Differential Antibiotic Susceptibility of Mycobacterium abscessus Variants in Biofilms and Macrophages Compared to That of Planktonic Bacteria

Bacterial strains. M. abscessus isogenic strains 390R, 390S, and 390V were used in these studies. Strain 390S has a smooth colony phenotype, while strains 390R and 390V have a rough phenotype (5, 8, 26). Briefly, 390R is a rough isolate from a patient which spontaneously dissociated to smooth variant 390S when it was subcultured (5). Strain 390V, a rough revertant, then arose from 390S on subculture (26). The rough variants are able to grow in macrophages and cause persistent infection in mice, while the 390S variant lacks these capabilities. Unlike the rough variants, the 390S variant expresses large amounts of GPL and is able to form biofilms (5, 26).

Bacteria were maintained as titered frozen stocks stored at −70°C with intermittent passage for 3 days on Middlebrook 7H11 agar, followed by flash freezing. All three variants have stable phenotypes with low rates of spontaneous reversion (5, 25, 26). The rate of spontaneous reversion of variant 390S to a rough phenotype is in the range of 1 in 10⁵ to 1 in 10⁶ (26).

Antibiotic MICs and MBCs.Amikacin (Sigma), cefoxitin (Sigma), and clarithromycin (USP Pharmacopeia) were prepared to a concentration of 1 mg/ml in Middlebrook 7H9 broth (Difco) supplemented with oleic acid-alubmin-dextrose-catalase and Tween 80 (7H9 medium). M. abscessus strains 390R, 390S, and 390V were grown on Middlebrook 7H11 (Difco) plates for 2 to 3 days until confluent growth was obtained. The bacteria were suspended in sterile phosphate-buffered saline, and the turbidity was adjusted to a 0.5 McFarland standard (approximately 1 × 10⁷ CFU/ml) with a Vitek colorimeter and a McFarland standard set. The bacteria were then diluted in 7H9 medium so that 100 μl contained approximately 1 × 10⁵ cells. The antibiotics were serially diluted to the desired final concentration, and 100 μl was added to the appropriate wells of a 96-well plate. One hundred microliters of the bacteria was added to each well, with the final antibiotic concentrations ranging from 1 to 128 μg/ml for amikacin, 2 to 500 μg/ml for cefoxitin, and 0.0625 to 256 μg/ml for clarithromycin. A positive growth control was included for each strain. The plates were incubated at 37°C in a humidifying chamber, and the MICs were visually determined at 96 h, with the MIC being recorded as the concentration in the first visually clear well (40). Experiments were also performed with cation-adjusted Mueller-Hinton broth at 37°C and 30°C, Sautons medium at 37°C and 30°C, and 7H9 medium at 30°C. Although MIC determinations are routinely used for susceptibility testing of NTM, determination of MBCs is not a standardized procedure and differences in interpretation between rapidly growing mycobacteria and slowly growing mycobacteria have been reported (22). For the purposes of this study, we determined MBCs by removing 200 μl from each well and plating serial dilutions onto 7H11 plates. The plates were incubated at 37°C for 4 to 5 days, and the MBC was recorded as the first antibiotic dilution which reduced M. abscessus growth by 99.9%.

