African green monkey kidney cells (Vero) were purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% heat-inactivated FBS (Invitrogen), 1% penicillin–streptomycin and 1% sodium pyruvate (Sigma-Aldrich) at 37°C in a 5% CO₂ atmosphere. Oxethazaine (CAS no. 126-27-2) and colistin sulfate salt (CAS no. 1264-72-8) were purchased from GlpBio and Sigma-Aldrich, respectively.

The standard strains for this study were purchased from the China General Microbiological Culture Collection Center (CGMCC), Escherichia coli SHP45 and other clinical strains were isolated and preserved by our laboratory. MG1655-ΔwaaC was previously constructed in our laboratory (Li et al., 2023).

The clinical and laboratory standards institute (CLSI 2021) guideline was used to determine the minimum inhibitory concentration (MIC) of oxethazaine and other antibiotics (CLSI, 2021). The combined effect of oxethazaine with other antibiotics was determined using the checkerboard method. Briefly, the single colonies were cultured in Mueller–Hinton broth (Qingdao Haibo Biotechnology Co., Ltd.) and then diluted to 10⁶ CFU/mL. Antibiotics were serial diluted 2-fold in Mueller–Hinton broth, and an equal volume of bacterial suspension (10⁶ CFU/mL) was added in a 96-well microtiter. The MIC values were defined as the lowest antibiotic concentrations with no visible bacterial growth after incubation at 37°C for 18 h. The FICI value is calculated from the MIC value of the drug alone. The specific calculation method of the FICI is as follows: FICI = FICI a + FICI b = MIC ab MIC a + MIC ba MIC b

MIC_a is the MIC of A alone, MIC_ab is the MIC of compound A in combination with compound B, MIC_b is the MIC of compound B alone, and MIC_ba is the MIC of compound B in combination with compound A. FICI ≤0.5 was deemed as synergistic (Song et al., 2020).

Sterile sheep red blood cells (RBC) were used to evaluate hemolytic activity as previously described with slight modifications (Liu et al., 2020). In brief, RBC were washed with PBS for three times, then 8% suspension of RBC was treated with colistin (0.12, 0.25, 0.5, 1, 4, 8 and 16 μg/mL) with or without oxethazaine (20 μg/mL). At the same time, PBS and Triton X-100 were used as the negative and positive control, respectively. After 1 h incubation at 37°C, the cells were centrifuged at 3,500 g for 8 min, and the absorbance of the supernatant was determined at 576 nm. The haemolysis rate was calculated according to the absorbance of positive control and the blank control. All experiments were carried out at least twice.

The cytotoxicity of Vero cells was detected by cell counting kit-8 (CCK-8) assay (Solarbio life sciences) based on previous report (Zhang et al., 2023). The cells were plated in a 96-well plate at a density of 3 × 10⁴ cells per well and treated with colistin (0.5, 1, 2, 4, 16, 32, 64 and 16 μg/mL) or the combination of colistin (0.5, 1, 2, 4, 16, 32, 64 and 16 μg/mL) and oxethazaine (20 μg/mL). After cultured 4 h at 37°C and 5% CO₂, 10% CCK-8 solution was added to 100 μL of medium in each well and the culture maintained for an additional 2 h, cells were analyzed with absorbance at 450 nm.

An overnight culture of E. coli ATCC 25922 and E coli SHP45 was diluted 1:1000 in fresh LB broth (Qingdao Haibo Biotechnology Co., Ltd.), and 2 mL bacterial suspension was placed in the 10 mL centrifuge tube. Then the bacteria were treated with 0.5×MIC of colistin, oxethazaine (20 μg/mL) or the combination of colistin and oxethazaine. After incubated at 37°C, 180 rpm for 0, 2, 4, 8, 12, 16, 20 and 24 h, 100 μL of bacterial suspension were collected and ten-fold serially diluted in PBS, and the suspensions were plated on LBA plates. After overnight cultivation at 37°C, bacterial colonies were calculated. Experiments were performed in biological triplicate.

A LIVE/DEAD BacLight bacterial viability kit was used to evaluate the synergistic antimicrobial effect of colistin and oxethazaine. An overnight culture of E. coli ATCC 25922 was adjusted to OD₆₀₀ of 0.5 and incubated with colistin, oxethazaine or their combination. After 30 min, the bacterial cells were washed with PBS and LIVE/DEAD dye was added. The images of E. coli were observed using a confocal fluorescence microscope (Leica, Germany). In addition, the dead cells were observed at excitation wavelength 535 nm and emission wavelength 615 nm with an EnSight multimode plate reader.

The integrity of the OM was evaluated using NPN fluorescent dye (Aladdin, China) (Liu et al., 2020; Song et al., 2020). After incubation overnight at 37°C, the E. coli ATCC 25922 were washed three times with 5 mM HEPES (5 mM glucose). After 20 min incubation with the NPN dye, the colistin alone, oxethazaine alone, or their combination was added to the bacterial suspensions. After treatment in the dark at 37°C for 30 min, the fluorescence intensity was determined with the excitation wavelength of 350 nm and the emission wavelength of 420 nm with an EnSight multimode plate reader.

