The MelJuSo human melanoma cell line (kindly provided by Jacques Neefjes, Leiden University Medical Center, Leiden, the Netherlands) was maintained in Gibco Iscove’s Modified Dulbecco’s Medium (IMDM) (Life Technologies, Bleiswijk, the Netherlands) supplemented with 10% fetal bovine serum (FBS, Greiner Bio-One, Alphen a/d Rijn, the Netherlands), 100 units/ml penicillin, and 100 μg/ml streptomycin (Life Technologies) at 37°C/5% CO₂. Peripheral blood mononuclear cells were isolated from anonymized healthy donor buffy coats obtained after written informed consent (Sanquin Blood Bank, Amsterdam, the Netherlands) by density gradient centrifugation over Ficoll Amidotrizoate (Pharmacy, LUMC, the Netherlands). This was approved by the Sanquin Ethical Advisory Board, in accordance with the Declaration of Helsinki, and according to Dutch regulations. CD14+ monocytes were isolated by magnetic cell sorting using anti-CD14-coated microbeads (Miltenyi Biotec, Bergisch Gladsbach, Germany) and differentiated for 6 days into pro-inflammatory (M1) or anti-inflammatory (M2) macrophages with 5 ng/ml of granulocyte-macrophage colony-stimulating factor (GM-CSF; Miltenyi Biotec) or 50 ng/ml macrophage-CSF (M-CSF; R&D Systems, Abingdon, UK), respectively, as previously reported (Verreck et al., 2006). Monocytes and macrophages were cultured in Gibco Dutch modified Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies) supplemented with 10% FBS, 2 mM L-alanyl-L-glutamine (PAA, Linz, Austria), and during differentiation with 100 units/ml penicillin and 100 μg/ml streptomycin at 37°C/5% CO₂.

Mav laboratory strain 101 (700898, ATCC, Virginia, the United States) and three clinical isolates denoted as Mav 100 (amikacin-resistant), (drug-susceptible) 568, and (clarithromycin-resistant) 918 strains [the clinical isolates were isolated from pulmonary infections and displayed different susceptibility profiles to antibiotics as indicated, according to the French guidelines (Comité de l’Antibiograme de la SFM V.1.0 Avril 2021, European Committee on Antimicrobial Susceptibility Testing)] were cultured in Difco Middlebrook 7H9 broth (Becton Dickinson, Breda, the Netherlands), containing 0.2% glycerol (Merck Life Science, Amsterdam, the Netherlands), 0.05% Tween-80 (Merck Life Science), and 10% Middlebrook albumin, dextrose, and catalase (ADC) enrichment (Becton Dickinson), which was supplemented with 100 μg/ml Hygromycin B (Life Technologies) for culturing the green fluorescently labeled Mav Wasabi strain.

Growth of Mav Wasabi in suspension at 37°C was evaluated by measuring the absorbance at an optical density of 600 nm (OD₆₀₀) using the OD₆₀₀ Ultrospec 10 Cell density meter (Amersham Biosciences). In parallel, growth was evaluated by enumerating bacterial colonies by an agar plate assay to determine the OD factor (defined as CFU/ml in a culture with an OD₆₀₀ of 1.0) for Mav Wasabi. Bacterial suspensions were therefore prepared using the estimated OD factor and plated on 7H10 square agar plates, containing Difco Middlebrook 7H10 broth (Becton Dickinson) supplemented with 10% Middlebrook oleic, albumin, dextrose and catalase (OADC) enrichment (Becton Dickinson) and 0.5% glycerol for a standard colony-forming unit (CFU) assay. Afterwards, the estimated OD factor was adjusted to the colonies counted to achieve the final OD factor. The doubling time (the time required for a population of bacteria to double in number) was calculated by first determining the doubling factor (i.e., the number of times the bacteria have doubled in numbers) by determining how many times the bacteria have doubled in numbers (c in the below equation) from early log-phase (OD₆₀₀ = 0.25; b in the equation) until late log-phase culture (OD > 3; a in the equation).

Doubling factor = (LOG(a) − LOG(b))/LOG(c)

(As an example: Doubling factor = (LOG(3.9) − LOG(0.25))/LOG(2) = 3.96. This number indicates how many times the bacteria have doubled in numbers. When this doubling factor is corrected for the amount of time that was used, say 96 h, the doubling time of the bacteria is determined: the doubling time = time required for doubling factor/doubling factor = 3.96/96 = 24.22 h. This number indicates the time required for one generation round.

