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Susceptibility

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

Rebecca Greendyke, Thomas F. Byrd
Rebecca Greendyke
1The University of New Mexico School of Medicine, Albuquerque, New Mexico 87131
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Thomas F. Byrd
1The University of New Mexico School of Medicine, Albuquerque, New Mexico 87131
2Department of Medicine, New Mexico Veterans Affairs Health Care System, Albuquerque, New Mexico 87108
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  • For correspondence: tbyrd@salud.unm.edu
DOI: 10.1128/AAC.00986-07
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ABSTRACT

Mycobacterium abscessus causes refractory pulmonary infections requiring surgery for cure. It exists as a smooth biofilm-forming phenotype which is noninvasive and a rough, non-biofilm-forming phenotype which can invade macrophages and cause persistent pulmonary infection in mice. We have postulated that the dissociation of the smooth phenotype to the rough phenotype may lead to invasive lung disease following initial colonization of the airways. Amikacin, cefoxitin, and clarithromycin are standard therapies for this infection. We determined the MICs of these antibiotics against this pathogen in biofilms and macrophages, the niches that it likely occupies in the human host. Our results demonstrate that even though the MICs indicate sensitivity to these antibiotics, the minimal bactericidal concentrations for amikacin and clarithromycin were substantially higher and were out of the range of the concentrations achievable in serum. Cefoxitin demonstrated only bacteriostatic activity. In addition, although amikacin had modest activity against M. abscessus in biofilms, clarithromycin demonstrated only minimal activity at the highest concentrations tested. Our results indicate that M. abscessus in mature biofilms is in a stationary-phase state and that clarithromycin is relatively inactive against stationary-phase M. abscessus. In human macrophages, all three antibiotics were only bacteriostatic for M. abscessus variants at 10 times their MICs. These results suggest why treatment failure with antibiotics alone is common in the clinical setting of M. abscessus pulmonary infection. Determination of the efficacies of new antibiotics should include an assessment of their activities against the smooth and rough M. abscessus morphotypes in biofilms and macrophages.

Mycobacterium abscessus is a nontuberculous mycobacterium (NTM) which causes a variety of infections involving many sites, including the lung, skin, and soft tissue (24). Lung infection can occur as a syndrome involving bronchiectasis in middle-aged women similar to what has been described with Mycobacterium avium (21, 31). In addition, M. abscessus is an emerging pathogen associated with colonization and infection of the lungs of cystic fibrosis patients (11, 17, 29, 35). It has also been reported to cause disseminated disease in immunocompromised patients (34) or those with underlying deficiencies in the gamma interferon signaling pathway (15, 33).

We have found that M. abscessus exists in a rough form and a smooth form (5, 25). The smooth form expresses glycopeptidolipid (GPL) and forms biofilms but is noninvasive. The rough form expresses minimal amounts of GPL and is unable to form biofilms but is able to invade macrophages and cause persistent infection in mice (5, 26). We have also demonstrated that M. abscessus strains can bidirectionally change from the rough to the smooth morphotype and the smooth to the rough morphotype (26). We have postulated that this ability allows M. abscessus to transition from a noninvasive colonizer residing in human airways to an invasive human pathogen capable of invading the lung and disseminating through the body (26).

Even though NTM are sensitive to a number of antibiotics, infections with these bacteria require long courses of antimicrobial therapy. Recommendations for the treatment of pulmonary infection include combination antimicrobial therapy for periods ranging from 6 to 12 months. Despite the use of appropriate antimicrobial therapy, M. abscessus pulmonary infection is usually progressive, often necessitating lung resection for cure (13, 21).

The choice of antibiotic to be used for the treatment of a bacterial infection is based on the MIC of the antibiotic against the bacterium (4). Minimum bactericidal concentration (MBC) determinations are not standardized for clinical use but provide information as to whether an antibiotic's mode of action is primarily bacteriostatic rather than bactericidal (22). However, in the case of many different bacterial species, the conditions used for these assays do not mimic the conditions of an infection occurring in vivo. For example, biofilm formation affords many bacterial species protection against antibiotics normally active against the same bacteria in the planktonic state (16). In addition, antibiotics active against bacteria in the planktonic state may be unable to reach the specific compartments within cells, such as macrophages, where the same bacteria reside if they are intracellular pathogens (18).

