ABSTRACT
Macrolide antibiotics are mainstays in the treatment of lung disease due to the Mycobacterium abscessus complex. Although previous studies have reported development of acquired macrolide resistance in this species, limited data are available on the outcomes of lung disease due to macrolide-resistant Mycobacterium abscessus subsp. abscessus. This study evaluated the clinical features, treatment outcomes, and molecular characteristics of macrolide-resistant isolates of M. abscessus subsp. abscessus. We performed a retrospective review of medical records and genetic analysis of clinical isolates from 13 patients who had acquired macrolide-resistant M. abscessus subsp. abscessus lung disease between November 2006 and March 2016. Eleven (85%) patients had the nodular bronchiectatic form of the disease, and two (15%) patients had the fibrocavitary form. When acquired macrolide resistance was detected, 10 (77%) patients were on antibiotic therapy for M. abscessus subsp. abscessus, and three (23%) patients were on therapy for lung disease due to other nontuberculous mycobacteria. The median treatment duration after detecting resistance was 24.0 months (interquartile range, 16.0 to 43.0 months). Treatment outcomes were poor, and final sputum culture conversion was achieved in only one (8%) patient, after resectional surgery. All 13 clinical isolates demonstrated point mutations at position 2058 (n = 10) or 2059 (n = 3) of the 23S rRNA gene, which resulted in acquired macrolide resistance. This study indicates that treatment outcomes are very poor after the development of acquired macrolide resistance in patients with M. abscessus subsp. abscessus lung disease. Thus, more effective measures are needed to prevent development and effectively treat macrolide-resistant M. abscessus subsp. abscessus lung disease.
KEYWORDS: nontuberculous mycobacteria, Mycobacterium abscessus, macrolides, drug resistance
INTRODUCTION
The prevalence of pulmonary disease caused by nontuberculous mycobacteria (NTM) is increasing worldwide (1, 2). Mycobacterium abscessus complex is the most important cause of pulmonary infections by rapidly growing mycobacteria in patients with chronic lung diseases, such as bronchiectasis and cystic fibrosis (3, 4). Currently, Mycobacterium abscessus complex can be divided into three subspecies: Mycobacterium abscessus subsp. abscessus, Mycobacterium abscessus subsp. massiliense, and Mycobacterium abscessus subsp. bolletii (5, 6). Of the three subspecies, M. abscessus subsp. abscessus is the most common pathogen, followed by M. abscessus subsp. massiliense and M. abscessus subsp. bolletii (7).
Of the rapidly growing mycobacterial pathogens, M. abscessus subsp. abscessus is the most difficult to treat because of its intrinsic and acquired multidrug resistance (8), and antibiotic treatment success rates are less than 50%, in contrast to the high treatment success rates (80 to 90%) with M. abscessus subsp. massiliense infection (9–13). Macrolides, such as clarithromycin and azithromycin, are considered the cornerstone for treatment of M. abscessus subsp. abscessus infections (3). The poor treatment outcomes in M. abscessus subsp. abscessus lung disease are attributed to inducible macrolide resistance, demonstrated by susceptibility to macrolides at day 3 but resistance at day 14 of drug susceptibility testing (DST); this type of resistance is conferred by the ribosomal methyltransferase gene erm(41) (14, 15). In addition to inducible macrolide resistance, acquired macrolide resistance, demonstrated by resistance to macrolides at day 3 of DST, can develop during macrolide antibiotic treatment due to mutations in the drug-binding pocket of the 23S rRNA gene (rrl) at nucleotide positions 2058 and 2059 (15, 16).
Although previous studies reported that acquired macrolide resistance developed in some patients with M. abscessus subsp. abscessus infections during macrolide-containing antibiotic treatment (17–19), no published data are available regarding detailed clinical characteristics and treatment outcomes of patients with acquired macrolide-resistant M. abscessus subsp. abscessus lung disease. The purposes of this study were to evaluate the clinical features and treatment outcomes of patients with acquired macrolide-resistant M. abscessus subsp. abscessus lung disease and to examine the molecular characteristics of their isolates.
RESULTS
Patient characteristics.