Electroporation of Mav 101 was performed using the pSMT3-Wasabi construct. The Wasabi gene, amplified from the pTEC15 plasmid (Addgene plasmid #30174) by PCR, was kindly provided by Herman Spaink (Leiden University, Leiden, the Netherlands) and cloned into the mycobacterial expression vector pSMT3 (Gaora, 1998). In this vector, expression of Wasabi is constitutive and controlled by the hygromycin resistance gene-containing hsp60 promoter. First, electrocompetent Mav was freshly prepared from a 50-ml log-phase culture by incubation with 1.5% glycine (Life Technologies) for 18 h at 37°C. Subsequently, bacteria were centrifuged at 1,934 rcf for 20 min and washed three times with 37°C deionized H₂O supplemented with 10% glycerol and 0.5 M sucrose (electroporation solution) followed by centrifugation at 2,120 rcf for 10 min. Electrocompetent bacteria were concentrated 100× in electroporation solution and 100 μl of bacteria was electroporated at room temperature with 5 μg of plasmid DNA using 0.2-cm-gap Gene Pulser electroporation cuvettes and the Gene Pulser Xcell Electroporation System (Bio-Rad) with the following settings: 1,000 Ω, 25 μF, 1.25 kV, and 2.5 V. Transformed bacteria were incubated overnight in 7H9 broth at 37°C in a shaking incubator, transferred to 7H10 agar plates under 100 μg/ml hygromycin selection, and incubated at 37°C/5% CO₂ for 7–10 days.

Expression of the Wasabi green fluorescent protein in individual clones of Mav Wasabi was analyzed by fixating samples in Falcon Round-Bottom Polystyrene Tubes with 1% paraformaldehyde at 4°C for at least 45 min before measuring samples at wavelength 518–548 nm on the BD Accuri C6 Plus flow cytometer (BD Biosciences). FlowJo v10 Software (BD Biosciences) was used for analysis. Resistance to hygromycin was validated by mixing early log-phase Mav Wasabi culture with either 100 μg/ml or 200 μg/ml hygromycin, 20 μg/ml rifampicin (Sigma-Aldrich, Zwijndrecht, the Netherlands) as positive control, or DMSO (Merck Life Science) as negative control. Plates were incubated at 37°C/5% CO₂ for 10 days. Once every 2 days, the wells were resuspended and the absorbance at 600 nm was measured using the EnVision Multimode Plate Reader (Perkin Elmer). Outgrowth of bacteria in the hygromycin condition was compared to the controls.

One day prior to infection, cultures of Mav Wasabi and the three clinical isolates of Mav were diluted to a density corresponding with early log-phase growth (OD₆₀₀ of 0.4). On the day of infection, bacterial suspensions were diluted in appropriate cell culture medium without antibiotics to reach the indicated multiplicity of infection (MOI). MOI of the inoculum was verified by preparing tenfold serial dilutions in 7H9 medium and plating 10-μl drops of each dilution on 7H10 agar plates. For experiments using the MGIT system, 125 μl of each dilution was transferred into MGIT tubes that contain a fluorescence-quenching oxygen sensor and prepared according to the manufacturer’s protocol. Subsequently, the inoculated tubes were incubated at 37°C in a BACTEC MGIT 960 instrument and were monitored automatically for oxygen utilization, which results in an increase in fluorescence. The number of days from inoculation until cultures reached a fluorescent intensity threshold was recorded as time to positivity (TTP). The TTP measurements were plotted against plate-counted log10 CFU using linear regression to be able to calculate bacterial loads ( Supplementary Figure 1 ). MelJuSo cells or primary human macrophages, seeded in flat-bottom 96-well plates at a density of 20,000 cells (2 × 10⁵ cells/ml) or 30,000 cells (3 × 10⁵ cells/ml) per well, respectively, in MelJuSo or macrophage culture medium without antibiotics 1 day before infection, were inoculated in triplicate or indicated otherwise with 100 μl of the bacterial suspension. Plates were centrifuged for 3 min at 129 rcf and incubated for 1 h at 37°C/5% CO₂. In order to monitor only intracellular bacteria following infection, cells were washed with culture medium containing 30 μg/ml gentamicin (Merck Life Science), which blocks extracellular Mav growth ( Supplementary Figure 2 ). Afterwards, cells were treated with fresh cell culture medium containing 5 μg/ml gentamicin and, if applicable, compounds of interest. Plates were incubated at 37°C/5% CO₂ until readout by flow cytometry, CFU or MGIT, as indicated.

