M. abscessus Bamboo is a clinical isolate from a patient with amyotrophic lateral sclerosis and bronchiectasis. The strain was provided by Wei Chang Huang (Taichung Veterans General Hospital, Taichung, Taiwan). The strain was previously whole-genome sequenced which showed that it belongs to M. abscessus subsp. abscessus and harbors an inactive, clarithromycin-sensitive erm(41) C28 sequevar (Yee et al., 2017). The strain was maintained in complete Middlebrook 7H9 broth (BD Difco 271310, Spark, MD, United States) supplemented with 0.2% glycerol (Fisher Scientific G31-1, Fair Lawn, NJ, United States), 0.05% Tween-80 (Sigma-Aldrich P1754-1L, St. Louis, MO, United States) and 10% Middlebrook albumin-dextrose-catalase (ADC) (BD BBL 212352, Sparks, MD, United States). CFU were determined using Middlebrook 7H10 agar (BD Difco 262710 Sparks, MD, United States) supplemented with 0.2% glycerol and 10% Middlebrook oleic acid-albumin-dextrose-catalase (OADC) (BD BBL 212351 Sparks, MD, United States). Antibiotics were purchased from Sigma-Aldrich St. Louis, MO, United States (with exception of imipenem which was purchased from Cayman Chemicals, Ann Arbor, MI, United States), dissolved in 100% DMSO (MP Biomedicals 190186, Solon, OH, United States). Amikacin, imipenem, moxifloxacin and cefoxitin were dissolved in water as recommended by manufacturer and sterilized using 0.2 μm PTFE membrane filters (Acrodisc PALL 4225, Ann Arbor, MI, United States) before use. The N-substituted indolemethylamine IMA6 N-[(6-Trifluoromethyl-1H-indol-2-yl)methyl]cyclooctanamine and the N¹,N³-dialkyl substituted dioxonaphthoimidazolium SA23 1-Butyl-2-methyl-3-octyl-4,9-dioxo-4,9-dihydro-1H-naphtho[2,3-d]imidazol-3-ium bromide were synthesized in-house in the context of M. tuberculosis drug discovery projects (Supplementary Figures S1, S2; Dick and Go, unpublished).

Exponentially growing Mab Bamboo precultures were generated from frozen stocks that were diluted into 12 mL 7H9 medium in 50 mL conical Falcon tubes (Corning 352070, Reynosa, TM, United States). Precultures were aerated with stir-bars at 450 rpm on magnetic stirring platforms at 37°C and grown overnight to mid-log phase (OD₆₀₀ = 0.4–0.6). To grow experimental aerated nutrient rich cultures, mid-log phase precultures were diluted to OD₆₀₀ = 0.05 (4.9 × 10⁶ CFU/mL) and 1 mL aliquots were transferred into 14 mL vented, round-bottom tubes (Thermo Fisher 150268, Rochester, NY, United States). The tubes were incubated on a tilted rack at 37°C on an orbital shaker at 220 rpm. Growth was measured by CFU determination. Serial dilution in phosphate buffered saline (Thermo Fisher 10010023) containing 0.025% Tween-80 (PBS/Tween-80) were plated on 7H10 agar. To determine the MIC of antibiotics, appropriate ranges of drug concentration were added at time point 0 h (Figure 1C) and incubated for 48 h with antibiotic before CFU were determined. The lowest concentration that prevented growth in CFU was defined as the MIC of the respective drug. Determination of MBC₉₀, the concentration of drug that reduces viable numbers 10-fold compared to the starting CFU/mL, were carried out the same way with increased concentrations of drug up to 100 μM.