Testing planktonic and biofilm cell resistance to killing.Although minimal media such as M63 minimal salts medium is able to support M. abscessus biofilm formation (data not shown), we have previously found that biofilm formation is optimal with Sautons medium (26); therefore, it was used in these experiments. The frozen stock of strain 390S was adjusted to 2.5 × 10⁵ cells/200 μl in Sautons medium, and 200 μl of this suspension was added to wells in an MBEC 96-well biofilm tray (Calgary biofilm device), as described previously (26). Within this tray, biofilms form on pegs protruding from the lid into individual wells containing bacteria and medium. Maximal biofilm growth on pegs is necessary so that factors such as nutrient limitation and antibiotic penetration can be adequately assessed, and the extent of growth varies among bacterial species (9). The trays were incubated for 3 days at 37°C in a humidifying chamber on a rotary shaker platform set at 150 rpm, and Sautons medium was replaced daily. After 96 h, the pegs were washed with sterile phosphate-buffered saline to remove any unattached bacteria. The pegs were aseptically removed from the tray lids, placed into Eppendorf tubes containing 1.0 ml Sautons medium, and sonicated in a cup horn sonicating device set at maximum power output for 30 s to dislodge the bacteria from the peg. Tenfold serial dilutions of bacterial sonicate from one set of triplicates was plated on Middlebrook 7H11 agar plates (100 15-mm bacteriological petri dishes) to determine the initial starting CFU on the pegs. At the same time point, the bacterial sonicate from another set of triplicate pegs was removed, placed in polypropylene culture tubes, and incubated at 37°C with or without antibiotics without agitation. Fresh medium with or without antibiotics was added to the remaining tray with intact biofilms, which was incubated at 37°C. After an additional 24 h, the bacteria from the biofilm pegs were dislodged. Serial dilutions of the sets of samples containing the 24-h resuspended biofilms and the sets of samples with resuspended biofilms from time zero cultured with or without antibiotics were plated on 7H11 plates. The plates were incubated at 37°C for 3 to 4 days, and the MBCs were determined. In some experiments, resuspended biofilms were incubated with clarithromycin for 24 h at various time points after removal from the pegs, corresponding to different phases of bacterial growth. The growth of these bacteria was compared to that of bacteria from the frozen stock.

Stationary-phase susceptibility.To generate spent medium for determination of the effect of nutrient limitation on the antibiotic MBCs for M. abscessus, 20 ml of 7H9 broth in a shaker flask was inoculated with 1.0 ml of medium containing 1 × 10⁷M. abscessus 390S (0.5 McFarland standard) and incubated at 37°C at 150 rpm. A control flask containing medium without bacteria was also incubated. After 3 days, the bacterial culture had reached stationary phase and the bacteria were removed by centrifugation. Both spent medium and replete medium (from the uninfected flask) were filtered through 0.2-μm-pore-size filters and stored at 4°C until they were used in subsequent experiments. The antibiotic MBCs for the M. abscessus variants were then determined by using both the spent medium and the replete medium.

MDM infection assay.Human mononuclear cells were isolated from buffy coats purchased from United Blood Services (Albuquerque, NM), as described previously (26), and cultured for 48 h in Teflon jars in Iscove's medium-5% normal human serum at 37°C in 5% CO₂ to facilitate monocyte maturation. Human monocytes were isolated by adherence to tissue culture wells (Falcon Primaria 24-well plate), followed by incubation for 24 h at 37°C in medium. Immediately prior to infection, monocyte-derived macrophage (MDM) monolayers were washed twice to remove nonadherent cells. A frozen stock of M. abscessus 390R, 390V, or 390S was added to the wells, resulting in a final concentration of 2.5 × 10⁵ bacteria/500 μl. The monolayers were incubated at 37°C in 5% CO₂ for 90 min, followed by three washes with medium to remove the bacteria not taken up by cells (a critical element of these experiments is the inclusion of human serum in the wash medium, which minimizes the adherence of 390S to the tissue culture wells and eliminates extracellular growth as an artifact). The lysis of the MDMs (26) was followed by plating the time zero counts of the macrophage-associated CFU on 7H11 plates. Antibiotics were then added to some of the remaining MDM-infected wells. At 24-h intervals, the wells to be plated were washed once with medium to remove extracellular bacteria, followed by the plating of the M. abscessus CFU from the MDM lysates on 7H11 plates. The remaining wells were washed twice with medium to minimize the potential for the extracellular growth of bacteria during the ensuing 24-h culture interval, followed by the readdition of antibiotics to the appropriate wells. In addition, the nuclear counts in replicate wells were determined at each time point, and the CFU was standardized to 10⁵ nuclei to account for any differences in macrophage numbers in the different monolayers (26). Viability was analyzed in replicate infected wells by trypan blue exclusion at each time point to ensure that the variants did not have a differential effect on macrophage viability independent of bacterial growth (26). In some experiments, infection was allowed to proceed for 24 h prior to addition of clarithromycin to determine the antimicrobial effect on M. abscessus cells actively growing in MDM monolayers.

Statistics.Data were compared by using Student's t test. Data were considered significant when the P value was <0.05.