A total of 13 patients were diagnosed with acquired macrolide-resistant M. abscessus subsp. abscessus lung disease during the study period. The clinical characteristics of the patients are summarized in Table 1. For four of the patients, some clinical data were included in recently published articles (12, 20); data on the remaining patients have not been previously reported. There were eight (62%) female patients, and the median age of all patients was 64 years (interquartile range [IQR], 55 to 78 years). Eight (62%) patients had a history of prior treatment for pulmonary tuberculosis. Six (46%) patients had a history of previous treatment for NTM lung disease: four of these patients had Mycobacterium avium complex (MAC) infection, one patient had M. abscessus subsp. massiliense infection, and one patient had MAC infection followed by M. abscessus subsp. massiliense infection.
TABLE 1.
Clinical characteristics of patients (n = 13) with acquired macrolide-resistant M. abscessus subsp. abscessus lung disease at diagnosis
| Clinical characteristicsa | Valueb |
|---|---|
| Female | 8 (62) |
| Age, years | 64 (55–78) |
| BMI, kg/m2 | 19.9 (18.2–22.0) |
| Nonsmoker | 11 (85) |
| Previous treatment for pulmonary tuberculosis | 8 (62) |
| Previous treatment for NTM lung disease | 6 (46) |
| Comorbidities | |
| COPD | 1 (8) |
| Bronchiectasis | 12 (92) |
| Chronic pulmonary aspergillosis | 1 (8) |
| Chronic heart disease | 1 (8) |
| Laboratory findings | |
| Positive sputum AFB smear | 13 (100) |
| ESR, mm/h | 39 (13–102) |
| CRP, mg/dl | 0.3 (0.1–1.6) |
| Radiologic findings | |
| Fibrocavitary form | 2 (15) |
| Nodular bronchiectatic form | 11 (85) |
| With cavity | 6 |
| Without cavity | 5 |
| Pulmonary function test | |
| FVC, liters | 2.33 (1.94–3.30) |
| FVC, % predicted | 75 (61–91) |
| FEV1, liters | 1.85 (1.31–2.56) |
| FEV1, % predicted | 80 (57–92) |
Abbreviations: BMI, body mass index; NTM, nontuberculous mycobacteria; COPD, chronic obstructive pulmonary disease; AFB, acid-fast bacilli; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; FVC, forced vital capacity; FEV1, forced expiratory volume in 1 s.
Data are presented as number (percentage of total) or as median (interquartile range).
Sputum smears were acid-fast bacillus (AFB) positive for all patients at the time acquired macrolide resistance was detected. Two (15%) patients had the fibrocavitary form of lung disease, and 11 (92%) patients had the nodular bronchiectatic form. Cavitary lesions were found on high-resolution computed tomography (HRCT) for 6 (55%) of 11 patients with the nodular bronchiectatic form.
Antibiotic therapy before detection of acquired macrolide-resistant M. abscessus subsp. abscessus.
For one (8%) patient, macrolide resistance was detected upon transfer to our hospital after long-term antibiotic treatment at another hospital. For 12 (92%) patients, macrolide resistance developed during antibiotic treatment at our institution. All patients received macrolide treatment, and the median duration of exposure to a macrolide was 19.0 months (IQR, 10.5 to 42.5 months) before detection of acquired macrolide resistance.
In 10 (77%) patients, macrolide resistance was detected during combined antibiotic therapy for M. abscessus subsp. abscessus lung disease, which consisted of intravenous (i.v.) amikacin, i.v. cefoxitin (or imipenem), and an oral macrolide, with or without fluoroquinolone, doxycycline, and clofazimine. In three (23%) patients, macrolide resistance was detected during antibiotic therapy for other NTM lung diseases: two patients with MAC infection had received an oral macrolide, rifampin, and ethambutol, and one patient with M. abscessus subsp. massiliense infection had received an oral macrolide, i.v. amikacin, and i.v. cefoxitin (Table 2).
TABLE 2.
