Infections caused by the difficult-to-treat bacterium Mycobacterium abscessus are increasing in frequency. Rifabutin, in contrast to rifampin, appears to be active in vitro against M. abscessus, especially against clarithromycin-resistant strains.
KEYWORDS: Mycobacterium abscessus, imipenem, in vitro synergy, macrolide resistance, rifabutin
ABSTRACT
Infections caused by the difficult-to-treat bacterium Mycobacterium abscessus are increasing in frequency. Rifabutin, in contrast to rifampin, appears to be active in vitro against M. abscessus, especially against clarithromycin-resistant strains. However, explorations for potential synergy between rifabutin and available antimicrobials are currently limited. In vitro synergism between rifabutin and 10 antimicrobials was evaluated in 31 mycobacterial strains by the checkerboard method. The fractional inhibitory concentration index (FICI) was calculated for each rifabutin-based combination. The colony morphology was recorded. Molecular methods for determination of the M. abscessus subspecies and analysis of macrolide resistance were performed by sequencing of the secA1, rpoB, hsp65, erm(41), and rrl genes. Rifabutin yielded an MIC50 of 16 mg/liter (range, 2 to 32 mg/liter) against 26 clinical M. abscessus isolates (comprising 13 M. abscessus subsp. abscessus and 13 M. abscessus subsp. massiliense isolates) and 5 reference strains, including M. abscessus subsp. abscessus ATCC 19977, M. abscessus subsp. bolletii BCRC 16915, M. abscessus subsp. massiliense BCRC 16916, M. chelonae ATCC 35752, and M. peregrinum ATCC 700686. Significant synergism, classified by an FICI of ≤0.5, was demonstrated for the combinations of rifabutin and imipenem in 100% of M. abscessus subsp. abscessus and 69% of M. abscessus subsp. massiliense isolates, and significant synergism for rifabutin and tigecycline was demonstrated in 77% of M. abscessus subsp. abscessus and 69% of M. abscessus subsp. massiliense isolates. Among the 6 clarithromycin-resistant (MICs ≥ 8 mg/liter) M. abscessus subsp. abscessus isolates, the combination of rifabutin and clarithromycin was 100% synergistic. Rifabutin showed promising in vitro synergism with first-line anti-M. abscessus agents, especially for macrolide-resistant M. abscessus subsp. abscessus isolates.
INTRODUCTION
Mycobacterium abscessus is a notorious multidrug-resistant pathogen which has emerged as a global threat among chronic lung disease, surgical, and neutralizing anti-interferon gamma autoantibody-producing patients (1–4). In addition to causing challenging and possibly lifelong infections among individuals, it has also caused large outbreaks in health care settings due to resistance to antiseptics and disinfectants (5–7). M. abscessus is intrinsically resistant not only to the classical antituberculosis drugs but also to most currently available antimicrobials (8). Of 1,040 FDA-approved drugs screened, only 7 compounds demonstrated activity with an MIC of ≤8 mg/liter against M. abscessus (9).
It was therefore surprising that a recent study showed rifabutin to be active in vitro against M. abscessus, especially against clarithromycin-resistant strains (10). Although clarithromycin has become the drug of choice for M. abscessus infections and therapeutic successes were reported in the 1990s, clarithromycin resistance has since been associated with primary and secondary treatment failures (11). In most M. abscessus subsp. abscessus isolates, a functional erythromycin ribosome methyltransferase gene [(erm)41] leads to intrinsic inducible macrolide resistance. In M. abscessus subsp. massiliense, however, truncation and, thus, a loss of function of the erm(41) gene often restores susceptibility to macrolides (12). Macrolide resistance could also be determined by the presence of a thymine rather than a cytosine nucleotide at position 28 in erm(41) (i.e., the T28 and not the C28 sequevar) and by point mutations (at positions A2058 and A2059) in a region of the rrl gene encoding the peptidyltransferase domain of the 23S rRNA (12–14). Any of the above-described mechanisms causing macrolide resistance renders cure of M. abscessus infections unlikely in individuals with cystic fibrosis or neutralizing anti-interferon gamma autoantibodies (3, 15).
The aims of this study were to evaluate the potential in vitro activity of rifabutin and the effect of combining rifabutin with agents currently recommended for treatment of infections caused by M. abscessus isolates, such as clarithromycin, imipenem, amikacin, and tigecycline. We also investigated the effect of combining rifabutin with agents with which less clinical experience has accumulated, such as clofazimine and ceftibuten, to explore alternative treatment regimens.
RESULTS
A total of 26 clinical isolates, which comprised 13 M. abscessus subsp. abscessus and 13 M. abscessus subsp. massiliense isolates, as determined by multilocus sequence analysis (MLSA), were included in this study. The isolates were sporadic, and phylogenetic analysis confirmed that the isolates were not clustered in an outbreak. The 26 M. abscessus clinical isolates had been obtained from the blood (n = 4), cerebrospinal fluid (n = 2), lymph nodes (n = 3), surgical wounds (n = 5), skin and soft tissue (n = 8), pleural fluid (n = 1), ascites (n = 1) and lung (n = 2) of 26 patients with active disease.
Morphotypes.
