofilms (Figure 2B). Although crystal violet staining indicated that Nifuratel was ineffective against preformed biofilms (Figure 2C), it significantly decreased the CFUs in both the supernatant and biofilm-associated cells at concentrations equal to or greater than 8 μg/mL (Figure 2D). These results suggest that the antibiofilm effects of Nifuratel are likely attributable to growth inhibition rather than suppression of extracellular matrix production. Consistently, fluorescence imaging using SYTO9/PI revealed that Nifuratel inhibited the biofilm development with decreased overall fluorescence intensity (Figure 2E). Despite unchanged total biomass, the mature biofilms treated with Nifuratel exhibited increased PI signals indicating enhanced cells damage in biofilms (Figure 2F). Furthermore, sub-MICs of Nifuratel inhibited S. aureus key virulence phenotypes including hemolytic activity (Figures 2G, H), agar surface diffusion capacity (Figure 2H), and Auto-aggregation (Figure 2I). These results collectively demonstrate the effectively antimicrobial activity of Nifuratel against S. aureus and its virulence factors.

Serial passage experiments revealed obvious differences in resistance development between Nifuratel and CIP. After 23 passages under sub-MIC, CIP induced a 32-fold increase in MIC against S. aureus ATCC 43300 whereas Nifuratel caused only a 2-fold MIC elevation (Figure 3A). The low resistance development probability by Nifuratel was also observed by ATCC 25923 and SA-11 (Supplementary Figure S4). Notably, the Nifuratel-exposed ATCC 43300 from the final passage exhibited reduced Staphyloxanthin production (Figure 3B), indicating suppression of virulence factor expression. After five passages in drug-free medium, the MIC values remained unchanged (Figure 3C), suggesting that Nifuratel/CIP-induced adaptations may involve genetic mutations rather than phenotypic changes. Furthermore, in single-step resistance selection assay, CIP generated resistant mutants at the concentration of 2–4× MIC, whereas Nifuratel produced no detectable resistant colonies (Figure 3D).

The postantibiotic effect (PAE) describes the delayed regrowth of bacteria following short-term antibiotic exposure, mainly caused by persistent cellular damage, such as disruptions in protein synthesis or DNA replication, that takes time to repair (Spangler et al., 1997). In comparison, the postantibiotic sub-MIC effect (PA-SME) prolongs this suppression by introducing sub-inhibitory antibiotic levels after the initial exposure. This further impedes bacterial recovery by continuously disrupting metabolic processes. While both effects enable longer dosing intervals, PA-SME specifically emphasizes how sustained sub-inhibitory drug concentrations can lead to more prolonged growth inhibition than PAE alone (Pankuch and Appelbaum, 2009). In our study, the PAE of Nifuratel was similar as CIP (Figure 3E), while its PA-SME surpassed CIP within 8h (Figure 3F). This indicates that the cellular damage inflicted by Nifuratel is comparable to that of a fluoroquinolone, but its unique property lies in its enhanced ability to suppress bacterial regrowth at sub-inhibitory concentrations. These data establish Nifuratel as an antimicrobial agent with negligible resistance development capacity and prolonged suppressor effects on bacterial regrowth.

As shown in Figure 4A, TEM revealed that untreated bacteria exhibited intact ultrastructures, whereas those treated with Nifuratel displayed blurred membrane contours, abnormal inward invaginations of the cytoplasmic membrane, and reduced density of both cell wall and membrane, which suggested possible “edema-like” changes in the periplasmic region. To further investigate the underlying mechanism, we observed that Nifuratel showed concentration-dependent reduction by DiSC3(5) fluorescence intensity (Figure 4B), indicating disruption of transmembrane potential-a main component of PMF. Similarly, using the BCECF-AM probe, we found that Nifuratel increased its fluorescence intensity similarly to the positive control glucose, demonstrating interference with the ΔpH, the other key component of PMF (Figure 4C). The formation of PMF relies on the dynamic compensation between ΔΨ and ΔpH. By exposing bacteria to varying pH conditions, the relative contribution of ΔΨ and ΔpH to the total PMF shifts, allowing us to infer the mechanism of action of Nifuratel based on changes in antibacterial efficacy. As shown in Figure 4D, the growth ability in culture medium was slightly influenced in the presence of varied pH, while obvious growth turbidity was still observed at control groups. As we expected, the antibacterial activity (including the MIC values) of Nifuratel was also notably influenced by external pH alteration (Figure 4D). This assay further supporting the PMF-dependent mechanism. Moreover, Nifuratel exhibited a partial synergy (FICI = 0.75) when combined with tetracycline (TCY) (Figure 4E) and doxycycline (DOX) (Supplementary Figure S3) against S. aureus, which was similar with the previous reports for known PMF inhibitors (Ejim et al., 2011). This partial synergism was also corroborated by time-kill curves using sub-MICs of Nifuratel and TCY (Figure 4F). Moreover, partial synergistic combinations were also observed between Nifuratel and some conventional antibiotics like daptomycin, ampicillin, and oxacillin, etc (Supplementary Figure S3). These effects may arise from mechanisms such as enhanced membrane permeability by the membrane-disrupting agents like daptomycin (Peñalba Arias et al., 2015). Additionally, β-lactam antibiotics can inhibit cell wall synthesis, compromising bacterial integrity and potentiating the activity of other drugs by improving access to targets (Dilworth et al., 2014). Collectively, these findings demonstrate that Nifuratel targeted PMF with boths bacterial transmembrane potential and ΔpH.