Treatment regimens before detection of macrolide-resistant M. abscessus subsp. abscessus lung diseasea,b
| Treatment regimen | Value | Duration of macrolide exposure, mo |
|---|---|---|
| Antibiotic therapy for M. abscessus subsp. abscessus lung disease | ||
| Macrolide + i.v. antibioticsc ± FQ ± DOX ± CFZ | 10 (77) | 25.5 (9.3–45.0) |
| Antibiotic therapy for other NTM lung disease | ||
| Macrolide + RIF + EMBd | 2 (15) | 17.0 (NA) |
| Macrolide + i.v. antibioticse | 1 (8) | 11.0 (NA) |
| Total | 13 (100) | 19.0 (10.5–42.5) |
The total number of patients in the study was 13. Abbreviations: i.v., intravenous; FQ, fluoroquinolone; DOX, doxycycline; CFZ, clofazimine; NTM, nontuberculous mycobacteria; RIF, rifampin; EMB, ethambutol; NA, not available.
Data are presented as number (percent) or as median (interquartile range).
Intravenous antibiotics included amikacin and cefoxitin (or imipenem).
Combination antibiotic therapy for M. avium complex lung disease.
Combination antibiotic therapy for M. abscessus subsp. massiliense lung disease.
Treatment and outcomes after detection of acquired macrolide-resistant M. abscessus subsp. abscessus.
The treatment regimens after detection of macrolide resistance and subsequent treatment outcomes are summarized in Table 3. Of 13 patients, two (15%) did not receive antibiotic therapy after detection of macrolide resistance: one patient had been observed at an outpatient clinic after completing antibiotic therapy for MAC infection, and another patient stopped receiving antibiotic therapy for M. abscessus subsp. abscessus lung disease after detection of macrolide resistance due to drug side effects. Another 11 (85%) patients received antibiotic therapy for macrolide-resistant M. abscessus subsp. abscessus lung disease. After detection of macrolide resistance, macrolides continued to be prescribed for all patients. Intravenous (i.v.) amikacin (n = 6 [46%]), i.v. cefoxitin or imipenem (n = 6 [46%]), an oral fluoroquinolone (n = 1 [8%]), or clofazimine (n = 10 [77%]) and amikacin inhalation (n = 7 [54%]) were used at the discretion of the attending physicians. The median duration of antibiotic therapy after detection of macrolide resistance was 24.0 months (IQR, 16.0 to 43.0 months). Two (15%) patients underwent surgical resections, consisting of lobectomy and wedge resection of another lobe at 6.9 months for one patient and pneumonectomy at 6.5 months for the second patient, after the detection of macrolide resistance.
TABLE 3.
Treatment modalities and outcomes after detection of macrolide-resistant M. abscessus subsp. abscessus lung diseasea,b
| Treatment modality | Value |
|---|---|
| Antibiotic therapy | |
| Amikacin | 6 (46) |
| Cefoxitin or imipenem | 6 (46) |
| Macrolide | 11 (85) |
| Fluoroquinolone | 1 (8) |
| Clofazimine | 10 (77) |
| Amikacin inhalation | 7 (54) |
| Surgical resection | 2 (15) |
| Total treatment duration, mo | 24.0 (16.0–43.0) |
| Treatment outcome | |
| Favorable outcome | 0 |
| Sputum culture conversion after surgery | 1 (8) |
The total number of patients in the study was 13.
Data are presented as number (percent) or as median (interquartile range).
Based on the occurrence and timing of sputum culture conversion (see Materials and Methods), no patient achieved a favorable outcome after antibiotic treatment alone. Sputum culture conversion was achieved only by the patient who had undergone surgical resection of the left upper lobe, with the conversion occurring subsequent to surgery. During the median follow-up period of 35.4 months (IQR, 13.8 to 59.9 months), two (15%) patients died, one at 3 months and the other at 8 months after the detection of macrolide resistance.
Genetic analysis of macrolide-resistant M. abscessus subsp. abscessus isolates.