M. abscessus MICs and MBCs.All three of our M. abscessus variants were susceptible to amikacin, clarithromycin, and cefoxitin, as determined by MIC testing. The MICs for amikacin, clarithromycin, and cefoxitin were 2 μg/ml, 0.25 μg/ml, and 8 μg/ml, respectively. The MICs were identical for the three variants. There were no differences in the MICs when the determinations were performed with 7H9 broth and when the determinations were performed with cation-adjusted Mueller-Hinton broth or Sautons medium or when the determinations were performed at 30°C (data not shown).

The MBCs for amikacin and clarithromycin were found to be substantially higher than their MICs and were identical for the three variants. The MBCs for amikacin and clarithromycin tested under standard conditions were 64 μg/ml and 16 μg/ml, respectively (Table 1). Unlike amikacin and clarithromycin, cefoxitin did not exhibit bactericidal activity against the M. abscessus variants when it was tested up to a concentration of 500 μg/ml.

M. abscessus 390S antibiotic susceptibility in biofilms and in nutrient-depleted medium.When it was grown as a biofilm, M. abscessus 390S was resistant to the bactericidal effects of amikacin and clarithromycin. Amikacin had modest activity against M. abscessus in biofilms, reducing the CFU by up to 1.1 log units, with maximal activity achieved at a concentration of 64 μg/ml (Fig. 1). In contrast, clarithromycin had a minimal effect on M. abscessus in biofilms, reducing the M. abscessus CFU by only 0.6 log unit at concentrations up to 512 μg/ml (Fig. 1). As in the planktonic state, cefoxitin had only bacteriostatic activity against M. abscessus in biofilms when it was tested at concentrations up to 1,024 μg/ml.

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FIG. 1.

M. abscessus 390S susceptibility to amikacin and clarithromycin in biofilms. M. abscessus 390S biofilms on pegs were incubated with various concentrations of amikacin or clarithromycin for 24 h, followed by sonication of the pegs to dislodge the bacteria and plating for CFU determination. Data represent the means of duplicate determinations ± standard deviations.

At the time that intact 390S biofilms were placed in antibiotics, replicate biofilms were sonicated to disrupt and resuspend the biofilm. Resuspended biofilms were then cultured with amikacin, clarithromycin, or cefoxitin at the same concentrations used with intact biofilms. We excluded an effect of sonication on growth in an experiment in which planktonic bacteria in mid-log phase were sonicated with the same energy and for the same duration of time as the bacteria dislodged from the biofilms and found that sonication had no effect on subsequent growth compared to the effect of no sonication on the growth of the controls (data not shown).

The MBC for amikacin against M. abscessus 390S from resuspended biofilms was regained at a concentration 64 μg/ml, equivalent to the standard MBC (Table 1). In the case of clarithromycin, the MBC of M. abscessus 390S from resuspended biofilms was much higher than the standard MBC (256 μg/ml versus 16 μg/ml), although it became more susceptible than bacteria within the biofilm (Fig. 1; Table 1). Consistent with the fact that cefoxitin has only a bacteriostatic effect against M. abscessus 390S in the planktonic state and in biofilms, the bacteria remained resistant to this antibiotic under these culture conditions.

We next sought to determine the reason for the difference in clarithromycin sensitivity between M. abscessus 390S planktonic bacteria and resuspended M. abscessus 390S biofilms (Table 1). There is evidence that the bacteria in biofilms have reached a stationary state due to nutrient limitation in the local environment (2, 19). It has been established that there is a close correlation between bacterial growth phase and killing by beta-lactam antibiotics, with bacteria being susceptible to the bactericidal effects of beta-lactam antibiotics in the logarithmic phase but not in the stationary phase (37, 39). A direct relationship between nutrient limitation, stationary phase, and decreased susceptibility to ampicillin and the fluoroquinolone antibiotic ciprofloxacin has been demonstrated for Klebsiella pneumoniae in biofilms (2). Although clarithromycin is a macrolide antibiotic, we postulated that nutrient limitation and the resultant stationary state might account for M. abscessus clarithromycin resistance within the biofilm and immediately after resuspension of the biofilm. To test this hypothesis, we determined the amikacin and clarithromycin MBCs for M. abscessus 390S cultured in spent 7H9 medium. Consistent with our hypothesis, there was an insignificant change in the MBC of amikacin (1 dilution) when planktonic bacteria in replete medium were compared to planktonic bacteria in depleted medium (Table 1). In contrast, the MBC of clarithromycin increased 4 dilutions when planktonic bacteria in replete medium were compared to planktonic bacteria in depleted medium. Notably, the clarithromycin MBC for planktonic bacteria in depleted medium was the same as the MBC for the resuspended biofilm (Table 1). This suggests that nutrient limitation in association with the stationary state may be playing a role in the decreased bactericidal activity of clarithromycin against M. abscessus 390S growing within biofilms.