Of the 13 M. abscessus subsp. abscessus isolates, 10 exhibited a smooth colony morphology and 3 exhibited rough morphotypes. Similarly, 9 of the 13 M. abscessus subsp. massiliense isolates exhibited smooth colonies and 4 exhibited rough colonies.
erm(41) and rrl partial gene sequencing.
Among the 13 M. abscessus subsp. abscessus isolates, there were 3 isolates harboring the C28 variant and 10 isolates harboring the T28 variant of the erm(41) gene, and no point mutations were found in the rrl gene. In contrast, all except 1 of the 13 M. abscessus subsp. massiliense isolates harbored a truncated erm(41) gene. The one M. abscessus subsp. massiliense isolate with the full erm(41) gene harbored the C28 variant. In addition, 2 M. abscessus subsp. massiliense isolates from 2 different patients harbored rrl mutations (one A2057G and one A2058G point mutation).
Antimicrobial susceptibility testing.
The single- and dual-drug (rifabutin-based) susceptibility test results for the 11 antimicrobial agents and 10 combinations are shown in Tables 1 and 2, respectively.
TABLE 1.
MICs of 11 antimicrobials tested individually against Mycobacterium abscessus subsp. abscessus and Mycobacterium abscessus subsp. massiliense isolatesa
| Subspecies and drug | MIC (mg/liter) |
Susceptibility breakpoint (mg/liter)b |
||||
|---|---|---|---|---|---|---|
| 50% | 90% | Range | Susceptible | Intermediate | Resistant | |
| M. abscessus subsp. abscessus (n = 13)c | ||||||
| Rifabutin | 16 | 32 | 8 to 32 | ≤2 (0, 0) | >2 (13, 100.0) | |
| Clarithromycin | 4 | 8 | 0.5 to 16 | ≤2 (5, 38.5) | 4 (2, 15.4) | ≥8 (6, 46.2) |
| Amikacin | 32 | 32 | 16 to 64 | ≤16 (2, 15.4) | 32 (10, 76.9) | ≥64 (1, 7.7) |
| Cefoxitin | 32 | 32 | 16 to 64 | ≤16 (1, 7.7) | 32–64 (12, 92.3) | ≥128 (0, 0) |
| Ceftibuten | 256 | 256 | 128 to 256 | ≤16 (0, 0) | 32–64 (0, 0) | ≥128 (13, 100.0) |
| Clofazimine | 1 | 2 | 0.5 to 2 | ≤2 (13, 100.0) | ≥8 (0, 0) | |
| Doxycycline | >64 | >64 | >64 | ≤1 (0, 0) | 2-4 (0, 0) | ≥8 (13, 100.0) |
| Imipenem | 16 | 16 | 8 to 64 | ≤4 (0, 0) | 8–16 (11, 84.6) | ≥32 (2, 15.4) |
| Linezolid | 32 | 32 | 4 to 64 | ≤8 (1, 7.7) | 16 (0, 0) | ≥32 (12, 92.3) |
| Moxifloxacin | 32 | 32 | 8 to 256 | ≤1 (0, 0) | 2 (0, 0) | ≥4 (13, 100) |
| Tigecycline | 0.5 | 1 | 0.25 to 1 | ≤0.5 (10, 76.9) | 1 (3, 23.1) | ≥2 (0, 0) |
| M. abscessus subsp. massiliense (n = 13)d | ||||||
| Rifabutin | 16 | 16 | 2 to 16 | ≤2 (1, 7.7) | >2 (12, 92.3) | |
| Clarithromycin | 0.25 | 2 | <0.03 to >256 | ≤2 (11, 84.6) | 4 (0, 0) | ≥8 (2, 15.4) |
| Amikacin | 32 | 32 | 8 to 64 | ≤16 (3, 23.1) | 32 (9, 69.2) | ≥64 (1, 7.7) |
| Cefoxitin | 32 | 64 | 16 to 128 | ≤16 (2, 15.4) | 32-64 (10, 76.9) | ≥128 (1, 7.7) |
| Ceftibuten | 256 | 256 | 128 to 256 | ≤16 (0, 0) | 32-64 (0, 0) | ≥128 (13, 100.0) |
| Clofazimine | 2 | 2 | 0.5 to 2 | ≤2 (13, 100.0) | ≥8 (0, 0) | |
| Doxycycline | >64 | >64 | 8 to >64 | ≤1 (0, 0) | 2-4 (0, 0) | ≥8 (13, 100.0) |
| Imipenem | 16 | 16 | 8 to 64 | ≤4 (0, 0) | 8–16 (11, 84.6) | ≥32 (2, 15.4) |
| Linezolid | 32 | 32 | 1 to 32 | ≤8 (2, 15.4) | 16 (4, 30.8) | ≥32 (7, 53.8) |
| Moxifloxacin | 32 | 32 | 2 to 128 | ≤1 (0, 0) | 2 (1, 7.7) | ≥4 (12, 92.3) |
| Tigecycline | 0.5 | 1 | 0.06 to 1 | ≤0.5 (10, 76.9) | 1 (3, 23.1) | ≥2 (0, 0) |
The results of broth microdilution susceptibility testing are for each antimicrobial tested alone.
Data in parentheses represent the number, percent, of isolates with the indicated result. Data in bold indicate the MIC breakpoints applied in this study.