Innovotech MBEC 96-well Biofilm Assay Plates (Innovotech 19111, Edmonton, AB, United States) were used, and the supplier’s manual was followed with minor modifications. Mid-log phase Mab Bamboo precultures (section Growth in Aerated Nutrient-Rich Broth and MIC, MBC₉₀ Determination) were spun down at 3200 × g for 10 min at 25°C and washed with 7H9 medium without Tween-80 (7H9nt). Bacteria were resuspended into 25 mL 7H9nt to an OD₆₀₀ = 0.0125 (1.0 × 10⁶ CFU/mL). One hundred and fifty microliter of culture was dispensed into each well of MBEC multi-titer plates and the polystyrene protrusions (pegs) of the MBEC lid were inserted into the culture-containing wells for 24 h at 37°C on an orbital shaker at 110 rpm to allow attachment of the bacteria to the pegs and initiation of biofilm growth. The lids with the pegs were transferred to a new MBEC multi-titer plate containing 150 μL of fresh 7H9nt medium per well without bacteria (time point 0 h in Figure 1D). After that, the pegs with growing biofilm were transferred once a day to a new multi-titer plate containing fresh 7H9nt medium. To measure growth of the biofilm formed on the peg, the pegs were washed in 200 μL of 7H9nt medium before they were aseptically removed and placed in 1.7 mL microcentrifuge tubes (VWR 87003-294, Rador, PA, United States) containing 500 μL PBS/Tween-80. The microcentrifuge tubes were vigorously vortexed at 2,000 rpm for 90 s at 25°C to detach the bacteria from the pegs before samples were serially diluted and plated for the determination of CFU/peg. To determine the biofilm MIC of antibiotics, appropriate drug concentrations were added at the 24 h timepoint (Figure 1D) and CFU were determined after 48 h of incubation with antibiotic. The lowest concentration that prevented increase in CFU compared to the value at 24 h, was defined as the biofilm MIC of the respective drug. Determination of biofilm MBC₉₀, the concentration of drug that reduces viable numbers 10-fold compared to the CFU/peg at 24 h, were carried out the same way with increased concentrations of drug up to 100 μM.

Exponentially growing Mab Bamboo precultures (section Growth in Aerated Nutrient-Rich Broth and MIC, MBC₉₀ Determination) were spun down at 3200 × g for 10 min at 25°C and washed three times with phosphate buffered saline containing 0.025% Tyloxapol (Sigma-Aldrich T0307-10G, St. Louis, MO, United States) (PBS/Tyloxapol). Washed culture was then diluted with PBS/Tyloxapol to OD₆₀₀ = 0.2 (1.8 × 10⁷ CFU/mL). One hundred milliliter of OD₆₀₀ = 0.2 cultures were incubated in roller bottles (Corning 430195, Oneonta, NY, United States) at 37°C and 2 rpm. CFU were determined by plating. For determination of MBC₉₀ against nutrient-starved non-replicating culture, 1 mL aliquots of 144 h old roller bottle cultures (Figure 1A) were transferred into 14 mL vented, round-bottom tubes (Thermo Fisher 150268, Rochester, NY, United States) and appropriate concentrations of drugs up to 100 μM were added. The tubes were incubated on a tilted rack at 37°C on an orbital shaker at 220 rpm for 48 h of incubation with antibiotics before plating and CFU enumeration.

Exponentially growing Mab Bamboo precultures (section Growth in Aerated Nutrient-Rich Broth and MIC, MBC₉₀ Determination) were diluted with fresh 7H9 medium to OD₆₀₀ = 0.02. 7 mL aliquots were transferred to 10 mL air-tight vacutainer tubes (BD 366430, Franklin Lakes, NJ, United States) containing elliptical stir bars (Radleys RR98096, Wood Dale, IL, United States). Tubes were incubated at 37°C on magnetic stirring platforms at 150 rpm. To monitor depletion of oxygen, methylene blue was added to cultures at a concentration of 1.5 μg/mL. The dye decolorized around day 5, indicating anaerobiosis. CFU were determined by plating. For determination of MBC₉₀ against anaerobic non-replicating culture, appropriate concentrations of drugs up to 100 μM were added using a 23G needle (BD 305194, Franklin Lakes, NJ, United States) to minimize the re-introduction of air at 144 h (Figure 1B), i.e., after reaching anaerobiosis, and cultures were incubated for 48 h with antibiotics before plating and CFU enumeration.

