The M. abscessus reference strain (ATCC 19977) was purchased from the American Type Culture Collection. Clinical isolates were purchased from the Korea Mycobacterium Resource Center (KMRC, Osong, Korea). All M. abscessus strains were grown at 37°C either in liquid or on solid media. Middlebrook 7H9 broth was used as the liquid medium, which contained 0.2% glycerol (v/v) and 10% albumin-dextrose-catalase (ADC) (v/v), while the solid medium was Middlebrook 7H10 agar that contained 0.5% glycerol (v/v) and 10% oleic acid-ADC (OADC) (v/v). Stock vials of each M. abscessus were preserved in a deep freezer at −80°C in 7H9 broth containing 40% glycerol (v/v), 0.05% Tween 80 (v/v), and 10% OADC (v/v).

Amikacin, ciprofloxacin, clarithromycin, doxycycline, cefoxitin, imipenem, linezolid, moxifloxacin, trimethoprim, sulfamethoxazole, and tobramycin were purchased from Sigma–Aldrich Co. (St Louis, MO, United States), and isoegomaketone was purchased from AA BLOCKS, INC (San Diego, United States). Minimum inhibitory concentrations (MICs) were determined according to CLSI guidelines, except for the composition of the medium and the concentration of isoegomaketone. To prepare the test MIC 96-well plate, drugs were mixed with 50 μL of 7H9 medium containing 10% OADC supplement in each well. The final concentrations of the tested drugs were as follows: amikacin (2 to 128 μg/mL), ciprofloxacin (0.25 to 16 μg/mL), clarithromycin (0.5 to 32 μg/mL), doxycycline (0.25 to 16 μg/mL), cefoxitin (4 to 256 μg/mL), imipenem (1 to 64 μg/mL), linezolid (1 to 64 μg/mL), moxifloxacin (0.125 to 8 μg/mL), trimethoprim/sulfamethoxazole (0.5/9.5 to 32/608 μg/mL), and tobramycin (1 to 8 μg/mL). For MIC determination, all Mabs strains were inoculated in 7H9 medium and incubated at 37°C for 3 to 5 days. Cultures were homogenized with 7H9 media and then adjusted to a McFarland of 0.5 with sterile saline. Then, the cells were diluted 100-fold for inoculation in 2X cation-adjusted Mueller-Hinton broth (CAMHB) containing 50 μg/mL 2,3-diphenyl-5-thienyl-(2)-tetrazolium chloride (STC; TCI, Tokyo, Japan) for mycobacterial staining, and 50 μL was inoculated into each well of the prepared MIC test 96-well plate. For amikacin, ciprofloxacin, doxycycline, cefoxitin, imipenem, linezolid, moxifloxacin, trimethoprim/sulfamethoxazole, and tobramycin, the MICs were determined after 3, 5, and 7 days of incubation. In addition, the plates were submitted to an extended incubation for clarithromycin MIC testing, which allowed the evaluation of macrolide-inducible resistance with readings taken after 3, 5, 7, 10, and 14 days of incubation at 30°C. The MIC was defined as the lowest concentration at which no visual bacterial growth occurred, indicating complete inhibition of bacterial growth. Susceptibility was assessed following the guidelines set by CLSI for breakpoints (Supplementary Table S1). The MIC of isoegomaketone was determined using the same method, with the final isoegomaketone test concentrations ranging from 1 to 1,024 μg/mL. MICs were determined after 3-and 5-day incubations. All assays were performed in triplicate.

Minimum bactericidal concentrations (MBCs) of isoegomaketone were determined for all M. abscessus strains to differentiate between bacteriostatic and bactericidal activities. After 4 days of incubation with isoegomaketone at different concentrations, the wells with drug concentrations that exceeded the MIC were resuspended. Then, 100 μL of medium from each well was serially diluted (by 10-fold) and inoculated into a 7H10 plate. The plates were incubated at 37°C for 5 days, then, the number of colonies was counted. The MBC value was the lowest drug concentration at which no colonies grew. Antibiotics were classified as bactericidal if the MBC/MIC ratio was ≤4 and as bacteriostatic if the ratio exceeded 4 (Levison, 2004; Benjamin et al., 2012).

The time-kill assays were performed by adding isoegomaketone, clarithromycin, or amikacin at 1X MIC antibiotic concentrations and the inoculum (density 1 × 10⁵ CFU/mL) into 1 mL of 7H9 broth with 10% OADC. As a control, aliquots containing sterile distilled water instead of antibiotics were included. All tubes were incubated at 37°C for 5 days. The bacteria were enumerated at 24 h intervals by plating serial dilutions on 7H10 agar plates. CFUs were counted after an additional 4 days of incubation at 37°C. A CFU count curve was drawn over time to characterize the effect of the antibiotics at different concentrations.