There were T28 (n = 10) and C28 (n = 3) sequevars among the 13 isolates. None of the isolates had rrl mutations.
A truncated erm41 (n = 12), the C28 sequevar (n = 1), and rrl mutations (A2057G, A2058G; n = 2) were found among the 13 isolates.
TABLE 2.
MICs of 10 antimicrobials tested in combination with rifabutin using the checkerboard broth microdilution method against Mycobacterium abscessus subsp. abscessus and Mycobacterium abscessus subsp. massiliense isolates
| Subspecies and drug | MIC (mg/liter) |
Categorical MIC breakpoints (mg/liter)a |
No. (%) of isolates for which the combination showed synergy (FICI ≤ 0.5) | ||||
|---|---|---|---|---|---|---|---|
| 50% | 90% | Range | Susceptible | Intermediate | Resistant | ||
| M. abscessus subsp. abscessus (n = 13)b | |||||||
| Rifabutin | 4* | 8* | 1 to 32 | ≤2 (4, 30.8) | >2 (9, 69.2) | ||
| Clarithromycin | 0.5* | 1* | 0.25 to 1 | ≤2 (13, 100.0) | 4 (0, 0) | ≥8 (0, 0) | 8 (61.5) |
| Amikacin | 8* | 16 | 1 to 32* | ≤16 (12, 92.3) | 32 (1, 7.7) | ≥64 (0, 0) | 7 (53.8) |
| Cefoxitin | 32 | 32 | 2 to 32 | ≤16 (6, 46.2) | 32-64 (7, 53.8) | ≥128 (0, 0) | 4 (30.8) |
| Ceftibuten | 64* | 128 | 8 to 128 | ≤16 (1, 7.7) | 32-64 (8, 61.5) | ≥128 (4, 30.8) | 8 (61.5) |
| Clofazimine | 1 | 2 | 0.5 to 2 | ≤2 (13, 100.0) | ≥8 (0, 0) | 0 (0) | |
| Doxycycline | >64 | >64 | 32 to >64 | ≤1 (0, 0) | 2–4 (0, 0) | ≥8 (13, 100.0) | 0 (0) |
| Imipenem | 1* | 2* | 0.5 to 8 | ≤4 (12, 92.3) | 8–16 (1, 7.7) | ≥32 (0, 0) | 13 (100.0) |
| Linezolid | 16 | 32 | 4 to 32 | ≤8 (5, 38.5) | 16 (3, 23.1) | ≥32 (5, 38.5) | 4 (30.8) |
| Moxifloxacin | 32 | 32 | 8 to 256 | ≤1 (0, 0) | 2 (0, 0) | ≥4 (13, 100) | 1 (7.7) |
| Tigecycline | 0.12* | 0.25* | 0.06 to 0.25 | ≤0.5 (13, 100.0) | 1 (0, 0) | ≥2 (0, 0) | 10 (76.9) |
| M. abscessus subsp. massiliense (n = 13)c | |||||||
| Rifabutin | 8 | 16 | 0.12 to 16 | ≤2 (3, 23.1) | >2 (10, 76.9) | ||
| Clarithromycin | 0.12 | 0.25* | <0.03 to >256 | ≤2 (12, 92.3) | 4 (0, 0) | ≥8 (1, 7.7) | 3 (23.1) |
| Amikacin | 16 | 16 | 4 to 32 | ≤16 (11, 84.6) | 32 (2, 15.4) | ≥64 (0, 0) | 2 (15.4) |
| Cefoxitin | 32 | 32 | 4 to 64 | ≤16 (4, 30.8) | 32–64 (9, 69.2) | ≥128 (0, 0) | 2 (15.4) |
| Ceftibuten | 128 | 128 | 32 to 256 | ≤16 (0, 0) | 32–64 (4, 30.8) | ≥128 (9, 69.2) | 2 (15.4) |
| Clofazimine | 2 | 2 | 0.5 to 2 | ≤2 (13, 100.0) | ≥8 (0, 0) | 0 (0) | |
| Doxycycline | >64 | >64 | 4 to >64 | ≤1 (0, 0) | 2-4 (1, 7.7) | ≥8 (12, 92.3) | 0 (0) |
| Imipenem | 2* | 8 | 0.25 to 32 | ≤4 (10, 76.9) | 8–16 (2, 15.4) | ≥32 (1, 7.7) | 9 (69.2) |
| Linezolid | 16 | 16 | 0.5 to 32 | ≤8 (6, 46.2) | 16 (5, 38.5) | ≥32 (2, 15.4) | 2 (15.4) |
| Moxifloxacin | 32 | 32 | 2 to 128 | ≤1 (0, 0) | 2 (1, 7.7) | ≥4 (12, 92.3) | 0 (0) |
| Tigecycline | 0.12* | 0.25* | 0.06 to 0.25 | ≤0.5 (13, 100.0) | 1 (0, 0) | ≥2 (0, 0) | 9 (69.2) |
Data in parentheses represent the number, percent, of isolates with the indicated result. Data in bold indicate the MIC breakpoints applied in this study. The results of the broth microdilution susceptibility testing for two-drug combinations read at the minimal fractional inhibitory concentration index (FICI) are marked by an asterisk (*) if a fourfold or more decrease was observed when the drug was tested in rifabutin-based combinations (as shown here) compared to when it was tested alone (Table 1). For the case of rifabutin, the two-drug combination results refer to those for rifabutin combined with clarithromycin.