Note: In the current work we use the term “persister assays” for the assays described under sections Growth as Biofilm and MIC, MBC₉₀ Determination, Survival and MBC₉₀ Determination Under Nutrient Starvation, and Survival and MBC₉₀ Determination Under Anaerobic Conditions. By this we mean assays that grow bacterial cultures that show a shift in MIC and/or MBC₉₀ to higher values when compared to the standard planktonic, aerated nutrient rich assay. Thus, these bacteria show “drug tolerance” or “phenotypic drug resistance.” For a detailed discussion (and various definitions) of these non-genetic resistance phenomena we refer to reviews by Gold and Nathan (Gold and Nathan, 2017) for mycobacteria and Balaban and colleagues (Balaban et al., 2019) for a consensus statement.

Genomic DNA of Mab Bamboo was extracted from bacterial culture samples following the Qiagen DNA extraction protocol and using the QIAamp 96 DNA Qiagen Kit (Sukumar et al., 2014). Samples were thawed and 100 μL was transferred to a 96 well plate (Qiagen 19585, Germantown, MD, United States) containing 180 μL of ASL buffer (Qiagen 19082, Germantown, MD, United States), 20 μL of proteinase K (Qiagen 19131, Germantown, MD, United States), 1 μL of reagent DX (Qiagen 19088, Germantown, MD, United States) and 250 μL of 0.1 mm zirconia-silica beads (MP Biomedicals 116540428, Solon, OH, United States) in each well. Samples were inactivated for 1 h at 80°C. The plate was centrifuged at 200 × g to remove foam and precipitate beads, then loaded in a QIAcube HT (Qiagen). Samples were mixed with 600 μL of 50% buffer AL (Qiagen 19075, Germantown, MD, United States) and 50% ethanol (Fisher Scientific A962-4, Fair Lawn, NJ, United States). 600 μL of supernatant from each well was transferred onto a 96 well plate silica column (Qiagen 28181, Germantown, MD, United States) and drawn through using a vacuum pump. DNA samples were cleaned with 3 consecutive wash steps: 600 μL of buffer AW1 (Qiagen 19081, Germantown, MD, United States), 600 μL of AW2 (Qiagen 19072, Germantown, MD, United States) and 600 μL of ethanol. DNA was then eluted with a total of 100 μL buffer AE (Qiagen 19077, Germantown, MD, United States). Mab Chromosome Equivalent (CEQ) was quantified using the previously described protocol (Munoz-Elias et al., 2005) with erm41 primer-probe combination [Primers: 5′-AAACCGTGAACGAAGGTGTC-3′ and 5′-TGGTATCCGCTCACTGATGA-3′ Probe: 5′-/56-FAM/CCGAATCCG/ZEN/GTGTTCGCTCA/3lABkFQ/-3′] (Integrated DNA Technologies, Coralville, IA, United States) adapted from Lin et al. (2014). CEQ quantification was achieved by building standard curves using serial dilutions of whole Mab genome prepared from broth culture. Real time quantitative PCR (qRT-PCR) reactions were performed in duplicate at dilution 1x, 3x, and 9x (for a total of six q-PCR measures per sample) on the Mx3005P (Stratagene) using TaqMan Universal Master Mix II (Life Technologies 4440038, Carlsbad, CA, United States).

To determine whether Mab Bamboo is capable of surviving nutrient starvation, aerated pre-cultures growing exponentially in nutrient rich broth were transferred to PBS devoid of any nutrients and CFU over time were measured. Figure 1A shows that nutrient starved cultures stopped growth and maintained viability for 10 days, the duration of the experiment. This result shows that Mab can survive in saline lacking nutrients without significant loss of viability.

To determine whether Mab is capable of surviving oxygen deprivation, we transferred exponentially growing Mab pre-cultures into sealed containers (containing nutrient-rich broth), thus limiting the total amount of oxygen available. Figure 1B shows that Mab grows for 1 day and then enters stationary phase. Growth termination occurred earlier and at a lower cell density when compared to aerated unsealed (thus providing unlimited oxygen supply) cultures in the same medium (Figure 1C), presumably because the oxygen concentration fell below a level supporting growth. Indeed, when the oxygen indicator dye methylene blue was added to the sealed cultures, fading of the color (indicating microaerobic conditions) was observed at day four and decolorization (indicating anaerobiosis) was observed around day five (Figure 1B). Anaerobic cultures maintained viability at least until day 10, the duration of the experiment. This result shows that Mab can survive anaerobiosis without significant loss of viability.