M. abscessus 390S clarithromycin susceptibility during different phases of bacterial growth.Stationary-phase bacteria placed into fresh medium will resume logarithmic growth after a lag phase. If M. abscessus clarithromycin resistance in biofilms is the result of nutrient limitation and the stationary state, then clarithromycin susceptibility should return when resuspended biofilms in fresh medium resume growth after a lag phase. In support of this hypothesis, bacteria in resuspended biofilms exhibited a lag phase that lasted approximately 48 h before the biofilms entered the logarithmic phase of growth (Fig. 2A), indicating that while these bacteria were in the biofilm they had been in a stationary state. Clarithromycin added at concentrations up to 128 μg/ml during this time for a 24-h period had a minimal effect on M. abscessus 390S viability (Fig. 2A). When clarithromycin was added during mid-logarithmic-phase growth, it decreased the M. abscessus 390S CFU by over 1 log unit (Fig. 2B). Since bacteria frozen at minus 70°C have entered a metabolically inactive state, they enter an initial lag phase when they are thawed and placed in fresh medium. In a parallel experiment, similar numbers of M. abscessus 390S from a frozen stock were incubated with clarithromycin at the same concentrations and time intervals as the bacteria removed from biofilms. A similar trend in terms of the magnitude of the effect of clarithromycin during different growth phases was noted (data not shown). These results support the concept that at least part of the M. abscessus 390S clarithromycin resistance in biofilms is due to stationary-state existence.

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FIG. 2.

M. abscessus 390S sensitivity to clarithromycin at various time points after resuspension of biofilms. The top panels show the bacterial CFU with no antibiotics, and the bottom panels show the log change in CFU with the addition of various concentrations of clarithromycin. (A) Addition of clarithromycin to bacteria resuspended from biofilms during the lag phase of bacterial growth; (B) addition of clarithromycin to bacteria resuspended from biofilms during logarithmic-phase growth. *, P < 0.05 for comparison of log bacterial CFU with no antibiotics to log bacterial CFU with clarithromycin at 120 h (all antibiotic concentrations). Data represent the means of duplicate determinations ± standard deviations.

M. abscessus antibiotic susceptibility in human MDMs.Our previous studies have demonstrated that a single clinical M. abscessus isolate can revert from a rough to a smooth phenotype during culture and can then revert back again to a rough phenotype (5, 26). We postulated that M. abscessus smooth variants such as variant 390S are able to colonize the lungs of patients with bronchiectasis by virtue of their biofilm-forming capability and that within the lung airways, smooth variants give rise to rough invasive mutants (26). In support of this, it has been demonstrated in a mouse model of infection that reversion of the smooth phenotype to a rough invasive phenotype can occur in vivo (8). M. abscessus 390V is one such spontaneous, invasive rough mutant that lacks a biofilm-forming capability and that expresses minimal amounts of GPL (26). The ability of M. abscessus 390V to replicate in human MDMs treated with antibiotics was examined. At 10 times their MICs, both amikacin and cefoxitin had only a bacteriostatic effect on M. abscessus 390V intracellular growth (Fig. 3A). In the case of cefoxitin and amikacin, this result is not surprising, since we demonstrate that cefoxitin is bacteriostatic against planktonic bacteria and aminoglycosides such as amikacin are known to act primarily against extracellular bacteria (32). Since clarithromycin is known to concentrate within mononuclear phagocytes (18), we sought to determine whether its effect would be bacteriostatic or bactericidal against both the 390V variant, which causes persistent infection in MDMs, and the 390S variant, which does not. At 10 times its MIC, clarithromycin had a bacteriostatic effect against the 390V variant in MDMs and caused a slight, but significant reduction in the bacterial CFU in macrophages infected with the 390S variant (Fig. 3B).

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FIG. 3.

