Differential antibiotic susceptibility of Mycobacterium abscessus variants in biofilms and macrophages compared to that of planktonic bacteria

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.

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.

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.

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.

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.

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.