Cells were infected as described above, and infection rates were determined by washing cells with PBS and subsequently trypsinized with Gibco 0.05% Trypsin-EDTA (Life Technologies). After trypsinization, appropriate cell culture medium containing FBS was added to the wells to inactivate trypsin and the monolayers were scraped. Harvested cells were centrifuged in Falcon Round-Bottom Polystyrene Tubes at 453 rcf for 5 min to remove the supernatant. Cells were fixated with 1% paraformaldehyde prior to measurement and analysis as described above.

To determine numbers of bacteria taken up during infection and the subsequent survival of bacteria after prolonged incubation, infected MelJuSo cells were lysed at 0 and 24 h and primary human macrophages also at 48, 72, and 144 h post-infection using 100 μl of lysis buffer (H₂O + 0.05% SDS). Cell lysates were serially diluted in multiple steps in 7H9 medium and 10-μl droplets were plated on 7H10 agar plates. After 7–10 days of incubation at 37°C/5% CO₂, plates were photographically scanned, and bacterial colonies were counted. CFU counts were averaged and corrected for dilution factors to give CFU count per sample.

The ability of the MGIT system to accurately predict CFU of Mav was determined by evaluating intracellular bacterial loads of experimental cell lysates obtained in the same way as for the CFU analysis. Of each cell lysate, 125 μl was transferred to MGIT tubes. The obtained TTP measurements were then converted into CFU counts by using linear regression and compared with the plate-counted values. The percentage of bacterial survival was defined as the fraction of CFU measured during prolonged incubation of the total CFU measured at uptake (=100%). As part of the validation of the MGIT assay, primary human macrophages exposed to Mav Wasabi (10:1) were treated for 24 h with 20 μg/ml rifampicin or 0.1% DMSO as negative control. After incubation, supernatant was removed, and cells were lysed with 100 μl of lysis buffer. Number of bacteria per cell lysate was measured by both the agar plate assay and the MGIT assay. The activity of the antibiotic was determined by calculating the fraction of bacteria observed in the rifampicin condition of the total CFU measured in control (=100%).

To determine the efficacy of antibiotics on extracellular bacteria, early log-phase Mav Wasabi culture was mixed in round-bottom 96-wells plates in duplicate with 1.29 μg/ml rifampicin, 1.74 μg/ml bedaquiline (kindly provided by Dirk Lamprecht, Janssen, Beerse, Belgium), or control (0.1% DMSO). These concentrations indicate the minimal inhibitory concentration (MIC) determined for each antibiotic by testing twofold serial drug dilutions against Mav Wasabi in liquid broth cultures ( Supplementary Figure 3 ). Plates were incubated at 37°C/5% CO₂ for 2 weeks. Once every 2 days, the wells were resuspended and absorbance at 600 nm was measured using the Envision Multimode Plate Reader (Perkin Elmer). For the determination of intracellular activity, primary human macrophages exposed to Mav Wasabi (10:1) in duplicate were treated for 24 h with 1.29 μg/ml rifampicin, 1.74 μg/ml bedaquiline, or control (0.1% DMSO). After treatment, supernatant was removed, and cells were lysed with 100 μl of lysis buffer. Cell lysates were further evaluated by the MGIT assay as described above. The activity of the antibiotics on bacteria was determined by calculating the fraction of bacteria observed in the rifampicin or bedaquiline conditions of the total CFU measured in control (=100%).

Normality of data was assessed using the Shapiro–Wilk test. For normally distributed paired datasets of more than two groups, we used repeated measures one-way ANOVA if data were determined by one independent variable, and repeated measures two-way ANOVA if two independent variables were involved. Paired and unpaired t-tests were used to evaluate differences in normally distributed datasets between two groups, whereas the Wilcoxon matched-pairs signed rank test was used for non-normally distributed paired data. To determine the strength of association between non-normally distributed datasets, the Spearman rank correlation test was used. Analyses were performed using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA), with p-values < 0.05 considered as significant.

The first step in developing the human cell-based in vitro infection models was the generation of a green fluorescent protein-expressing Mav strain. This was achieved by electroporating a hygromycin resistance conferring plasmid, pSMT3-Wasabi, into wild-type laboratory strain Mav 101. Successful transfection was confirmed by expression of the Wasabi fluorescent protein using flow cytometry ( Figure 1A ), and resistance to hygromycin by observing outgrowth ( Figure 1B ).