There were T28 (n = 10) and C28 (n = 3) sequevars among the 13 isolates. None of the isolates had rrl mutations.
A truncated erm41 (n = 12), the C28 sequevar (n = 1), and rrl mutations (A2057G, A2058G; n = 2) were found among the 13 isolates.
Single-drug susceptibility testing.
The clarithromycin MIC50 and MIC90 were 4 and 8 mg/liter, respectively, for the M. abscessus subsp. abscessus isolates and 0.25 and 2 mg/liter, respectively, for the M. abscessus subsp. massiliense isolates. Phenotypic macrolide resistance was observed, which is in line with the presence of an intact erm(41) or T28 sequevar for the former isolates and the rrl point A2058G mutation for the latter isolates. The proportion of clarithromycin-resistant isolates was 46.2% and 15.4% for the M. abscessus subsp. abscessus and M. abscessus subsp. massiliense isolates, respectively.
Apart from the clear differences in macrolide susceptibility between the M. abscessus subsp. abscessus and M. abscessus subsp. massiliense isolates, the MIC50/MIC90 of amikacin, ceftibuten, doxycycline, moxifloxacin, and tigecycline tested alone for both subspecies were exactly the same at 32/32 mg/liter, 256/256 mg/liter, >64/>64 mg/liter, 32/32 mg/liter, and 0.5/1 mg/liter, respectively. There were also few intersubspecies differences in the MIC50 and MIC90 values for rifabutin, clofazimine, cefoxitin, and imipenem. In other words, all M. abscessus isolates, regardless of the subspecies, had clofazimine MICs of ≤2 mg/liter, and all except one M. abscessus subsp. massiliense isolate had a rifabutin MIC of >2 mg/liter. The majority of isolates were intermediately susceptible to cefoxitin (92.3% versus 76.9% for the M. abscessus subsp. abscessus and M. abscessus subsp. massiliense isolates, respectively) and imipenem (84.6% versus 84.6%), but linezolid resistance was greater among the M. abscessus subsp. abscessus isolates than among the M. abscessus subsp. massiliense isolates (92.9% versus 53.8%, respectively).
The colony morphology did not affect the susceptibility of the M. abscessus isolates to amikacin, cefoxitin, ceftibuten, doxycycline, and moxifloxacin (data not shown). However, lower MIC50/MIC90 values for rough versus smooth morphotypes for clofazimine (0.5/1 versus 2/2 mg/liter), rifabutin (4/16 versus 16/32 mg/liter), linezolid (16/32 versus 32/64 mg/liter), and tigecycline (0.25/0.25 versus 0.5/1 mg/liter), in contrast to higher MIC50/MIC90 values for imipenem (16/64 versus 8/16 mg/liter), were observed. The two rough colony-forming M. abscessus subsp. abscessus T28 variants had an unexpectedly low clarithromycin MIC of 1 mg/liter, whereas the other smooth colony-forming T28 variants were clarithromycin resistant, with clarithromycin MICs ranging from 4 to 16 mg/liter.
Dual-drug (rifabutin-based) susceptibility testing by the checkerboard method and FIC determination.
Synergy could be demonstrated for the combinations of rifabutin and imipenem in 100% and 69.2% of M. abscessus subsp. abscessus isolates and M. abscessus subsp. massiliense isolates, respectively. Remarkably, in combination with rifabutin and when the result was read at the minimum fractional inhibitory concentration (FIC), the MIC50 and MIC90 of imipenem and the proportion of imipenem-nonsusceptible isolates decreased from 16 to 1 mg/liter, from 32 to 2 mg/liter, and from 100% to 7.7%, respectively, for M. abscessus subsp. abscessus. The reciprocal decrease in the MIC50 and MIC90 of rifabutin (in combination with imipenem and when the result was read at the minimum FIC) and the proportion of isolates with rifabutin MICs of >2 mg/liter was from 16 to 1 mg/liter, from 32 to 2 mg/liter, and from 100% to 7.7%, respectively, for M. abscessus subsp. abscessus. The combination of rifabutin and imipenem was synergistic for all other standard strains of rapidly growing mycobacterial isolates, including M. abscessus subsp. bolletii, M. chelonae, and M. peregrinum (Table 3).
TABLE 3.
