AZM and BDQ are both purchased by Beijing Solarbio Science & Technology Co., Ltd.(Beijing, China), and WX-081 is provided by Shanghai Jiatan Biotechnology Co., Ltd. (Shanghai, China). Both drugs were dissolved in dimethyl sulfoxide (DMSO), and the drug solution was prepared according to the suggestions provided by the Institute of Clinical and Laboratory Standards (CLSI) (Woods et al., 2011). The standard strain of M. abscessus ATCC 19977, and 36 clinical strains of M. abscessus were cultured on solid lwenstein-Jensen medium at 37°C for 4-6 days. The MICs of AZM, BDQ and WX-081 were determined by adding drugs to M. abscessus cultured in 96-well plates according to the recommended CLSI broth microdilution method. The broth was diluted twice, and the concentrations of AZM ranged from 0.5-256μg/mL, both BDQ and WX-081 ranged from 0.015-7.68μg/mL. A bacterial inoculum with turbidity equivalent to 0.5 McFarland standard dilution of 1∶200 was prepared for each strain. The MIC of M. abscessus was determined after 3 days of culture at 37°C together with antibiotics. Thereafter, 70 μl of Alamar Blue solution (Sirotec, 20μL Alamar Blue+50μL 5% Tween 80 of 5% Tween 80) was added to each well, and then the plates were incubated for another 24 hours. The color change from blue to pink indicates bacterial growth (Cowman et al., 2016). MIC is defined as the lowest drug concentration that no color change, that is, the lowest concentration that can inhibit the visible growth of the test isolate. Explain the results of drug sensitivity test (DST) according to the breakpoint recommended by CLSI.

For species with enough isolates and for which WX-081 demonstrated good inhibitory activity, The epidemiological cut-off (ECOFF) was determined according to the distribution profile of the MIC values. ECOFF is defined as the concentration that can inhibit > 95% of the bacterial population for the unimodal MIC distribution curve. For the bimodal MIC distribution curve, ECOFF is set between the two peaks.

This study was approved by the Ethics Committee of Beijing Chest Hospital, Capital Medical University(2021-020). The wild-type AB zebrafish strain selected for the study was propagated via natural paired mating and reproduction and maintained in unchlorinated fresh water at 28°C and fed commercial fish food. M. abscessus ATCC19977 bacilli with smooth (S) morphology were incubated for 5 to 7 days at 30°C in Middlebrook 7H9 broth (Becton Dickinson) containing 10% OADC (Becton Dickinson) and 0.05% Tween 80 (Sigma-Aldrich). When cultures reached mid-log-phase growth, they were centrifuged then pellets were washed and resuspended in phosphate-buffered saline (PBS) containing 0.05% Tween 80. Next, the bacterial suspensions were homogenised and sonicated then the tubes were allowed to stand without shaking to allow bacteria to settle to the bottoms of tubes for 5 to 10 min. After the liquid in tubes was carefully removed, the settled bacterial cells were collected and resuspended in PBS then DiO green fluorescent dye was added to the cell suspensions to fluorescently label the bacilli. Thereafter, the bacteria were transplanted into zebrafish of the wild-type AB strain at 2 days post-fertilisation (dpf) via micro-venous injection. Zebrafish were anesthetized using 3-aminobenzoic acid ethyl ester methanesulfonate (C₉H₁₁NO₂·CH₄O₃S, MESAB). The MESAB was prepared by mixing MESAB and Na₂HPO₄·12H₂O in a total mass ratio of 1:5 to make a 4 mg/mL solution, which was stored at 4°C. For use, it was diluted with standard dilution water, with a final anesthetic concentration of 0.16 mg/mL. Then the zebrafish was injected bacteria by intravenous microinjection with each fish receiving approximately 3.6×10³ colony forming units (CFUs) of the transplant to establish the zebrafish model of M. abscessus infection (Guo et al., 2020; Nie et al., 2020).