Since it has been demonstrated that M. abscessus variants are able to change from a smooth biofilm-forming phenotype to a rough invasive phenotype (8, 26), this study was undertaken to compare the susceptibility of these bacteria in the planktonic state (as would occur during MIC and MBC assays in the laboratory) to their susceptibility within biofilms and macrophages (as would occur in vivo). Our results demonstrate the limitations of these assays in the assessment of M. abscessus antibiotic susceptibility vis à vis the preferred host niches that this organism likely occupies.

MATERIALS AND METHODS

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.

RESULTS

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.

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TABLE 1.

Amikacin and clarithromycin MBCs for M. abscessus 390S grown under different culture conditions

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.

FIG. 1.
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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.

FIG. 2.
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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).

FIG. 3.
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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).

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.

FIG. 5.
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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.

DISCUSSION

Both rough and smooth variants of M. abscessus occur as clinical isolates, with the rough phenotype predominating in patients with chronic pulmonary infection and with the smooth phenotype predominating in patients with wounds and contaminating environmental isolates (27; unpublished data). We have speculated that initial airway colonization occurs via biofilm formation by smooth variants which spontaneously give rise to rough variants possessing an invasive phenotype (26). Isolates with both the rough phenotype and the smooth phenotype have been isolated from the same patient simultaneously and have been found to belong to a single strain, supporting this hypothesis (27; unpublished data). The observation that many chronically infected patients harbor only a rough strain (27; unpublished data) in their lungs may be explained by the finding that smooth variants stimulate a minimal innate immune response, in contrast to the robust innate immune response triggered by rough variants (8; unpublished data). We speculate that a rough variant that arises from an airway-colonizing smooth strain would stimulate a strong innate immune response by resident alveolar macrophages. This would lead to an influx of effector cells, such as newly emigrated blood monocytes, which would clear the noninvasive smooth variant but which would be permissive for the growth of the rough variant, allowing it to persist and multiply.

Our study was undertaken to compare the susceptibilities of our M. abscessus variants in the planktonic state, as determined from the standard MICs and MBCs, to their susceptibilities within biofilms (390S smooth variant) and human mononuclear phagocytes (390R and 390V rough variants), the niches that they likely occupy in the human host. The MICs of amikacin, clarithromycin, and cefoxitin to our M. abscessus strains demonstrate susceptibility based on published values (40) and are well within the concentrations achievable in serum (1). In contrast, the amikacin and clarithromycin MBCs for the M. abscessus variants fall outside of this range, indicating that these antibiotics have only bacteriostatic activity against M. abscessus in serum. Cefoxitin demonstrates no MBC and demonstrates only bacteriostatic activity up to a concentration of 500 μg/ml, the highest concentration tested. Cefoxitin has been used to treat infections caused by rapidly growing NTM (23). Although the MICs for cefoxitin against various rapidly growing NTM have been reported, there is only one report indicating MBC testing, and this was with M. fortuitum. This study reported MICs for 13 M. fortuitum strains which ranged from 6.25 to 25 μg per ml. Cefoxitin was not bactericidal against any of the strains at concentrations up to 100 μg/ml (12). The bacteriostatic effect of cefoxitin against M. abscessus was observed under all conditions tested in our study, supporting its utility as an adjunctive agent and not as a primary antibiotic in treatment regimens.

One consideration in determining the efficacies of antibiotics against M. abscessus lung infection is the relationship of antibiotic MICs and MBCs to the drug concentrations achievable in the lung parenchyma and airways. Initial colonization of the lung airways in cystic fibrosis patients is likely facilitated by M. abscessus biofilm formation. None of these antibiotics could eradicate M. abscessus 390S from established biofilms, and MBCs could not be achieved in the biofilm. However, amikacin was able to reduce the M. abscessus CFU within the biofilm by a maximum of 1.1 log units, with its activity reaching a plateau at concentrations equal to or greater than 64 μg/ml. In one study that used single daily dosing, a mean peak serum amikacin level of 121.4 μg/ml resulted in measured concentrations in sputum of only 10 μg/ml (6), below the 64-μg/ml threshold that we found to be necessary to cause a substantial reduction in the M. abscessus CFU in biofilms. Despite the relatively low concentrations in sputum achieved with systemic administration, amikacin can be nebulized and administered as an aerosol directly into the lung. This results in much higher concentrations in the lung airway, with a reduction in the toxicity associated with systemic administration (10). In one illustrative case report, a cystic fibrosis patient with progressive cavitary M. abscessus pneumonia received conventional therapy with amikacin, cefoxitin, and clarithromycin for 6 months. This resulted in clinical and radiographic improvements, although sputum cultures remained positive for M. abscessus. Only after addition of maintenance nebulized amikacin did sputum cultures become negative (11). Thus, the results of our study support the use of clinical treatment strategies which utilize nebulized amikacin for pulmonary M. abscessus infection (14), particularly when GPL-expressing strains are isolated from the sputum.