The OD factor of Mav [the number of colony-forming units per ml (CFU/ml) in a culture with an OD₆₀₀ value of 1.0] was determined to be able to prepare bacterial suspensions and infect cells with standardized MOI. To this end, growth kinetics of Mav were determined by measuring the optical density (OD₆₀₀) and enumerating CFU of Mav Wasabi cultures at 0, 24, 48, 72, and 96 h after the start of the culture ( Figure 1C ). Starting in early log-phase (OD₆₀₀ = 0.1), the bacterial culture reached an OD₆₀₀ value of 1.0 after 48 h. At the same time point, the number of CFU/ml was obtained and verified in multiple inocula to obtain the definitive OD factor of 2.4 × 10⁸ CFU/ml.

Ultimately, the bacteria grew to an OD₆₀₀ value of 2.2 within 96 h ( Figure 1C ). The doubling time was calculated for multiple Mav cultures and was determined to be 23 h on average (range: 17–33 h) ( Figure 1D ), which is in line with the slow replication rate reported in literature (Ratnatunga et al., 2020).

In order to investigate NTM infections at the intracellular bacterial level, we developed human cell-based infection models for Mav, adapted from our previously reported infection models for Mtb (Korbee et al., 2018; Vrieling et al., 2019). First, we evaluated the capacity of MelJuSo cells to engulf Mav and optimized the level of infection by adjusting the MOI to reach an infection percentage comparable to what we observed previously in our MelJuSo-Mtb infection model (Korbee et al., 2018). In Mav-infected MelJuSo cells, an MOI-dependent increase in infection was observed, as reflected by an increase in infection rate (% of infected cells) and intracellular bacterial loads directly after infection as determined by flow cytometry and CFU analysis, respectively ( Figures 2A, B ). By infecting cells for 1 h with an MOI of 10, 8% of the cells were infected as determined by flow cytometry, reflected in intracellular Mav counts of 1.2 × 10⁴ ± 2 × 10³ CFU. In contrast, Mtb-MelJuSo cells reached an infection rate of near 30% at an MOI of 10 ( Figure 2A ) (Korbee et al., 2018). Cells exposed to an MOI of 20, 50, or 100 of Mav showed a mean infection rate of 11%, 18%, or 22% and CFU counts of 2.5 × 10⁴ ± 8 × 10³, 5.3 × 10⁴ ± 2 × 10⁴, or 1.1 × 10⁵ ± 3 × 10⁴, respectively. After 24 h of incubation, intracellular bacterial loads were similar to bacterial loads directly after infection ( Figure 2B ), suggesting a steady state infection during the first 24 h.

In addition to the MelJuSo-Mav infection model, we also developed a Mav infection model using primary monocyte-derived human macrophages, differentiated into two diametrically opposed subsets, namely, GM-CSF-driven classically activated pro-inflammatory macrophages (M1), and M-CSF-driven alternatively activated anti-inflammatory macrophages (M2), which represent the two main phenotypes of human alveolar macrophages (Mitsi et al., 2018; Hu and Christman, 2019). A clear MOI-associated increase in infection was observed for both M1 and M2 ( Figure 2C ); using an MOI of 1, 10, and 100, M1 showed infection percentages of 6%, 22%, and 60%, respectively, while 7%, 64%, and 93% of M2 were infected. Using a similar model, the infection rates for MOI 10 Mtb-infected macrophages were reported to be 41% and 67% for M1 and M2, respectively ( Figure 2C ) (Korbee et al., 2018). No differences were observed in flow cytometry-based infection levels between M1 and M2, and also no consistent significant differences in numbers of CFU were observed between these cells ( Figure 2D ). In addition to the laboratory Mav strain, we also evaluated the phagocytosis capacity of the macrophages for the three Mav clinical isolates 100, 568, and 918. The uptake by M1 and M2 of these clinical isolates during infection at MOI 10 was in the same magnitude (3.3 × 10⁴ ± 5 × 10³, 2.4 × 10⁴ ± 4 × 10³ and 3.5 × 10⁴ ± 1 × 10³ CFU) as observed for the laboratory strain ( Figure 2E ).