For biofilm formation, 500,000 CFUs of the reference and clinically isolated M. abscessus strains were inoculated in 7H9 medium containing 10% OADC in U-bottom 96-well plates. Isoegomaketone at final concentrations of 4, 16, or 64 μg/mL was then added to the wells in quintuplicate. Control wells received an equal volume of DMSO and were incubated at 37°C for approximately 10 days without shaking. After the formation of the bacterial biofilm, the medium was removed from wells by pipetting underneath the biofilm at the interface. Biofilms were dried in a biosafety cabinet; then, the biofilms were incubated with 200 μL of 1% crystal violet for 30 min. The stain was removed, and the biofilm was gently washed three times with PBS. The bound crystal violet was then extracted by a 10 min incubation at 37°C with 200 μL of 95% ethanol. The absorbance of the extracted crystal violet was measured at 570 nm on a spectrophotometer.

The viability of BMDMs and THP-1 cells treated with isoegomaketone was evaluated using a CCK-8 assay kit (Dojindo, Japan). BMDMs were obtained by flushing the femurs and tibias of eight-week-old female C57BL/6 J mice purchased from DBL (Gyeonggi-do, Pyeongtaek-si, Korea) with DMEM (Welgene, Gyeongsan-si, Gyeongsangbuk-do, Korea). Bone marrow cells were differentiated into BMDMs by exposure to 10 ng/mL recombinant mouse macrophage colony-stimulating factor (M-CSF; PeproTech) at 37°C in a 5% CO2 incubator for 6 days. THP-1 cells were obtained from the American Type Tissue Collection (ATCC) and were maintained in DMEM with 10% FBS. THP-1 differentiation was induced by adding 100 nM phorbol 12-myristate 13-acetate (PMA; Sigma) and incubating at 37°C in a 5% CO2 incubator overnight. BMDMs and differentiated THP-1 cells were seeded in a 96-well plate at a density of 1 × 10⁵ cells per well. Isoegomaketone was added to the cells at various concentrations, and the samples were incubated at 37°C for 48 h. For the cell viability assay, 20 μL of CCK-8 reagent (10% media volume) was added to each well, and the incubation was continued for 1.5 h. The resulting color was assessed at 450 nm. A SpectraMax^® M3 Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) was used for each assay.

To assess the activity of isoegomaketone against intracellular M. abscessus, BMDMs and differentiated THP-1 cells (1.5 × 105 cells/well) were dispensed into 48-well plates (SPL, New York, NY, USA) and then infected with the reference or clinically isolated M. abscessus strains at a multiplicity of infection (MOI) of 1:1. After 4 h of infection, the cells were washed with PBS to remove the uninfected bacteria and incubated with DMEM containing 20 μg/mL amikacin for 1.5 h to kill the remaining extracellular M. abscessus. The cells were then washed with PBS and a broth medium with various concentrations of isoegomaketone was added. After 24 h or 48 h of incubation at 37°C and 5% CO2, BMDMs and differentiated THP-1 cells were extensively washed with PBS, lysed with 0.05% Triton X-100, and then serially diluted with PBS. The number of CFUs was quantified by plating serial dilutions of lysates on M7H10 agar plates.

The animals were housed in an SPF barrier room under controlled conditions on a 12 h light–dark cycle and a constant temperature (25°C). The experiments were performed in accordance with the Animal Care and the Guiding Principles for Animal Experiments Using Animals were approved by the University of Konyang Animal Care and Use Committee (21-07-E-01).

All experiments were performed in triplicate. Statistical analyses were performed using GraphPad Prism 6.0 software. For the biofilm formation assay, cell viability assay, and intracellular bactericidal assay, one-way ANOVA was employed to identify significant differences between the groups. In the case of time–kill assays, a two-way ANOVA was utilized to determine significant differences between the groups. Error bars represent the standard deviation (SD). Differences were considered statistically significant at a p value of <0.05.

A total of 10 Mabs strains, including 2 reference strains and 8 clinically isolated strains, were studied. Morphological observation indicated that 5 out of the 8 Mabs isolates had smooth morphotypes, while 3 exhibited rough morphotypes. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and MBC/MIC ratio of iEMK in the reference and clinically isolated strains are shown in Table 1. iEMK showed effective activity against most Mabs strains. The two reference and two clinical strains had MICs of 128 μg/mL, and the 6 other clinical strains had MICs of ≤64 μg/mL. Additionally, there was no difference in MIC based on morphological differences. A total of 10 Mabs strains had MBCs of 128 to 256 μg/mL, and the MBC/MIC ratio ranged from 2 to 4. Since iEMK had MBC/MIC ratios ≤4 against the strains, the bactericidal effect was evaluated in these strains. The results of antimicrobial susceptibility testing, in accordance with the recommendations from The Clinical & Laboratory Standards Institute (CLSI) 2011, for both the reference and clinically isolated strains are shown in Table 2. All of the strains were susceptible or intermediate susceptible to amikacin, cefoxitin, imipenem, linezolid, and tigecycline. Eight of the strains were resistant to moxifloxacin, and six were resistant to ciprofloxacin and tobramycin. Furthermore, all strains showed resistance to doxycycline and trimethoprim/sulfamethoxazole. All nine strains were susceptible to clarithromycin, except for a single clinically isolated strain (KMRC 00136–61,203) that showed resistance. Therefore, iEMK is an effective drug candidate for clarithromycin, doxycycline, and trimethoprim/sulfamethoxazole resistant isolates.