Antimicrobial susceptibility testing of reference strains of rapidly growing mycobacteria by checkerboard broth microdilution methoda
| Mycobacterial strain | Rifabutin + clarithromycin |
Rifabutin + amikacin |
Rifabutin + cefoxitin |
Rifabutin + ceftibuten |
Rifabutin + clofazimine |
Rifabutin + doxycycline |
Rifabutin + linezolid |
Rifabutin + moxifloxacin |
Rifabutin + imipenem |
Rifabutin + tigecycline |
||||||||||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| MIC (mg/liter) |
FICI | MIC (mg/liter) |
FICI | MIC (mg/liter) |
FICI | MIC (mg/liter) |
FICI | MIC (mg/liter) |
FICI | MIC (mg/liter) |
FICI | MIC (mg/liter) |
FICI | MIC (mg/liter) |
FICI | MIC (mg/liter) |
FICI | MIC (mg/liter) |
FICI | |||||||||||||||||||||||||||||||
| A | a | B | b | A | a | B | b | A | a | B | b | A | a | B | b | A | a | B | b | A | a | B | b | A | a | B | b | A | a | B | b | A | a | B | b | A | a | B | b | |||||||||||
| M. abscessus subsp. abscessus ATCC 19977 | 32 | 32 | 0.5 | 0.5 | 2 | 32 | 4 | 32 | 8 | 0.38 | 32 | 32 | >256 | >256 | 2 | 32 | 16 | 256 | 128 | 1 | 32 | 16 | 2 | 1 | 1 | 32 | 32 | >64 | >64 | 2 | 32 | 16 | 64 | 32 | 1 | 32 | 32 | 16 | 32 | 3 | 32 | 8 | 16 | 4 | 0.5 | 32 | 8 | 0.5 | 0.12 | 0.49 |
| M. abscessus subsp. bolletii BCRC 16915 | 16 | 2 | 4 | 0.25 | 0.19 | 16 | 8 | 32 | 16 | 1 | 16 | 16 | 32 | 32 | 2 | 16 | 4 | 256 | 128 | 0.75 | 16 | 16 | 2 | 2 | 2 | 16 | 16 | >64 | >64 | 2 | 16 | 8 | 64 | 32 | 1 | 16 | 16 | 32 | 32 | 2 | 16 | 1 | 16 | 0.5 | 0.09 | 16 | 2 | 0.5 | 0.06 | 0.25 |
| M. abscessus subsp. massiliense BCRC 16916 | 8 | 2 | 0.5 | 0.25 | 0.75 | 8 | 4 | 32 | 16 | 1 | 8 | 2 | 64 | 32 | 0.75 | 8 | 2 | 256 | 128 | 0.75 | 8 | 8 | 2 | 2 | 2 | 8 | 1 | 2 | 0.25 | 0.25 | 8 | 4 | 32 | 16 | 1 | 8 | 8 | 32 | 32 | 2 | 8 | 0.25 | 64 | 4 | 0.09 | 8 | 0.5 | 2 | 0.25 | 0.19 |
| M. peregrinum ATCC 700686 | 16 | 8 | 0.5 | 0.25 | 1 | 16 | 4 | 1 | 0.25 | 0.5 | 16 | 4 | 16 | 2 | 0.38 | 8 | 8 | 256 | 256 | 2 | 16 | 8 | 1 | 0.5 | 1 | 8 | 1 | 0.5 | 0.25 | 0.63 | 8 | 4 | 8 | 4 | 1 | 8 | 8 | <0.25 | <0.25 | 2 | 8 | 1 | 4 | 0.25 | 0.19 | 8 | 4 | 0.12 | 0.06 | 1 |
| M. chelonae ATCC 35752 | 16 | 16 | 0.25 | 0.25 | 2 | 8 | 2 | 64 | 16 | 0.5 | 16 | 16 | >256 | >256 | 2 | 16 | 16 | 256 | 256 | 2 | 16 | 16 | 2 | 2 | 2 | 16 | 4 | 32 | 4 | 0.38 | 8 | 8 | 2 | 2 | 2 | 16 | 8 | 0.5 | 0.25 | 1 | 16 | 1 | 4 | 0.5 | 0.19 | 16 | 2 | 0.5 | 0.015 | 0.16 |
| M. chelonae (clinical) | 32 | 32 | 1 | 2 | 3 | 32 | 4 | 64 | 16 | 0.38 | 32 | 32 | >256 | >256 | 2 | 32 | 32 | 256 | 256 | 2 | 32 | 32 | 1 | 1 | 2 | 32 | 4 | 1 | 0.25 | 0.38 | 32 | 32 | 32 | 32 | 2 | 32 | 32 | 16 | 16 | 2 | 32 | 8 | 32 | 8 | 0.5 | 32 | 8 | 0.5 | 0.12 | 0.49 |
A, MIC of rifabutin (drug A) tested alone; a, MIC of rifabutin (drug A) tested with the second drug in the combination (drug B); B, MIC of the standard antimycobacterial drug B tested alone; b, MIC of the standard antimycobacterial drug B tested in combination with rifabutin; FICI, fractional inhibitory concentration index. FICIs of ≤0.5, indicating synergism, are in boldface.
Synergy could be demonstrated for the combination of rifabutin and tigecycline in 76.9% of M. abscessus subsp. abscessus isolates and 69.2% of M. abscessus subsp. massiliense isolates. The combination of rifabutin and tigecycline lowered the MIC50 and MIC90 of tigecycline and the proportion of isolates with tigecycline MICs of >0.5 mg/liter from 0.5 to 0.12 mg/liter, from 1 to 0.25 mg/liter, and from 23.1% to 0%, respectively, for both subspecies of M. abscessus. The reciprocal decrease in the MIC50 and MIC90 of rifabutin (in combination with tigecycline) and the proportion of isolates with rifabutin MICs of >2 mg/liter was from 16 to 4 mg/liter, from 32 to 8 mg/liter, and from 93.3% to 61.5%, respectively, for both subspecies of M. abscessus. Stratified by intrinsic macrolide resistance, rifabutin-tigecycline synergy was observed in 3 of 5 (60%) clarithromycin-susceptible and in 7 of 8 (88%) of clarithromycin-nonsusceptible M. abscessus subsp. abscessus isolates. This combination was also synergistic for the reference strains of M. abscessus subsp. bolletii and M. chelonae but not for M. peregrinum (Table 3).