Together, these results show that obligate aerobe and chemo-organo-heterotroph Mab is capable of surviving the absence of oxygen as well as nutrients for extended periods of time in an apparent non-replicating state.

The observed constant CFU/mL in the nutrient and oxygen-starved cultures could be due to a balance of bacterial death and “cryptic” growth or the bacteria may enter a state of “non-replicating persistence,” i.e., stop growing but maintaining viability. To differentiate between these two possibilities, chromosome equivalents per mL (CEQ/mL) over time were determined. If the observed constant CFU/mL is due to balanced death and growth, CEQ/mL of culture should increase over time due to the accumulation of the chromosomes from dead bacteria. If, however, the bacteria adopt a state of non-replicating persistence, CEQ/mL should be constant. Figure 2 shows that in both, nutrient-starved and oxygen-deprived conditions, CEQ/mL of culture did not change in a major way over time. This suggests that the bacteria under both culture conditions enter a state of non-replicating persistence, i.e., the bacteria largely stop replication and maintain viability under these adverse conditions.

To determine whether Mab is capable of growing as biofilm, aerated (planktonic) pre-cultures growing exponentially in nutrient rich broth were transferred into the wells of multi-titer plates containing nutrient broth. A surface for biofilm growth was provided by pegs – attached to the specialized lid of the multi-titer plates – that were inserted into the cultures present in the wells. Bacteria in the broth of the wells were allowed to attach to the surface of the pegs for 1 day after which the lids with the pegs were transferred to new micro-titer plates containing nutrient-rich medium without bacteria. Growth of bacteria attached to the surface of the pegs was determined by recovering the bacterial films attached to the surface of the pegs and CFU enumeration. Figure 1D shows that bacteria attached to the pegs and grew exponentially on this surface for 2 days before entering stationary phase. These results show that Mab is capable of growing as biofilm on this plastic surface.

Taken together, these experiments show that Mab Bamboo is able to survive nutrient and oxygen starvation in a non-replicating state and can grow as biofilms. The described culture models are reproducible and can thus be used for testing for activity of anti-bacterials against the different physiological forms of Mab generated under these culture conditions.

Before determining the effect of being in a non-replicating state or growing as biofilm on drug susceptibility we established a baseline by measuring the standard MIC and MBC₉₀ for clinically used anti-Mab drugs against Mab growing under standard conditions. First, exponentially growing, aerated nutrient-rich broth, planktonic Mab cultures were treated with increasing concentrations of drug to determine the concentration that inhibited growth (measured by CFU enumeration). Table 1 shows that most drugs show potent MICs. Smaller than 5 μM MIC values were displayed by the macrolides clarithromycin, azithromycin, the aminoglycoside amikacin, the glycylcycline tigecycline, the beta-lactam imipenem, the oxazolidinone linezolid, the fluoroquinolone moxifloxacin and the riminophenazine clofazimine. Only the tetracycline minocycline and the beta-lactam cefoxitin showed non-favorable MICs in the range of 20–30 μM against Mab Bamboo. To evaluate bactericidal activity of anti-Mab drugs under these standard conditions MBC₉₀ values were measured. Again, Mab cultures exponentially and planktonically growing in aerated, nutrient-rich broth were treated with increasing concentrations of drug (starting with the MICs of the drugs and up to a maximum of 100 μM) to determine the effect on viability of the cultures (measured by CFU enumeration). Only four of the drugs – amikacin, imipenem, cefoxitin, and moxifloxacin showed appreciable bactericidal activity with MBC₉₀ values between 3 and 25 μM. For clarithromycin, azithromycin, minocycline, tigecycline, linezolid, and clofazimine – 100 μM, the highest concentration tested, resulted in less than a 10-fold reduction of viable counts. Thus, these antibiotics did not display any significant bactericidal activity. This result shows that about half of the commonly used drugs are not bactericidal against rapidly growing planktonic cultures.