MD simulations revealed distinct binding behaviors of Nifuratel toward two types of mixed lipid membranes. As shown in Figures 4G, Nifuratel stably bound to and inserted into the DOPC: DOPG bilayer during the simulations, while it rapidly dissociated from the DOPC: Cholesterol membrane after 105 ns. Quantitative analysis indicated that the contact surface area (CSA) (Supplementary Figure S5A) and number of atomic contacts (Supplementary Figure S5C) increased rapidly after 25 ns and stabilized around 3.5 nm² and 250, respectively, for the DOPC: DOPG system. Although a brief dissociation occurred between 350–400 ns, Nifuratel spontaneously re-embedded into the membrane. In contrast, interaction with the DOPC: Cholesterol membrane was transient and unstable (Supplementary Figure S5B, C). As shown in Figures 4H–K, Van der Waals (Figure 4H) and electrostatic interactions (Figure 4I) were the main driving forces, with hydrogen bonding (Figure 4J) playing a minor role. Distance analysis corroborated deep and stable embedding of Nifuratel into the DOPC: DOPG bilayer, unlike its temporary binding to the cholesterol-containing membrane (Supplementary Figure S6).

By metabolomic analysis, Nifuratel treatment could induce extensive significant alterations in the metabolite profile of S. aureus (Figure 4L and Supplementary Table S1). Key differentially abundant metabolites are summarized in Figure 4M. Upregulated metabolites included 5-Hydroxylysine, Hydroxymuconate semialdehyde, N-Acetyl-Cadaverine, et al, while downregulated metabolites consisted of Cyclohexane-1-carboxylic acid, Deoxyguanosine, Acetyl-Glycine, et al. The pronounced accumulation of polyamine derivatives (e.g., N-Acetyl-Cadaverine) coupled with a sharp decline in nucleotide pools (e.g., Adenosine and Deoxyadenosine) points to a profound disruption of cellular energy and biosynthetic homeostasis. These coordinated metabolic shifts are consistent with the collapse of the proton motive force (PMF), which critically governs bacterial energetics and transmembrane transport (Yang et al., 2023). Notably, the concurrent pattern of nucleotide depletion alongside polyamine buildup delineates a metabolic stress signature that may represent a previously uncharacterized bacterial adaptive response to PMF impairment.

To evaluate the antibacterial efficacy of Nifuratel in vivo, we employed both abscess and wound infection models. In the abscess model, Nifuratel treatment significantly reduced the viable bacterial load compared to the vehicle group (Figure 5A), which was in accordance with the representative images of the abscesses (Figure 5B). Histological examination via H&E staining revealed a substantial reduction in both abscess size and total inflammatory infiltration in Nifuratel-treated mice (Figure 5C). Additionally, immunohistochemical analysis showed markedly reduced overall expression of cytokines IL-1β, IL-6, and TNF-α after treated with Nifuratel (Figure 5C). Similarly, in the wound infection model, a 7-day period observation demonstrated obvious wound closure in the groups treated with Nifuratel or fusidic acid (positive control), in contrast to the vehicle group (Figures 5D, E). Quantification of viable bacterial cells in wounds on day 1, 3, 5 and 7 indicated that Nifuratel exhibited time-dependent antibacterial activity, reaching efficacy comparable to fusidic acid by day 7 (Figure 5F). Meanwhile, H&E staining of the wounds displayed obvious reduced inflammatory cell infiltration in both Nifuratel and fusidic acid treated groups relative to the Vehicle group (Figure 5G). Together, these findings demonstrate that Nifuratel robustly diminishes bacterial loads and alleviates infection-associated inflammation in vivo, highlighting its therapeutic potential for treating S. aureus skin and soft tissue infections.

To assess the biosafety of Nifuratel, both in vitro and in vivo toxicity studies were conducted. As shown in Figure 6A, Nifuratel exhibited extremely low hemolytic activity even at concentrations up to 32 μg/mL. Nifuratel also demonstrated low cytotoxicity toward human keratinocyte (HaCaT) and human skin fibroblast (HSF) cell lines with the CC₅₀ values of 27.22 μg/mL and > 32 μg/mL, respectively (Figure 6B). For in vivo toxicity evaluation, mice treated intraperitoneally with 30 mg/kg Nifuratel showed no remarkable changes in routine blood parameters (Figure 6C) or specific biomarkers of liver (Figure 6D) and kidney function (Figure 6E). Consistent with these observations, histopathological examination via H&E staining revealed no apparent pathological alterations in major organs (Figure 6F). Collectively, these results indicate that Nifuratel, repurposed as an antimicrobial agent, possesses a high safety profile in vivo, supporting its potential for therapeutic application.