In addition to the MIC results, in vitro time-kill assays were conducted using iEMK and the first-line drug clarithromycin or amikacin (Figure 1). In general, time–kill curves for the Mabs reference strains showed a greater decrease in bacterial population size after 2 to 4 days than what was observed for the clinically isolated strains when exposed to 1 X MIC iEMK. All Mabs strains exhibited a slow decrease in population size in response to treatment with positive controls, such as clarithromycin or amikacin, over a 5-day period. However, when exposed to iEMK, the reference Mabs strains exhibited only a slight decrease in population size, similar to what was seen with the positive control treatment, whereas in clinically isolated strains, iEMK had bacteriostatic activity.

The phenomenon of macrolide-inducible resistance poses a formidable challenge in the clinical management of Mabs infections. This resistance significantly undermines the efficacy of standard macrolide therapy, which is essential for treating these infections (Nash et al., 2009; Brown-Elliott et al., 2012). Given the critical nature of this issue, our study aimed to elucidate the potential of iEMK in addressing macrolide-inducible resistance. In our extended 14-day in vitro time-kill assays, we assessed the effects of combining iEMK with CLR on Mabs strains exhibiting this type of resistance. Our results, depicted in Figure 2, demonstrate that CLR at 8 μg/mL initially suppressed bacterial growth in strains with macrolide-inducible resistance. Notably, by day 14, these strains began to exhibit regrowth. In contrast, the combination of iEMK with CLR prevented such regrowth, effectively showcasing iEMK’s ability to inhibit the development of macrolide-inducible resistance.

Biofilms are thought to play an essential role in Mabs infections, and there is evidence of microcolony formations in patient lungs that resemble biofilms (Qvist et al., 2015; Fennelly et al., 2016). Moreover, in vitro biofilm models of Mabs have reduced susceptibility to several first-line antibiotics, including cefoxitin, amikacin, and clarithromycin (Greendyke and Byrd, 2008; Marrakchi et al., 2014). Thus, we examined the antibiofilm effects of iEMK. Eight of the reference and clinically isolated Mabs strains had average OD570 values ranging from 0.52 to 2.43 (Figure 3). The remaining clinically isolated strains (61,041 and 61,309) had OD570 values below 0.20 and were considered non-biofilm-forming isolates. Our investigation into iEMK’s antibiofilm activity demonstrated a dose-dependent reduction in biofilm formation across the tested strains when treated with concentrations of 4, 16, or 64 μg/mL (Figure 3). This observation was particularly notable as iEMK effectively inhibited biofilm formation at significantly reduced concentrations, showcasing its potential as an effective antibiofilm agent against both reference and clinically isolated Mabs strains, including the notably resistant 61,040 strain (Figure 3).

To determine whether iEMK induces cell death, cell viability at different concentrations of iEMK was assessed 48 h after treatment using the CCK assay. The results showing the effects of iEMK on the viability of murine bone marrow-derived macrophages (BMDMs) and differentiated human monocytic cells (THP-1) are presented in Figure 4. When the concentration of iEMK was 16 μg/mL, the survival rates of the BMDMs were nearly 100% after 48 h of exposure. In contrast, the differentiated THP-1 cells exhibited reduced cell viability compared to the control group when exposed to iEMK concentrations higher than 8 μg/mL.

The efficacy of a drug against Mabs in a broth medium may not always align with its effectiveness against intracellular Mabs (Rastogi et al., 1987). In addition, Mabs can invade and replicate within macrophages, which indicates its ability to evade antimicrobial defenses in macrophages (Oberley-Deegan et al., 2010; Abdalla et al., 2015). Consequently, it is important to examine iEMK inhibition of Mab growth in intracellular environments. Thus, we tested the activity of iEMK in terms of intracellular bacterial replication. We infected BMDMs and THP-1 cells with the reference and clinically isolated Mabs strains, and 4 h later, we washed the cells to remove extracellular Mabs and supplemented the media with iEMK. After treatment, we estimated intracellular Mabs growth by plating and counting CFUs at 24 and 48 h postinfection. Our analysis demonstrated a dose-dependent decrease in CFUs of reference Mabs strains treated with iEMK within BMDMs and THP-1 cells, particularly noted at 48 h postinfection (Figures 5A, 6A). Moreover, iEMK showed significant growth inhibition for both reference and clinically isolated Mabs strains at 24 and 48 h post-treatment. Specifically, growth inhibition for both reference and clinically isolated Mabs strains was observed in BMDMs at iEMK concentrations ranging from 1/8 to 1/2 MIC (Figures 5B,C). In a similar vein, bacterial growth was significantly reduced in THP-1 cells treated with iEMK at concentrations from 1/32 to 1/8 MIC (Figures 6B,C).