Lack of antimicrobial bactericidal activity in Mycobacterium abscessus

Abstract

Antibiotic therapy of infections caused by the emerging pathogen Mycobacterium abscessus is challenging due to the organism's natural resistance toward most clinically available antimicrobials. We investigated the bactericidal activity of antibiotics commonly administered in M. abscessus infections in order to better understand the poor therapeutic outcome. Time-kill curves were generated for clinical M. abscessus isolates, Mycobacterium smegmatis, and Escherichia coli by using antibiotics commonly categorized as bactericidal (amikacin and moxifloxacin) or bacteriostatic (tigecycline and linezolid). In addition, the impact of aminoglycoside-modifying enzymes on the mode of action of substrate and nonsubstrate aminoglycosides was studied by using M. smegmatis as a model organism. While amikacin and moxifloxacin were bactericidal against E. coli, none of the tested compounds showed bactericidal activity against M. abscessus. Further mechanistic investigations of the mode of action of aminoglycosides in M. smegmatis revealed that the bactericidal activity of tobramycin and gentamicin was restored by disruption of the chromosomal aac(2') gene in the mycobacterial genome. The lack of bactericidal antibiotics in currently recommended treatment regimens provides a reasonable explanation for the poor therapeutic outcome in M. abscessus infection. Our findings suggest that chromosomally encoded drug-modifying enzymes play an important role in the lack of aminoglycoside bactericidal activity against rapidly growing mycobacteria.

FIG 1

Concentration- and time-dependent activity of 4 antimicrobials against E. coli strain AS19. Liquid cultures (2.0 × 10⁵ to 3.6 × 10⁵ CFU/ml) were exposed to linezolid (A), tigecycline (B), amikacin (C), and moxifloxacin (D) at 2-fold increasing drug concentrations (from 1× to 32× the MIC). Cell suspensions were plated on drug-free media for CFU determination after 0, 30, 60, and 120 min of incubation at 37°C.

FIG 2

Concentration- and time-dependent activity of 4 antimicrobials against M. abscessus subsp. abscessus clinical isolate 500042/08 (rrs and rrl wild type). Liquid cultures (10.4 × 10⁵ to 18.2 × 10⁵ CFU/ml) were exposed to linezolid (A), tigecycline (B), amikacin (C), and moxifloxacin (D) at 2-fold increasing drug concentrations (from 1× to 32× the MIC). Cell suspensions were plated on drug-free media for CFU determination after 0, 6, 12, and 24 h of incubation at 37°C.

FIG 3

Time-kill kinetics of amikacin against three clinical isolates of each M. abscessus subsp. abscessus (A to C), M. abscessus subsp. bolletii (D to F), and M. abscessus subsp. massiliense (G to I). All strains were rrs and rrl wild type, as confirmed by DNA sequencing. Liquid cultures (0.7 × 10⁵ to 17.2 × 10⁵ CFU/ml) were exposed to amikacin at 2-fold increasing drug concentrations (from 1× to 32× the MIC). Cell suspensions were plated on drug-free media for CFU determination after 0, 6, and 12 h of incubation at 37°C.

FIG 4

Concentration- and time-dependent activity of selected antimicrobials against M. smegmatis mc² 155. Liquid cultures (5.6 × 10⁵ to 14.2 × 10⁵ CFU/ml) were exposed to linezolid (A), tigecycline (B), amikacin (C), and moxifloxacin (D) at 2-fold increasing drug concentrations (from 1× to 32× the MIC). Bacterial suspensions were plated on drug-free media for CFU determination after 0, 6, and 12 h of incubation at 37°C.

FIG 5

Time-kill kinetics of aminoglycosides against M. smegmatis mc² 155 (A, C, and E) and M. smegmatis EP10 (B, D, and F). Liquid cultures (9.2 × 10⁵ to 16.5 × 10⁵ CFU/ml) were exposed to 2-fold increasing drug concentrations of amikacin (A and B), tobramycin (C and D), and gentamicin (E and F). Bacterial suspensions were plated on drug-free media for CFU determination after 0, 6, and 12 h of incubation at 37°C.