M. abscessus subsp. abscessus isolates were available from all patients for genetic analysis, and all isolates had very high clarithromycin MICs (≥64 μg/ml) at day 3 of DST. All M. abscessus subsp. abscessus isolates showed rrl mutations at position 2058 (10/13 [77%]) or 2059 (3/13 [23%]), with mutations of adenine to guanine (7/13 [54%]), adenine to cytosine (5/13 [38%]), or adenine to thymine (1/13 [8%]) (Table 4). The rrl genotypes were 100% concordant with antibiotic susceptibility testing phenotypes, and the 13 isolates with rrl mutations were clarithromycin resistant. Eleven isolates contained the T28 sequevar of the erm(41) gene, whereas the other two isolates contained the erm(41) C28 sequevar. The characteristics of the M. abscessus subsp. abscessus isolates from the 13 patients after the development of macrolide resistance are summarized in Table 5.
TABLE 4.
Genetic analysis of macrolide-resistant M. abscessus clinical isolates
| Mutation or sequevar | No. (%) of patients with mutationa |
|---|---|
| Presence of point mutation in rrlb | 13 (100) |
| Adenine → guanine | 7 (54) |
| A2058G | 4 |
| A2059G | 3 |
| Adenine → cytosine | 5 (38) |
| A2058C | 5 |
| A2059C | 0 |
| Adenine → thymine | 1 (8) |
| A2058T | 1 |
| A2059T | 0 |
| 28th sequevar of erm(41)c | |
| T28 | 11 (85) |
| C28 | 2 (15) |
The total number of patients in the study was 13.
Nucleotides 2058 and 2059 (Escherichia coli numbering) of rrl, for which the wild-type sequence is AA. A, adenine; G, guanine; C, cytosine; T, thymine.
Numbering system of erm(41), with the GTG start codon as 1.
TABLE 5.
Characteristics of isolates from the 13 patients studied
| Patient | Isolate | No. of mo since first isolatea | DSTb | MIC, μg/ml (day 3/day 14)c | Colony morphotyped | erm(41)e | rrlf |
|---|---|---|---|---|---|---|---|
| 01 | 01-1 | 0 | R | >64 | Mixed (R) | T28 | GA |
| Mixed (S) | T28 | AA | |||||
| 02 | 02-1 | 0 | R | >64 | Mixed (R) | T28 | AA |
| Mixed (S) | T28 | CA | |||||
| 02-2 | 16 | R | >64 | S | T28 | CA | |
| 03 | 03-1 | 0 | R | >64 | S | T28 | AG |
| 04 | 04-1 | 0 | R | >64 | R | T28 | CA |
| 05 | 05-1 | 0 | R | >64 | R | C28 | GA |
| 05-2 | 4 | R | >64 | R | C28 | GA | |
| 05-3 | 6 | R | >64 | R | C28 | GA | |
| 05-4 | 8 | R | >64 | R | C28 | GA | |
| 05-5 | 11 | R | >64 | R | C28 | GA | |
| 05-6 | 19 | R | >64 | R | C28 | GA | |
| 05-7 | 25 | R | >64 | R | C28 | GA | |
| 05-8 | 31 | R | >64 | S | C28 | GA | |
| 06 | 06-1 | 0 | R | >64 | S | C28 | CA |
| 06-2 | 6 | R | 64 | S | C28 | CA | |
| 06-3 | 10 | R | 64 | Mixed (R) | C28 | CA | |
| Mixed (S) | C28 | CA | |||||
| 07 | 07-1 | 0 | R | >64 | R | T28 | TA |
| 07-2 | 3 | R | >64 | R | T28 | GA | |
| 07-3 | 5 | R | 64 | R | T28 | GA | |
| 07-4 | 7 | R | >64 | R | T28 | GA | |
| 07-5 | 14 | R | >64 | R | T28 | GA | |
| 08 | 08-1 | 0 | R | >64 | R | T28 | CA |
| 08-2 | 8 | R | >64 | R | T28 | CA | |
| 08-3 | 9 | R | >64 | R | T28 | CA | |
| 08-4 | 13 | R | >64 | R | T28 | CA | |
| 09 | 09-1 | 0 | R | >64 | Mixed (R) | T28 | CA |
| Mixed (S) | T28 | CA | |||||
| 09-2 | 2 | R | >64 | Mixed (R) | T28 | CA | |
| Mixed (S) | T28 | CA | |||||
| 09-3 | 4 | R | >64 | Mixed (R) | T28 | CA | |
| Mixed (S) | T28 | CA | |||||
| 10 | 10-1 | 0 | R | 64 | R | T28 | AG |
| 10-2 | 2 | R | 64 | Mixed (R) | T28 | AG | |
| Mixed (S) | T28 | AG | |||||
| 11 | 11-1 | 0 | R | >64 | R | T28 | GA |
| 11-2 | 1 | R | 32 | R | T28 | GA | |
| 12 | 12-1 | 0 | R | >64 | R | T28 | AG |
| 12-2 | 1 | R | >64 | S | T28 | CA | |
| 12-3 | 5 | R | >64 | S | T28 | CA | |
| 12-4 | 15 | R | >64 | S | T28 | CA | |
| 12-5 | 18 | R | >64 | S | T28 | CA | |
| 13 | 13-1 | 0 | R | >64 | R | T28 | GA |
| 13-2 | 2 | R | >64 | R | T28 | CA | |
| 13-3 | 7 | R | >64 | R | T28 | CA |
The first isolate was a clinical isolate that was firstly confirmed to be resistant to macrolide on DST.