When clarithromycin was combined with rifabutin, synergy was seen in 61.5% (n = 8) of M. abscessus subsp. abscessus isolates and in 23.1% (n = 3) of M. abscessus subsp. massiliense isolates. Stratified by initial clarithromycin susceptibility, rifabutin-clarithromycin synergy was observed in 1 of 5 (20%) clarithromycin-susceptible and in 6 of 6 (100%) clarithromycin-resistant M. abscessus subsp. abscessus isolates and in 2 of 11 (18%) clarithromycin-susceptible and in 1 of 2 (50%) clarithromycin-resistant M. abscessus subsp. massiliense isolates.
For M. abscessus subsp. abscessus isolates, the MIC50 and MIC90 for clarithromycin (when combined with rifabutin and when the result was read at the FIC index [FICI]) decreased from 4 to 0.5 mg/liter and from 8 to 0.5 mg/liter, respectively, and the proportion of clarithromycin-resistant isolates decreased from 61.5% to 0%. While for M. abscessus subsp. massiliense isolates the MIC50 and MIC90 for clarithromycin (in combination with rifabutin and when the result was read at the FICI) decreased from 0.25 to 0.12 mg/liter and from 2 to 0.25 mg/liter, respectively, and only one clarithromycin-resistant isolate remained. The reciprocal decrease in the MIC50 and MIC90 of rifabutin (in combination with clarithromycin) and the proportion of isolates with rifabutin MICs of >2 mg/liter was from 16 to 4 mg/liter, from 32 to 8 mg/liter, and from 100% to 69.2%, respectively, for the M. abscessus subsp. abscessus isolates. The MIC50 and MIC90 of rifabutin (in combination with clarithromycin) for M. abscessus subsp. massiliense isolates did not change much and went from 16 to 8 mg/liter and from 16 to 16 mg/liter, respectively, and the proportion of M. abscessus subsp. massiliense isolates with rifabutin MICs of >2 mg/liter from 92.3% to 76.9%.
The combination of rifabutin with doxycycline or with clofazimine was indifferent for all M. abscessus isolates. In fact, the MICs of the individual drugs for these mycobacteria remained the same when they were tested alone or in combination. However, the combination of rifabutin with doxycycline was synergistic for M. chelonae ATCC 35752 (Table 3).
The combination of rifabutin with amikacin, cefoxitin, ceftibuten, or linezolid was synergistic in ≤60% M. abscessus isolates, with more synergy being demonstrated for M. abscessus subsp. abscessus than for M. abscessus subsp. massiliense. Rifabutin with amikacin was synergistic for the M. peregrinum ATCC 700686 and M. chelonae ATCC 35752 isolates. Rifabutin with moxifloxacin was synergistic for only one M. abscessus subsp. abscessus strain and was indifferent for all other rapidly growing mycobacteria tested in this study. None of the tested combinations demonstrated antagonism (FICI > 4) for any isolates.
The colony morphology also appeared to affect in vitro synergism. Isolates with rough morphotypes demonstrated less synergism than isolates with smooth morphotypes with the combinations of rifabutin with clarithromycin (14.3% versus 62.5%), imipenem (57.1% versus 93.8%), tigecycline (28.6% versus 87.5%), and ceftibuten (28.5% versus 50.0%) but better synergism with the combinations of rifabutin with cefoxitin (71.4% versus 6.3%), linezolid (57.1% versus 12.5%), and amikacin (57.1% versus 31.2%).
DISCUSSION
Patients with M. abscessus infections are routinely treated with clarithromycin along with two other antibiotics, usually amikacin, imipenem, or tigecycline (8, 15). The clinical utility of these antibiotic combinations is limited by the induction of resistance to clarithromycin and by their respective toxicities (16). Our present study confirmed that rifabutin holds promising activity against M. abscessus, and combinations comprising this drug with the core recommended treatment for clarithromycin-resistant M. abscessus are synergistic (10, 15).
We showed that for the M. abscessus subsp. abscessus isolates harboring inducible macrolide resistance, determined by the presence of an intact erm(41) with a thymine rather than a cytosine nucleotide at position 28, the combination of rifabutin and clarithromycin was reliably (100%) synergistic in vitro. For the highly macrolide-resistant M. abscessus subsp. massiliense isolate harboring the acquired A2058G point mutation in the rrl gene, the combination of rifabutin and clarithromycin was not significantly more active than either drug tested alone; however, for the phenotypically macrolide-resistant M. abscessus subsp. massiliense isolate with a truncated erm(41) gene and a wild-type rrl gene, the combination was significantly more active than either drug tested alone. Practically speaking, regardless of subspecies, if the initial clarithromycin MIC on the 3rd to 5th day was in the range of 0.5 to 16 mg/liter, the presence or absence of inducible macrolide resistance did not alter the synergism observed between rifabutin and clarithromycin. However, if the initial clarithromycin MIC was >256 mg/liter, point mutations in the rrl gene were more likely to underlie such an excessively high level of macrolide resistance and addition of rifabutin to clarithromycin was not considered synergistic and could no longer lower clarithromycin MICs to the susceptible range. If the in vitro synergism between rifabutin-clarithromycin is clinically validated, this may circumvent the need for extending incubation periods to 14 days for antimicrobial susceptibility testing or routine molecular determination of erm(41) and the M. abscessus subspecies.