In contrast to amikacin, clarithromycin had a minimal effect on M. abscessus survival in biofilms, even though it was active against bacteria in the planktonic state. Our results demonstrate that this is likely because M. abscessus 390S in biofilms is in a stationary phase of growth. Consistent with this, the susceptibility of planktonic M. avium to clarithromycin has been reported to be dependent on the growth phase, with log-phase bacteria being the most susceptible (3). The inability of clarithromycin to have a significant impact on M. abscessus 390S growing in biofilms is consistent with the findings described in numerous reports which demonstrate that a variety of bacteria in biofilms are resistant to antibiotics, even though they are susceptible to the same antibiotics in the planktonic state (19). In addition, clarithromycin has been found to have no significant effect on established M. avium biofilms, although it is able to inhibit new M. avium biofilm formation at concentrations less than the MIC (7).

All three antibiotics had only a bacteriostatic effect against M. abscessus within macrophages, which may also account for the refractoriness of invasive pulmonary infection to antibiotic therapy (21). Amikacin, an aminoglycoside antibiotic, acts primarily extracellularly due to its limited penetration into macrophages (32); in our assay it allowed intracellular replication to occur but limited the spread of M. abscessus to uninfected MDMs. An unexpected result of our study was the finding that clarithromycin was bacteriostatic but not bactericidal against both rough (390R and 390V) and smooth (390S) M. abscessus variants in human MDMs. Unlike many antibiotics, clarithromycin concentrates within the lung, achieving much higher levels than those that are concomitantly found in the serum (30). In particular, clarithromycin has been demonstrated to concentrate approximately 15- to 20-fold within macrophages; however, this does not lower the MIC required to inhibit the growth of M. avium in macrophages compared to that required to inhibit the growth of M. avium in broth medium (18). Similar to our results, others have demonstrated that clarithromycin exerts only a bacteriostatic effect on M. avium growth in macrophages (18). Our data, obtained by using MDMs, demonstrate only a bacteriostatic effect on intracellular M. abscessus with extracellular clarithromycin concentrations up to 16 μg/ml. At 16 μg/ml, the MBC for our M. abscessus variants in the planktonic state, the clarithromycin concentration within MDMs would greatly exceed the MBC. One explanation for the lack of intracellular killing may be that the bacteria reside in an intracellular compartment which is distinct from lysosomes, where clarithromycin is concentrated, as has been postulated for M. avium (18). Another explanation may relate to the optimal pH for clarithromycin activity. Mycobacterial phagosomes have a pH range of 6.3 to 6.5 (38). Phagolysosomes containing intracellular pathogens have been reported to have pHs in the range of 4.7 to 5.2 (28). The pH at which clarithromycin has optimal antimicrobial activity is 7.4, with significant reductions in activity at the lower pH values in the intracellular environments in which these pathogens reside (20). Consistent with our findings, it has been demonstrated that clarithromycin monotherapy for pneumonia results in symptomatic improvement and a favorable microbiologic response, but in the majority of patients it does not result in the clearance of M. abscessus from the sputum (13). This was the case, even though the isolates remained sensitive to clarithromycin (13).

ACKNOWLEDGMENTS

This work was supported by the U.S. Department of Veterans Affairs and the DeSouza Research Award from the American Lung Association (to T.F.B.).

FOOTNOTES

    • Received 30 July 2007.
    • Returned for modification 14 September 2007.
    • Accepted 24 March 2008.
  • Copyright © 2008 American Society for Microbiology

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Differential Antibiotic Susceptibility of Mycobacterium abscessus Variants in Biofilms and Macrophages Compared to That of Planktonic Bacteria
Rebecca Greendyke, Thomas F. Byrd
Antimicrobial Agents and Chemotherapy May 2008, 52 (6) 2019-2026; DOI: 10.1128/AAC.00986-07

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Differential Antibiotic Susceptibility of Mycobacterium abscessus Variants in Biofilms and Macrophages Compared to That of Planktonic Bacteria
Rebecca Greendyke, Thomas F. Byrd
Antimicrobial Agents and Chemotherapy May 2008, 52 (6) 2019-2026; DOI: 10.1128/AAC.00986-07
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KEYWORDS

Anti-Bacterial Agents
biofilms
macrophages
Mycobacterium Infections, Nontuberculous
nontuberculous mycobacteria
Plankton

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