For DST: R, resistance.
The microdilution method was used to determine the MIC for clarithromycin.
For colony morphotype: S, smooth; R, rough.
Numbering system of erm(41), with the GTG start codon as 1.
Nucleotides 2058 and 2059 (E. coli numbering) of rrl, for which the wild-type sequence is AA. A, adenine; G, guanine; C, cytosine; T, thymine.
DISCUSSION
This is the first study to investigate the detailed clinical characteristics and treatment outcomes of patients with acquired macrolide-resistant M. abscessus subsp. abscessus lung disease, as well as the molecular characteristics of the disease isolates. Overall, the treatment outcome was very poor, with limited effective treatment options. Of 13 patients, only one (8%) achieved sputum culture conversion, which occurred after surgical resection. During the median follow-up period of 35.4 months after detection of macrolide resistance, two (15%) patients died.
Macrolide resistance in M. abscessus subsp. abscessus can be intrinsic or acquired depending on the resistance mechanism. Intrinsic macrolide resistance is associated with inducible resistance, involving the presence of a functional erm(41) gene, which encodes a type of methyltransferase known to methylate 23S rRNA (14). Acquired macrolide resistance involves spontaneous mutations in the rrl gene, which encodes 23S rRNA, that are selected during macrolide-containing antibiotic therapy (15). Whereas M. abscessus subsp. abscessus strains with inducible macrolide resistance show low MICs at day 3 and require longer incubation times (up to 14 days) for the induction of resistance, M. abscessus subsp. abscessus strains with acquired macrolide resistance show high MICs by day 3 (21).
Treatment success rates after antibiotic therapy for M. abscessus subsp. abscessus lung disease are less than 50% (9–13), and these low rates are attributed to inducible macrolide resistance associated with erm(41) or to the acquisition of resistance due to rrl mutations. Although many previous studies have reported on inducible macrolide resistance in M. abscessus subsp. abscessus (9–13), very few studies have evaluated acquired macrolide resistance in M. abscessus subsp. abscessus (17–19). Clinically acquired mutational resistance in M. abscessus subsp. abscessus occurs relatively infrequently (17). In our previous study, acquired macrolide resistance associated with rrl gene mutations developed in only 13% of patients who had persistently positive sputum cultures after more than 12 months of antibiotic therapy (12). Recently, Maurer et al. reported the acquisition of macrolide resistance in M. abscessus subsp. abscessus isolates in three of five patients with chronic M. abscessus subsp. abscessus lung disease who received long-term macrolide antibiotic therapy (18). In addition, Rubio et al. reported that on follow-up, three patients who initially had M. abscessus subsp. abscessus strains with the wild-type rrl sequence eventually developed acquired macrolide resistance (19). In those studies, acquired macrolide resistance was associated with macrolide resistance mutations in the 23S rRNA gene, which occurred regardless of the presence of an inducible erm(41) methylase. However, detailed clinical characteristics and long-term prognoses were not available in the previous studies.