Of note, although an FICI of ≤0.5 for clarithromycin-susceptible M. abscessus strains could not be achieved since it was difficult to determine a further 4-fold decrease in isolates with low baseline clarithromycin MICs (0.25 to <0.03 mg/liter), even for these isolates, a 4-fold or more synergistic decrease in the MICs of rifabutin in combination with clarithromycin could be demonstrated for most isolates (e.g., from 16 mg/liter to 2 mg/liter or from 8 mg/liter to 0.5 mg/liter). This lowered in vitro MIC of rifabutin would fall to concentrations that would be achievable in the lung (2 mg/liter) and in the serum (1 mg/liter) (17, 18).
In addition, imipenem and rifabutin were reliably synergistic for 100% of the M. abscessus subsp. abscessus isolates, for 69% of the M. abscessus subsp. massiliense isolates, and for the M. abscessus subsp. bolletii, BCRC 16915 isolate. The combination of tigecycline and rifabutin also demonstrated reliable synergism against 77% of the M. abscessus subsp. abscessus isolates, against 69% of the M. abscessus subsp. massiliense isolates, and against the M. abscessus subsp. bolletii BCRC 16915 isolate.
Our results are supported by those of two smaller studies recently published online. Rifabutin-clarithromycin synergism was demonstrated in one study in which synergism (FICI ≤ 0.5) was demonstrated by the checkerboard method for 3 of 6 (50%) clinical isolates of M. abscessus (all of which were clarithromycin susceptible with an MIC of ≤2 mg/liter and not delineated to the subspecies level) alongside the reference M. abscessus ATCC 19977 strain, and additivity (FICI = 0.63 to 0.75) was demonstrated for the remaining 3 isolates (19). In the same study, rifabutin-tigecycline synergism with an FICI of ≤0.5 was also observed for 3 of the 6 (50%) clinical isolates, while additive effects with an FICI of 0.75 were shown for the remaining isolates (19). In another study, where only a single strain of M. abscessus (ATCC 19977) and its β-lactamase-deficient derivative were studied, the rifabutin-imipenem combination was also shown to be synergistic by both the checkerboard method and a time-kill curve assay (20).
However, more convincingly in that study, rifabutin-imipenem was more effectively synergistic and bactericidal in a macrophage model (20). A 9-fold intracellular accumulation of rifabutin in human polymorphonuclear leukocytes has been reported; hence, our modest in vitro effects may, if anything, underestimate the effects in vivo (21).
Taken together, the inclusion of rifabutin as a frontline partner in combination therapy against M. abscessus infections should be investigated in therapeutic trials. Since amikacin activity is antagonized by clarithromycin-induced resistance genes via the expression of whiB7, a global regulator of intrinsic resistance genes which causes the upregulation of eis2 (which confers resistance to amikacin) (22) and which is also antagonized by tigecycline via yet uncharacterized mechanisms (23), the replacement of amikacin, an intravenous, potentially nephrotoxic and ototoxic agent, with rifabutin, an oral agent with better tolerability, intracellular accumulation, and reliable synergism in vitro with all three frontline therapies (clarithromycin, imipenem, and tigecycline across subspecies), seems to be rational, especially in the event of acquired macrolide resistance. The influence of subspecies and the mechanism underlying macrolide resistance on the extent of rifabutin-based synergism suggested by this study deserves further corroboration.
In addition, we found that the colony morphology, which can be routinely observed in clinical laboratories, influences the susceptibility of M. abscessus to selected antimicrobials and their combinations. Similar to previous findings, we showed that rough morphotypes had lower MICs to tigecycline (24) but higher MICs to imipenem than smooth morphotypes (25). We also showed that rough morphotypes were more susceptible to linezolid and rifabutin, which target the mycobacterial machinery involved in RNA and protein synthesis. This may be plausible, given that rough morphotypes correspond to cord-forming M. abscessus isolates associated with more invasive and persistent disease and smooth morphotypes correspond to non-cord-forming, biofilm-forming, and glycopeptidolipid-rich M. abscessus isolates (26–28). Putatively, therefore, rough morphotypes may require several redox proteins to resist oxidative attack from host macrophages due to their unmasking (29–31). Clofazimine also appears to be more active against rough morphotypes, possibly by interfering with the redox system, wherein it becomes reduced on interaction with NDH-2 and then subsequently oxidized in the presence of molecular oxygen, cycling between these two states and siphoning off electrons from the bacterial electron transport chain (32).
To the best of our knowledge, our investigation is the first to study rifabutin in combination with ceftibuten and linezolid, and these combinations were synergistic for a proportion of the M. abscessus isolates tested in our study. Given the requirement for prolonged therapy (6 to 12 months) for most cases of M. abscessus disease and the restricted availability of sophisticated molecular methods for determination of M. abscessus subspecies, the availability of oral combinatorial partners that can be applied based on easily made distinctions, such as colony morphology, is worth exploring. For example, the combination of rifabutin and ceftibuten was more favorably synergistic for smooth morphotypes than for rough morphotypes (50% versus 29%), whereas for rifabutin and linezolid, the combination was more synergistic for rough morphotypes than for smooth morphotypes of M. abscessus (57% versus 12.5%).