The above results show that primary macrophages are more readily infected with Mav compared to MelJuSo cells. Using an MOI of 10 in the macrophage Mav model or an MOI of 50 in MelJuSo model will allow detection of at least a 3-log reduction (i.e., bacterial survival from 100% down to 0.1%), in intracellular bacterial load, which will be sufficient to identify efficacious (HDT) compounds, while at the same time not overloading the cells with bacteria.

To determine how effective macrophages are in controlling Mav infection, clearance of Mav Wasabi by M1 and M2 exposed to MOI 10 was assessed 24, 48, 72, and 144 h post-infection ( Figure 2F ). Numbers of CFU decreased in both M1 and M2, with M2 seemingly better in controlling the infection. At the last time point, 144 h post-infection, 65 ± 20% and 86 ± 12% of intracellular bacteria were eliminated in M1 and M2, respectively ( Figure 2F ).

Additionally, we compared the intracellular elimination of Mav by macrophages with Mtb over time. We previously described kinetic analysis of intracellular Mtb survival in a similar M2 model, which showed a rapid reduction in Mtb bacterial load (Vrieling et al., 2019). These cells eliminated Mtb by at least 85% after 24 h, implying that Mtb is instantly controlled after infection, while this was less profound for Mav (39 ± 17%, Figure 2F ). Mav was, however, controlled to a similar extent as Mtb eventually (86 ± 12% and 97.8% elimination, respectively).

To increase throughput and to enhance objectivity (since CFU agar plate assays are known to result in inter-observer variation when enumerating colonies), the BACTEC MGIT 960 system was used to quantify bacteria by measuring bacterial metabolic activity as a surrogate for bacterial loads.

Intracellular bacterial loads of Mav-infected macrophages estimated by the MGIT significantly correlated with the CFU counted from plates (Spearman r: 0.78; p-value = 0.011) and intra-assay variation for data obtained with the MGIT seemed to be smaller (coefficient of variation: 36% compared to 51% for plate-counted CFU analysis; p-value = 0.109) ( Figure 3A ).

To obtain further insight into the usefulness of our infection model, we compared the MGIT system to determine the activity of first-line antibiotic rifampicin on intracellular Mav to the classical CFU assay ( Figure 3B ). Rifampicin-induced effects determined by MGIT are in concordance with the classical CFU assay for both M1 and M2. This indicates that the MGIT system, which showed a trend of higher CFU numbers possibly due to the liquid medium as an inherent characteristic, was able to observe a compound-induced effect. Additionally, the intra-assay variation in MGIT seemed to be smaller compared to the classical CFU assay (coefficient of variation: 32% versus 78%, respectively; p-value = 0.170), as observed in Figure 3A . Based on these data, we considered the MGIT system as a viable alternative to plate-counting CFU analysis for the determination of intracellular bacterial loads.

Currently, the gold standard to evaluate antibacterial activity of chemical compounds is by monitoring the growth of bacteria in the extracellular space (i.e., broth microdilutions) (Brown-Elliott et al., 2012). Also identified in this way was the first new tuberculosis drug in several decades, bedaquiline, which showed bactericidal activity against (multi-drug resistant) Mtb but has also shown promising results against extracellular Mav and other NTM in vitro (Mahajan, 2013; Aguilar-Ayala et al., 2017; Brown-Elliott et al., 2017; Vesenbeckh et al., 2017). Interestingly, cases of bedaquiline resistance have also been reported (Philley et al., 2015; Alexander et al., 2017; Veziris et al., 2017). Here, we applied the MGIT system to drug susceptibility testing (DST) by determining the susceptibility to both rifampicin and bedaquiline of intracellular Mav (within M1) in comparison to extracellular bacteria (in liquid broth).

While a concentration of 1.29 μg/ml rifampicin significantly impaired growth of extracellular bacteria (97% as compared to untreated controls), only a 31% reduction was observed in intracellular bacteria ( Figure 4A ). In line, bedaquiline treatment (1.74 μg/ml) impaired extracellular bacterial growth completely, while intracellular bacteria were only reduced by 17% as compared to untreated controls ( Figure 4B ). These findings show the higher susceptibility of extracellular bacteria to antibiotics, indicating that extracellular drug testing might overestimate bacterial susceptibility to treatments during the course of intracellular infection in vivo. Taken together, our Mav macrophage model facilitates screening of antibacterial agents against intracellular Mav and emphasizes the importance of measuring the intracellular compartment on antibiotic susceptibility.