In the present study, acquired macrolide resistance was detected during antibiotic therapy for M. abscessus subsp. abscessus lung disease in 10 patients (10/13 [77%]). After an intensive phase of combination i.v. antibiotic therapy for M. abscessus subsp. abscessus lung disease, the continuation phase usually consists of oral antibiotics such as macrolides, fluoroquinolones, and doxycycline, as described in our previous studies (9, 12, 22). Our current study showed that acquired macrolide resistance can occur in M. abscessus subsp. abscessus lung disease after macrolide monotherapy or weak macrolide-containing antibiotic regimens, consistent with previous findings (17). To prevent mutational macrolide resistance, a stronger regimen should be considered at the outset for treatment of M. abscessus subsp. abscessus lung disease. Such a regimen might include the recently proposed clofazimine or inhaled amikacin (4, 20, 23, 24), which we administered to some of our patients following the identification of acquired macrolide resistance.
In our study, acquired macrolide resistance was detected during antibiotic therapy for other NTM lung diseases in three patients (3/13 [23%]), with two cases of MAC and one case of M. abscessus subsp. massiliense lung disease. It is not uncommon to isolate M. abscessus subsp. abscessus during treatment of MAC lung disease, and microbiologic and clinical follow-up is important to determine the significance of M. abscessus subsp. abscessus isolation in such cases (25). As MAC treatment usually consists of macrolide, rifampin, and ethambutol, M. abscessus subsp. abscessus infection during MAC treatment could result in exposure to macrolide monotherapy for M. abscessus subsp. abscessus because neither rifampin nor ethambutol is effective in M. abscessus subsp. abscessus infection (26, 27). Macrolide-resistant M. abscessus subsp. abscessus lung disease also developed during antibiotic treatment of M. abscessus subsp. massiliense lung disease in one of our patients. An oral macrolide can be effective in the continuation phase of treatment of M. abscessus subsp. massiliense lung disease (28), but this raises the possibility that new infections with M. abscessus subsp. abscessus may be exposed to this monotherapy and acquire macrolide resistance.
In M. abscessus subsp. abscessus lung disease, the rates of achieving negative sputum culture conversion were reported as only 25 to 42% (9–13). In this study, no patients with acquired macrolide-resistant M. abscessus subsp. abscessus lung disease showed negative sputum culture conversion after antibiotic therapy alone, even after use of oral clofazimine and/or inhaled amikacin, which was reported to have clinical efficacy in patients with refractory M. abscessus subsp. abscessus lung disease (20, 23, 24). This implies that M. abscessus subsp. abscessus lung disease might be much more difficult to treat after acquisition of acquired macrolide resistance, regardless of intrinsic inducible macrolide resistance. Further research is needed to establish optimal treatment regimens for macrolide-resistant M. abscessus subsp. abscessus lung disease.
During macrolide-based antibiotic treatment, M. abscessus subsp. abscessus can develop acquired macrolide resistance through point mutations at position 2058 or 2059 in rrl (15, 16, 18). Mutations at these positions were found in the M. abscessus subsp. abscessus isolates of all patients in the present study, with the isolates resistant to clarithromycin at a high level (MICs of ≥64 μg/ml at day 3). The frequencies of mutations at position 2058 or 2059 differ between studies. Some studies showed that the majority of mutational changes involved A2059 (17, 29), whereas other studies found that most mutations involved A2058 (18, 30). In our study, 23% involved A2059, with the remaining 77% involving A2058, and there was no difference in clarithromycin MICs based on mutation position, although only small numbers of isolates were studied.
Acquired resistance occurs not only in M. abscessus subsp. abscessus with the macrolide-susceptible erm(41) C28 sequevar but also in M. abscessus subsp. abscessus with the intrinsically macrolide-resistant erm(41) T28 sequevar (15, 18, 19, 30, 31). In our study, of the 13 isolates with rrl mutations, 85% had the T28 sequevar and 15% had the C28 sequevar of erm(41). A recent study of mutants selected in vitro for acquired macrolide resistance found that only 19% of mutants derived from the T28 sequevar had rrl mutations, whereas 100% of mutants derived from the C28 sequevar had rrl mutations (29). In clinical isolates, rrl mutations were observed more frequently in isolates of the T28 sequevar than in isolates of the C28 sequevar (18, 30). If macrolide treatment pressure continues, M. abscessus subsp. abscessus is likely to develop a stable resistant lineage. Notably, none of the resistance-conferring mutations in rrl was found to affect bacterial fitness to a major degree; although the A2059G mutation demonstrated a small but significant fitness cost (2.4 to 2.6% per generation), the A2058G mutation had a nonsignificant cost (0.5 to 1.4% per generation) (32). Nevertheless, the existence of the inducible erm(41) gene suggests that acquisition of an rrl mutation confers some biofitness disadvantage in the absence of macrolide antibiotics.