In conclusion, remarkable synergy against M. abscessus was observed, in order of diminishing frequency, for rifabutin-based combinations with imipenem, tigecycline, clarithromycin, ceftibuten, amikacin, cefoxitin, and linezolid. Colony morphology, rrl point mutations, and subspecies identity but not inducible macrolide resistance impacted the rifabutin-based synergy. These findings merit clinical and mechanistic validation.
MATERIALS AND METHODS
Mycobacterial isolates.
A total of 26 nonduplicate M. abscessus clinical isolates from 26 patients with skin and soft tissue, lymph node, pulmonary, central nervous system, and bloodstream infections at the National Taiwan University Hospital in Taipei, Taiwan, between January 2009 and December 2015 were studied. Isolates were submitted by clinicians to the research laboratory with a request for susceptibility testing. Five reference strains were also tested, including M. abscessus subsp. abscessus ATCC 19977, M. abscessus subsp. bolletii BCRC 16915, M. abscessus subsp. massiliense BCRC 16916, M. peregrinum ATCC 700686, and M. chelonae ATCC 35752. The smooth or rough morphology of these mycobacterial colonies was observed and recorded (26).
secA1, rpoB, hsp65, erm(41), and rrl partial gene sequencing.
Genomic DNA was extracted from mycobacterial cultures for identification and molecular typing based on the sequences of the genes secA1, rpoB, and hsp65 (33). Details of the PCR method used were previously described by Zelazny et al. (34). Sequencing of erm(41) and rrl was performed using the methods described by Bastian et al. (14) and Maurer et al. (35). Sequences were analyzed and compared with those in the NCBI database using a BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
Antimicrobial susceptibility testing.
Broth microdilution in cation-adjusted Mueller-Hinton broth for rapidly growing mycobacteria, as suggested by the Clinical and Laboratory Standards Institute (CLSI), was used for drug susceptibility testing (36). The antimicrobial agents tested included rifabutin, clarithromycin, amikacin, cefoxitin, ceftibuten, doxycycline, imipenem, linezolid, moxifloxacin, clofazimine, and tigecycline. Susceptible, intermediately susceptible, and resistant breakpoints followed the CLSI guidelines for rapidly growing mycobacteria, with the exception of the breakpoints for (i) rifabutin, which were adopted from CLSI guidelines for M. marinum (36); (ii) tigecycline, which were adopted from a clinical study for M. abscessus (37), and (iii) clofazimine, which were adopted from a molecular resistance study for M. abscessus (38). For ceftibuten, an oral cephalosporin, no interpretative criteria for mycobacteria exist; hence, MIC breakpoints of ≤16, 32 to 64, and ≥128 mg/liter for susceptible, intermediately susceptible, and resistant, respectively, in line with the breakpoints for cefoxitin for descriptive purposes only, were used (36).
The MICs of the individual drugs were determined first. The checkerboard titration method was used to test the combination of rifabutin with another antimicrobial agent (39). Rifabutin was serially diluted along the ordinate, while the second drug was diluted along the abscissa. Concentrations ranging from 4 to 8 times the expected MIC to at least 1/8 to 1/16 the expected MIC were included to determine the occurrence and magnitude of synergism or antagonism. The MICs of the agents in combination were read between the 3rd and 5th day for all drugs if the control growth was positive. The fractional inhibitory concentration (FIC) was calculated using the formula MICa/MICA + MICb/MICB, where MICa represents the MIC of rifabutin (drug A) tested in combination, MICA represents the MIC of rifabutin tested alone, MICb represents the MIC of the other antimycobacterial drug (drug B) tested in combination, and MICB represents the MIC of other antimycobacterial drugs tested alone. The minimum FIC for each combination was defined as the FIC index (FICI). Synergy was defined as an FICI of ≤0.5, an FICI of between 0.5 and 4 was considered indifferent, and a FIC index of >4 indicated an antagonistic association (40). In addition, Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27853 were used as quality control strains according to the CLSI M24-A2 guidelines (36).
ACKNOWLEDGMENTS
We thank Po-ren Hsueh and the Department of Laboratory Medicine, National Taiwan University Hospital, for storage of and access to the mycobacterial isolates.
A.C. conceived of and designed the study, analyzed the results, and wrote the manuscript. Y.-T.T. conducted the experiments and analyzed the results. S.-Y.C. collected the mycobacterial isolates and helped execute the study. H.-Y.S. provided critical analysis and a review of the manuscript. U.-I.W. helped collect mycobacterial isolates and execute the study. W.-H.S. provided technical expertise and critiqued and reviewed the manuscript. Y.-C.C. coordinated the study and reviewed the manuscript. S.-C.C. provided technical expertise and critiqued and reviewed the manuscript.
There are no conflicts of interests to declare for the authors.
This study was funded by Taiwan’s Ministry of Science and Technology (grant no. 105-2628-B-002-019-MY3) and the Taiwan Ministry of Health and Welfare (MOHW107-TDU-B-211-113002).
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