Propidium iodide PI (Beyotime, China) was used to evaluated the cytoplasmic membrane permeability (Liu et al., 2020). Briefly, overnight cultures of E. coli ATCC 25922 were washed three times with PBS and resuspended with PBS to approximately an OD₆₀₀ nm of 0.5. The bacterial suspensions were cultured with propidium iodide PI at 37°C for 20 min, followed by the addition of colistin (0, 1, 2 and 4 μg/mL) alone or combined with 20 μg/mL oxethazaine. After incubation for 30 min at 37°C, fluorescence intensity was monitored using the EnSight^® Multimode Plate reader at an excitation wavelength of 535 nm and an emission wavelength of 615 nm. Experiments were performed in biological triplicate.

The fluorescent probe DiSC₃ (Silvestri and van Saene, 2010) (Aladdin, China) was used to analyze the membrane potential of E. coli ATCC 25922 (Song et al., 2021). In brief, bacteria were grown on LB broth to reach the exponential phase, then washed twice with PBS and adjusted to OD₆₀₀ of 0.5. After incubation of the bacterial suspension with DiSC₃ (Silvestri and van Saene, 2010) (0.5 µM) for 20 min, varying concentrations of colistin (0, 1, 2 and 4 μg/mL) with or without oxethazaine were added, and the fluorescence intensity was determined with the excitation wavelength at 622 nm and emission wavelength at 670 nm with an EnSight multimode plate reader. All tests included biological replicates.

The levels of ROS in E. coli ATCC 25922 were measured using the fluorescence probe 2′,7′-dichlorodihydro-fluorescein diacetate (DCFH-DA) (Beyotime, China) with the excitation wavelength at 488 nm and the emission wavelength at 525 nm (Song et al., 2020). In brief, the bacteria were resuspended in PBS to an OD₆₀₀ of 0.5, then the DCFH-DA probe was added to a final concentration of 10 µM. After the bacteria were incubated at 37°C for 20 min, different concentrations of colistin (0, 1, 2 and 4 μg/mL) in the presence or absence of 20 μg/mL oxethazaine was added and the fluorescence intensity was detected.

The bacterial morphology was observed by the SEM. Briefly, E. coli ATCC 25922 in mid-log phase was washed three times with PBS, and resuspended to 10⁷ CFU. Colistin (0.5×MIC), oxethazaine (20 μg/mL) or their combination (0.5×MIC colistin+20 μg/mL oxethazaine) were added and incubated at 37°C for 4 h. Then samples were washed with PBS and fixed overnight in 2.5% glutaraldehyde solution at 4°C. The bacteria were dehydrated with different gradients of ethanol, and the bacterial morphology was observed by the SEM (Hitachi, Japan) after vacuum drying.

The LC–MS/MS assay was prepared according to described earlier, with minor modifications (Lee et al., 2013). A C18 column (5 μm, 150 mm × 2.0 mm I.D.) was used for chromatography and maintained at 40°C. After the bacteria were treated with 20 μg/mL oxethazaine alone or combined with 1 μg/mL colistin, the accumulation of oxethazaine in E. coli was performed on the LCMS-8050 system (Shimadzu, Kyoto, Japan).

The synergistic antibacterial effect of oxethazaine and colistin was evaluated in the Galleria mellonella larvae infection model. The larvae of G. mellonella were randomly divided into four groups (n = 10 per group), and injected with 10 μL of 9.8 × 10⁴ CFUs E coli SHP45 suspension. After 1 h, G. mellonella were subjected to antibiotic treatment, including 20 mg/kg oxethazaine, 2 mg/kg colistin or the combination of 20 mg/kg oxethazaine and 2 mg/kg colistin. At the same time, PBS was used as the control group. Survival of G. mellonella in each group was observed.

6–8 weeks old female BALB/C mice (20 ± 2 g) were used for mouse peritonitis-sepsis model. Mice randomly assigned into five groups (n = 8 per group), mice were acclimated for 7 days before infection in the Laboratory Animal Center of South China Agricultural University. Then mice were infected with 200 μL of 1.8 × 10⁸ CFU E coli SHP45 suspension via intraperitoneal injection. After 1 h post infection, mice were treated with PBS, colistin (2 mg/kg), oxethazaine (20 mg/kg), or the combination of 2 mg/kg colistin with 20 mg/kg oxethazaine. The number of survival mice in each group was observed. Once the infected mice died, the liver, spleen, and kidney were removed and analyzed bacterial loading and subjected to histological analysis. After 5 days of experimentation, the remaining animals were euthanized by cervical dislocation according to animal welfare related guidelines. The organs were collected under aseptic conditions, one part of the organs was used for homogenization and other for histological analysis (Valiante et al., 2015; Xie et al., 2020).

GraphPad Prism 9.0 software was used for statistical analyses. All data are expressed as a mean ± standard deviation (SD). p-values were calculated by one-way ANOVA among multiple groups or t-test between two groups. *p < 0.05, **p < 0.01.