Our study has several limitations. First, it was conducted at a single referral center and included a small number of patients. Second, treatment regimens were chosen at the discretion of attending physicians, without an established institutional protocol. Further studies with a larger number of patients are needed to evaluate the efficacy of antibiotic therapy in treating macrolide-resistant M. abscessus subsp. abscessus lung disease.
In conclusion, we found that acquired macrolide resistance can develop in patients with M. abscessus subsp. abscessus lung disease after macrolide monotherapy or with weak antibiotic regimens in the continuation phase after initiation phases that include multiple intravenous antibiotics. Treatment outcomes are poor after the development of macrolide resistance, and therefore, prevention of macrolide resistance is important, with more effective therapies needed to treat M. abscessus subsp. abscessus lung disease both before and after the development of macrolide resistance.
MATERIALS AND METHODS
Study population.
We reviewed the medical records for all patients who had macrolide-resistant M. abscessus subsp. abscessus lung disease between November 2006 and March 2016, as identified from the NTM Registry of Samsung Medical Center (a 1,979-bed referral hospital in Seoul, South Korea) (12). All patients fulfilled the diagnostic criteria of NTM lung disease (3). This retrospective study was approved by the institutional review board (IRB) of Samsung Medical Center (IRB application no. 2016-07-137). Patient information was anonymized and deidentified prior to analysis; therefore, requirements for informed consent were waived.
Radiographic and microbiologic examination.
Chest radiography and high-resolution computed tomography (HRCT) were available at the time of detection of acquired macrolide-resistant M. abscessus subsp. abscessus in all patients. The fibrocavitary form of the disease (previously called the upper lobe cavitary form) was defined by the presence of cavitary opacities mainly in the upper lobes. The nodular bronchiectatic form was defined by the presence of bronchiectasis and multiple nodules on chest HRCT, regardless of the presence of small cavities in the lungs (12, 33).
Sputum acid-fast bacillius(AFB) smears and cultures were obtained using standard methods (34). M. abscessus subsp. abscessus was identified and differentiated from M. abscessus subsp. massiliense and M. abscessus subsp. bolletii by a PCR and restriction fragment length polymorphism method based on the rpoB gene or by reverse blot hybridization assay of the rpoB gene (9, 12, 28), followed by multilocus sequencing analysis of rrs, hsp65, and rpoB (35). Each isolate was also genotyped with regard to the presence of a T-to-C mutation at position 28 of the erm(41) gene, as the C28 polymorphism abrogates inducible resistance (15).
DST was performed at the Korean Institute of Tuberculosis using a broth microdilution method (21). The MIC of clarithromycin was determined at days 3 and 14 after incubation; M. abscessus subsp. abscessus isolates were considered susceptible (MIC ≤ 2 μg/ml at days 3 and 14), resistant (MIC ≥ 8 μg/ml at day 3), or inducibly resistant (susceptible at day 3 but resistant at day 14) to clarithromycin (21). MICs for azithromycin were not determined, as clarithromycin is the class drug for macrolides (21).
Once macrolide resistance was detected, M. abscessus subsp. abscessus isolates were stored at −80°C for further analysis. To detect point mutations at position 2058 or 2059 (Escherichia coli numbering) in the 23S rRNA gene, PCR was performed to amplify the region corresponding to domain V of the 23S rRNA gene, according to a method described previously (36). Primers 23SF1 and 23SRIII were used for PCR and sequencing (36).
Antibiotic therapy and treatment outcomes.