Zebrafish at 3 dpf were viewed under a microscope then they were randomly allocated to six-well plates (30 zebrafish per well) in 3 mL of water/well and maintained at 35°C throughout the experiment. AZM, BDQ, and WX-081 stock solutions were each prepared in 20.0 mg/mL DMSO solutionand stored at −20°C until needed. For experiments, drugs were diluted in water to generate 2-fold serial dilutions of AZM (62.5 μg/mL, 125 μg/mL, 250 μg/mL, 500 μg/mL, and 1000 μg/mL), BDQ (3.91 μg/mL, 7.81 μg/mL, 15.6 μg/mL, 31.2 μg/mL, and 62.5 μg/mL), and WX-081 (15.7 μg/mL, 31.3 μg/mL, 62.5 μg/mL, 125 μg/mL, 250 μg/mL, 500 μg/mL, and 1000 μg/mL). Zebrafish of drug-treated groups (designated AZM group, BDQ group, and WX-ZM group) received M. abscessus injections followed by additions of diluted drug treatments added directly to plate wells. Concurrently, blank control group and model group zebrafish were maintained using the same protocol, but the blank control group received no M. abscessus injection and no drug treatment, while model group zebrafish each received one M. abscessus injection and no drug treatment. After zebrafish were treated with control (no drug) or experimental (drug) treatments for 48 h at 35°C, MTCs of control groups (blank control, model) and experimental groups (AZM, BDQ, and WX-081 groups) were determined.

According to previously reported procedures, zebrafish at 3 dpf were viewed under a microscope then randomly assigned to 6-well plates (30 zebrafish/well). Drug stocks were diluted with water to create diluted AZM, BDQ, and WX-081 solutions in 3-mL volumes at concentrations of 62.5 μg/mL for AZM, 15.6 μg/mL for BDQ, and 1.95 μg/mL, 3.91 μg/mL, 7.81 μg/mL, 15.6 μg/mL, 31.2 μg/mL, and 62.5 μg/mL for WX-081. Control group and model group samples were set up at the same time in 3 mL of water per well. Live zebrafish was used for imaging. The zebrafish was anesthetized and transferred onto methyl cellulose using a Pasteur pipette. An electrically controlled, continuously variable magnification fluorescence microscope (AZ100, Nikon, Japan) equipped with a green fluorescence channel was used at a magnification of 30x. After 48 h of drug treatment at 35°C, 10 randomly selected zebrafish in each experimental group were digitally photographed under a fluorescent microscope. Image processing software (NIS-Elements D 3.20) was used to collect and analyse image-based data to determine drug efficacies for inhibiting the M. abscessus growth in vivo based on zebrafish whole-body fluorescence intensity data.

In order to evaluate whether AZM, BDQ, and WX-081 can prolong the survival time of M. abscessus-infected zebrafish and the difference in the prolonged time between them, the number of zebrafish surviving and dying in each experimental group should be observed and recorded every day, and the dead individuals should be removed in time until 9 dpf. Zebrafish at 3 dpf were viewed under the microscope then were randomly transferred to 50-mL beakers (experimental group) (60 zebrafish/beaker). AZM, BDQ, and WX-081 were diluted to generate working drug solutions of AZM (62.5 μg/mL), BDQ (15.6 μg/mL), and WX-081 (1.95 μg/mL, 3.91 μg/mL, and 7.81 μg/mL) in a 20-mL volume/beaker. Control and model groups were set up at the same time in a 20-mL volume per beaker. All groups were incubated at 35°Cduring the treatment period (48 h). Each day, numbers of dead zebrafish were recorded and dead zebrafish were removed. Survival analysis was performed on the survival time and survival quantity.

Data were statistically analysed using SPSS 26.0. Fluorescence intensities were expressed as the mean ± SE. Student’s t or t’ tests for independent samples were used for comparisons between two groups if data were normally distributed; non-parametric tests were used if data were non-normally distributed. Kaplan-Meier survival analysis was performed using the log-rank test to determine survival rates of WX-081-treated zebrafish at different drug concentrations. CFU counts obtained for different groups were analysed and compared using one-way ANOVA, with a p value of <0.05 indicating statistically significant intergroup differences were found.

MICs of WX-081 against M. abscessus ATCC19977, and 36 clinical isolates ranged from 0.12 μg/mL to 0.96 μg/mL, with a MIC₅₀ of 0.48 µg/mL and a MIC₉₀ of 0.96 µg/mL observed ( Figure 1 ). Ultimately, WX-081 exerted strong antibacterial activity against almost all tested M. abscessus isolates, as evidenced by MICs that were generally much lower than 1 µg/mL. The ECOFF value of WX-081 against M. abscessus was 0.96 µg/mL. The MIC of M. abscessus ATCC19977, MIC range, MIC₅₀ and MIC₉₀ of AZM, BDQ and WX-081 is showed in Table 1 .