Firstly, the potential efficacy of oxethazaine and existing antibiotics was evaluated using checkerboard dilution assays. Interestingly, we observed synergistic activity between oxethazaine and colistin against both colistin-resistant E. coli SHP45 and colistin-sensitive ATCC 25922, whereas oxethazaine alone had no antibacterial activity (Figures 1C,D). To evaluate whether the synergistic antibacterial activity was specific, six species of Gram-negative bacteria were used to determine the synergistic effect of oxethazine and colistin. As expected, oxethazaine combined with colistin showed a strong synergistic effect against Gram-negative bacteria (Figure 1B, Supplementary Table S1), suggesting that oxethazaine was a potent antibiotic adjuvant to restore the antibacterial activity of colistin.

To further confirm the synergistic antibacterial activity of oxethazaine and colistin, the time-killing experiments were performed on mcr-positive or -negative E. coli. We found that either 20 μg/mL oxethazaine alone or 0.5×MIC colistin alone showed little bactericidal activity, whereas the combination of oxethazaine and colistin rapidly reduced the number of bacteria with more than 2log₁₀ CFU (Figures 2A,B). Furthermore, the synergistic effect was also confirmed by LIVE/DEAD staining, which stains dead bacteria red and viable bacteria green. Compared with colistin or oxethazaine alone, the proportion of dead bacteria increased in the combination group (0.5×MIC colistin+20 μg/mL oxethazaine) (Figures 2C,D).

Toxicity is one of the key factors limiting the clinical application of combination therapy. Therefore, we subsequently evaluated whether the addition of oxethazaine would increase toxicity. Surprisingly, we found no significant increase in hemolytic activity of Sterile sheep red blood cells (RBC) after treatment with the combination of colistin and oxethazaine (Supplementary Figure S1). Moreover, Vero cells were also used to evaluate the cytotoxicity of colistin with or without oxethazaine. Compared to colistin alone, no increased cytotoxicity was observed in the Vero cells after exogenous addition of oxethazaine (Figure 2E). These results indicated that oxethazaine exhibited a robust potentiating effect on the colistin activity without increased toxicity.

Bacterial structural morphology is critical for bacterial viability (Srivastava et al., 2020), thus the morphological changes was evaluated by scanning electron microscopy (SEM) analysis. After treatment with either oxethazaine or colistin alone, a slight morphological change was observed. In comparison, the cell surface appeared strongly sunken and shrinkage after treated with the oxethazaine combined with colistin (Figure 3A). Therefore, we hypothesized that the combination might have an effect on the bacterial membranes. To confirm this hypothesis, we first evaluated the effect on the OM using the fluorescent dye N-phenyl-1-naphthylamine (NPN) (Song et al., 2020). As expected, the fluorescence value of colistin was significantly increased in the presence of oxethazaine (p < 0.05), whereas only subtle changes in fluorescence intensity was detected after treated with oxethazaine alone (Figure 3B) (p > 0.05). In addition, oxethazaine restored antibacterial activity against LPS-deleted strain MG1655-ΔwaaC (Table 1), indicating that the OM acts as a permeability barrier to prevent oxethazaine from entering the cell. Thus, the intracellular accumulation of agents was detected by LC-MS/MS (Figure 4C, Supplementary Table S2), and we found that colistin significantly promoted the accumulation of oxethazaine (Figure 4A) (p < 0.05). Moreover, cytoplasmic membrane (CM) integrity and membrane potential was significantly dissipated in the combination group (Figures 3C,D) (p < 0.05).

The integrity of the membrane structure is critical for several bacterial biosynthetic processes (Wang et al., 2022). Notably, we observed that the accumulation of bacterial reactive oxygen species (ROS) was enhanced in a dose-dependent manner after the addition of oxethazaine (Figure 3E). Moreover, exogenous addition of N-acetylcysteine (NAC, 6 mmol/L) can reduce the synergistic effect of oxethazaine combined with colistin (Figure 4B), revealing that ROS play a critical role in their synergistic process. These results indicated that oxethazaine enhanced the antibacterial activity of colistin by multiple modes of action (Figure 5).

Given the strong potentiation of oxethazaine on colistin in vitro, we further assessed whether their combination had potential efficacy in vivo infected with E. coli SHP45 using the G. mellonella model and the mouse peritonitis/sepsis model. As expected, the survival rate of infected larvae was increased after treatment with the combination of oxethazaine and colistin (20 mg/kg+2 mg/kg), whereas the monotreatment only displayed a slight therapeutic effect (Figure 6A). Accordingly, the significant therapeutic efficacy was also confirmed in the mouse model. 75.0% of the mice were protected in the therapy of 20 mg/kg oxethazaine combined with 2 mg/kg colistin. In contrast, all mice in the PBS and oxethazaine monotherapy groups died within 3 days (Figure 6B). Besides, the pathological changes in each organ were alleviated accordingly (Figure 6C). Moreover, compared with the monotherapy groups, combinational therapy significantly reduced the bacterial burden in various organs of mice (Supplementary Figure S2). These findings revealed that oxethazaine effectively rescued colistin activity to combat the MDR E. coli in vivo.