M. abscessus antibiotic susceptibility in human MDMs. (A) Human MDM monolayers were infected with M. abscessus 390V, which is able to replicate in human mononuclear phagocytes, followed by incubation with amikacin or cefoxitin (20 and 80 μg/ml, respectively). *, P < 0.05 for comparison of growth with and without antibiotics. (B) Human MDM monolayers were infected with M. abscessus 390V or M. abscessus 390S, which is unable to replicate in macrophages. Following infection, the macrophages were incubated with clarithromycin (2.5 μg/ml), which is known to concentrate in macrophages, to determine whether it has a bacteriostatic or a bactericidal effect against intracellular M. abscessus. *, P < 0.05 for comparison of growth with and without antibiotics. The numbers of intracellular CFU were determined at the indicated time points. Data represent the means of duplicate determinations ± standard deviations by using M. abscessus 390V (A and B) and the means of two experiments done in duplicate ± standard errors of the means for M. abscessus 390S (B). Error bars not visible fall within the time point markers.

We next sought to determine whether a bactericidal effect might occur if infected MDMs were incubated with higher concentrations of clarithromycin. It has been reported that bacteriostatic or bactericidal antibiotic activity against mycobacteria in macrophages can be determined by observing the effects of various antibiotic concentrations on intracellular growth at 48 h (36). A bacteriostatic effect is evidenced by diminished growth but the stable recovery of intracellular bacterial CFU over a range of increasing antibiotic concentrations, while a bactericidal effect is evidenced by the recovery of decreasing numbers of intracellular bacterial CFU as the antibiotic concentration increases (36). Although there was a significant decrease in the variant 390V and 390S intracellular CFU in macrophages treated with clarithromycin, the intracellular CFU did not decrease further with increasing clarithromycin concentrations. This demonstrates a bacteriostatic effect against both variants over a wide range of antibiotic concentrations (Fig. 4).

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FIG. 4.

M. abscessus 390S and 390V susceptibility in human MDMs to a wide range of clarithromycin concentrations. Human monocytes were infected with M. abscessus 390S or 390V and incubated with or without clarithromycin over a range of concentrations by following a previously described paradigm (36) used to determine the mode of activity of antibiotics against intracellular bacteria. For M. abscessus 390V there were 4.3 × 10⁵ ± 1.5 × 10⁵ CFU per 10⁵ monocytes and for M. abscessus 390S there were 7.3 × 10⁵ ± 2.6 × 10⁵ CFU per 10⁵ monocytes at time zero immediately after infection and before addition of clarithromycin. The numbers of intracellular CFU were determined at 48 h after infection. *, P < 0.05 for comparison of the growth of both 390S and 390V with various concentrations of clarithromycin to their growth without clarithromycin. Data represent the means of triplicate determinations ± standard deviations.

Human MDMs were infected with bacteria that had been aliquoted and stored as a frozen stock. Our results indicate that clarithromycin had a relatively small effect on planktonic M. abscessus 390S cells which were not actively replicating (Fig. 2A) but demonstrated enhanced activity as the bacteria entered logarithmic-phase growth (Fig. 2B). We thus sought to determine the effect of clarithromycin on M. abscessus variants actively growing in macrophages by adding clarithromycin 24 h after initial infection, during the phase of active intracellular replication. In contrast to our observations for M. abscessus 390S in the planktonic state, addition of clarithromycin to infected macrophages at 24 h had only a bacteriostatic effect on 390R (Fig. 5A) and 390V (data not shown) and had a minimal effect on 390S (Fig. 5B). These results demonstrate that the efficacy of clarithromycin against M. abscessus in macrophages remains bacteriostatic throughout the growth cycle within human macrophages.

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FIG. 5.

M. abscessus (Mabs) susceptibility to clarithromycin (CLR) during active growth in human MDMs. Human MDMs were infected with M. abscessus, followed by addition of clarithromycin (4.0 μg/ml) immediately after infection or 24 h after infection. The top panels show the bacterial CFU with no antibiotic, and the bottom panels show the log change in CFU with the addition of clarithromycin. (A) M. abscessus 390R. *, P < 0.05 for comparison of the log bacterial CFU with no antibiotics to the log bacterial CFU with clarithromycin. (B) M. abscessus 390S. *, P < 0.05 for comparison of the log bacterial CFU with no antibiotics to the log bacterial CFU with clarithromycin. Data represent the means of duplicate determinations.