Next, we determine the bactericidal activity of the same drugs against non-replicating cultures, either induced by nutrient or by oxygen starvation. Table 1 shows that all the standard growth assay non-bactericidal drugs were also non-bactericidal against the non-replicating bacterium. Importantly, the antibiotics that had shown bactericidal activity against growing culture lost this activity completely against non-replicating culture (MBC₉₀ > 100 μM). These results show that the non-replicating state of Mab displays extreme tolerance against currently used “bactericidal” antibiotics. It is interesting to note that the non-bactericidal clofazimine (MBC₉₀ in broth culture > 100 μM) gained some bactericidal activity against oxygen starved bacteria (MBC₉₀ = 31 μM). The mechanism of action of this riminophenazine is not known and the reason for the gain of bactericidal activity against anaerobic non-replicating Mab remains to be determined.

Above, we determined the effect of Mab entering non-replicating states on the bactericidal activity of drugs. Next, we asked whether growth as biofilm, as opposed to growth as planktonic culture, affects the MIC and the MBC₉₀ of drugs. Table 1 shows that biofilm MICs increased for most drugs around 3–4-fold. This result suggests that Mab growing as biofilm is less susceptible to growth inhibition when compared to Mab growing in suspension culture. The determination of MBC₉₀ for drugs against growing biofilm shows that this trend also holds true for bactericidal activities. Amikacin, imipenem, cefoxitin and moxifloxacin, the drugs that showed appreciable bactericidal activities against exponentially growing planktonic bacteria showed 2–8-fold shifts to higher MBC₉₀ values against exponentially growing biofilm bacteria. Interestingly, the “outlier” was again clofazimine, showing an MBC₉₀ of 12.5 μM against biofilm-growing bacteria whereas it was not bactericidal in broth culture (MBC₉₀ > 100μM).

Together, these results show that about half of the anti-Mab drugs are not bactericidal even under standard aerated, nutrient-rich culture conditions which allow the bacteria rapid growth. The few drugs that show bactericidal activity against growing planktonic cultures lose this activity completely against non-growing forms of the bacterium, whether induced by nutrient starvation or oxygen depletion. This shows that non-replicating Mab displays extreme resistance to killing by our clinically used drugs. Finally, we find that Mab growing as biofilm displays tolerance to growth inhibition when compared to Mab growing in suspension culture. Furthermore, biofilm-growing bacteria show reduced killing by drugs that display bactericidal activity against planktonically growing Mab.

The new “persister” culture models we developed here are intended to be used as secondary whole cell assays in Mab drug discovery flowcharts to complement standard MIC and MBC₉₀ profiling of new anti-mycobacterial agents. The mycobacterial membrane and its associated functions recently gained interest as target space (Chen et al., 2018). For instance, several diverse chemotypes were found to show attractive potency against M. tuberculosis and Mab by inhibiting the essential mycolic acid transporter MmpL3 (Li et al., 2018). To determine the attractiveness of this target for Mab “persister” states we tested a novel N-substituted indolemethylamine (IMA6, Supplementary Figure S1) in our assays. IMA6 was identified in a parallel drug discovery project on M. tuberculosis and shown to target MmpL3 in this pathogen (Dick and Go, unpublished). Data displayed in Table 1 confirm that the compound also shows attractive MIC and bactericidal activity against Mab growing under standard conditions. Interestingly, bactericidal activity of IMA6 was retained against nutrient-starved and oxygen-depleted non-replicating states, and against biofilm-growing Mab. These results suggest MmpL3 as an attractive “anti-persister” target. As a second example we tested a novel N¹,N³-dialkyl substituted dioxonaphthoimidazolium, SA23 (Supplementary Figure S2), which we are currently characterizing for activity against M. tuberculosis. In M. tuberculosis SA23 appears to act via redox cycling involving the electron transport chain (Dick and Go, unpublished). Table 1 shows that this agent displayed attractive MIC and exerts bactericidal activity against both oxygen deprivation as well as nutrient starvation induced non-replicating Mab. These results suggest that the described “persister assays” can be employed for the identification of novel chemical entities displaying bactericidal activity against Mab “persisters.”