Table 2 shows MTC values for AZM, BDQ, and WX-081 groups of 62.5 µg/mL, 15.6 µg/mL, and 62.5 µg/mL, respectively. Notably, when AZM was administered at 125 μg/mL, zebrafish health began to deteriorate; at 500 μg/mL, blood stasis in the heart was observed; at 1000 μg/mL, the zebrafish mortality rate reached 33.33%. When the concentration of BDQ was increased from 3.91 μg/mL to 15.6 μg/mL, no obvious change was observed relative to the model group; at 31.2 μg/mL, four zebrafish had renal edema, two had pericardial edema, and one had blood stasis; at 62.5 μg/mL, only one zebrafish survived. When the concentration of WX-081 was 62.5 μg/mL (the MTC), zebrafish were healthy; when the concentration was increased to 125 μg/mL, zebrafish exhibited signs of heart congestion and their bodies became bent, although no deaths of zebrafish were observed; when the WX-081 concentration was increased from 250 μg/mL to 500 μg/mL, the zebrafish mortality rate increased from 10.00% to 100.00%.

AZM, BDQ, and WX-081 MTCs and WX-081 serial 2-fold MTC dilutions (1/32, 1/16, 1/8, 1/4, 1/2) are shown below with corresponding zebrafish whole-body and head fluorescence intensity values ( Table 3 ). Pairwise comparisons of zebrafish whole-body fluorescence intensity values revealed lower values for AZM and BDQ groups vs. that of the model group (433684 ± 11910 vs. 671089 ± 22305, respectively, p < 0.001; 438648 ± 8280 vs. 671089 ± 22305, respectively, p < 0.001), with similar zebrafish head fluorescence intensity trends observed (78397 ± 4815 vs. 132412 ± 9989, p < 0.001; 79664 ± 5809 vs. 132412 ± 9989, p < 0.001, respectively).

An analysis of whole-body fluorescence intensities revealed a lower fluorescence intensity at the WX-081 MTC than at 1/32, 1/16, 1/8, 1/4, and 1/2 dilutions of the MTC. Fluorescence intensities of groups treated with the AZM MTC (62.5 μg/mL) and BDQ MTC (15.6 μg/mL) were each higher than that of the WX-081 MTC group (433684 ± 11910 vs. 375347 ± 11359, p < 0.01: 438648 ± 8280 vs. 375347 ± 11359, p< 0.001, respectively. By contrast, analysis of head fluorescence intensities revealed a lower fluorescence intensity at the WX-081 MTC than at 1/32, 1/16, and 1/8 dilutions of the WX-081 MTC that did not differ statistically from fluorescence intensities of AZM and BDQ MTC groups (78397 ± 4815 vs. 70976 ± 5726, respectively, p > 0.05. 79664 ± 5809 vs. 70976 ± 5726, respectively, p > 0.05). Figure 2 shows in vivo fluorescence intensity distributions for zebrafish treated with different drugs. Increased WX-081 concentration was associated with lower bacterial burdens in both whole-body and head of zebrafish as determined by bacterial CFU ( Figure 3 ).

The survival rate of zebrafish in AZM MTC group at 4-9dpf was 88.33% higher than model group (Untreated M. Abscessus-Infected ZebraFish) 66.67% (p<0.01), however, the survival rate in the BDQ MTC group was lower than model group (48.33% vs.66.67%, p<0.05). When the concentration of WX-081 increased from 1.95μg/mL to 7.81μg/mL, the survival rate decreased from 90.00% to 81.67%, but it was still higher than that of the model group ( Table 4 ). Figure 4 shows survival curves of zebrafish treated with different concentrations of AZM, BDQ, and WX-081. The survival experiment was conducted after confirming the effectiveness of the antibacterial experiment. the highest concentration in the survival experiment was determined based on the MTC observed in zebrafish treated for 48 hours. Although the deaths caused by high drug concentrations were indeed due to the drug itself, the results at medium and low concentrations adapted for demonstrating the ability of the drug to inhibit bacterial growth and extend the survival time of zebrafish.