Although the initial treatment regimens for M. abscessus subsp. abscessus lung disease are standardized at our institution (9, 12, 22), treatment regimens for macrolide-resistant M. abscessus subsp. abscessus lung disease were not standardized during the study period. Patients with mild symptoms at the time macrolide resistance was detected received oral antibiotics at the outpatient clinic. Patients with severe symptoms were hospitalized and received intravenous (i.v.) amikacin (15 mg/kg [of body weight]/day in two divided doses or 15 mg/kg/day with adjustment of a once-daily dose to target peak serum drug level of 55 to 65 μg/ml), plus cefoxitin (200 mg/kg/day; maximum of 12 g/day in three divided doses) or imipenem (750 mg three times per day) for 2 to 4 weeks (9, 12, 22), along with oral antibiotics. For oral antibiotics, treatment with a macrolide (clarithromycin at 1,000 mg/day or azithromycin at 250 mg/day) was continued, and additional drugs, such as a fluoroquinolone (ciprofloxacin at 1,000 mg/day or moxifloxacin at 400 mg/day), clofazimine (100 mg/day), or inhaled amikacin (250 to 500 mg/day), were used at the discretion of the attending physicians.
Treatment outcomes were assessed by sputum culture conversion after the detection of macrolide-resistant M. abscessus subsp.abscessus lung disease; conversion was defined as three consecutive negative cultures, with the time of conversion defined as the date of the first negative culture (9, 12). A favorable outcome was defined as sputum culture conversion within 12 months after treatment initiation and maintenance for ≥12 months with treatment.
Statistical analysis.
Data are presented as the median and interquartile range (IQR) for continuous variables and as frequency (percentage) for categorical variables. All statistical analyses were performed using SPSS Statistics for Windows, version 23.0 (IBM, Armonk, NY).
ACKNOWLEDGMENTS
Charles L. Daley has received grants from Insmed Inc. not associated with this article. Otherwise, we have no conflicts of interest to declare.
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future Planning (NRF-2015R1A2A1A01003959) and by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare, Republic of Korea (HI15C2778).
REFERENCES
- 1.Prevots DR, Marras TK. 2015. Epidemiology of human pulmonary infection with nontuberculous mycobacteria: a review. Clin Chest Med 36:13–34. doi: 10.1016/j.ccm.2014.10.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Stout JE, Koh WJ, Yew WW. 2016. Update on pulmonary disease due to non-tuberculous mycobacteria. Int J Infect Dis 45:123–134. doi: 10.1016/j.ijid.2016.03.006. [DOI] [PubMed] [Google Scholar]
- 3.Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C, Gordin F, Holland SM, Horsburgh R, Huitt G, Iademarco MF, Iseman M, Olivier K, Ruoss S, von Reyn CF, Wallace RJ Jr, Winthrop K. 2007. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 175:367–416. doi: 10.1164/rccm.200604-571ST. [DOI] [PubMed] [Google Scholar]
- 4.Floto RA, Olivier KN, Saiman L, Daley CL, Herrmann JL, Nick JA, Noone PG, Bilton D, Corris P, Gibson RL, Hempstead SE, Koetz K, Sabadosa KA, Sermet-Gaudelus I, Smyth AR, van Ingen J, Wallace RJ, Winthrop KL, Marshall BC, Haworth CS. 2016. US Cystic Fibrosis Foundation and European Cystic Fibrosis Society consensus recommendations for the management of non-tuberculous mycobacteria in individuals with cystic fibrosis. Thorax 71(Suppl 1):i1–i22. doi: 10.1136/thoraxjnl-2015-207360. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Griffith DE, Brown-Elliott BA, Benwill JL, Wallace RJ Jr. 2015. Mycobacterium abscessus. “Pleased to meet you, hope you guess my name….” Ann Am Thorac Soc 12:436–439. doi: 10.1513/AnnalsATS.201501-015OI. [DOI] [PubMed] [Google Scholar]
- 6.Tortoli E, Kohl TA, Brown-Elliott BA, Trovato A, Leão SC, Garcia MJ, Vasireddy S, Turenne CY, Griffith DE, Philley JV, Baldan R, Campana S, Cariani L, Colombo C, Taccetti G, Teri A, Niemann S, Wallace RJ Jr, Cirillo DM. 2016. Emended description of Mycobacterium abscessus, Mycobacterium abscessus subsp. abscessus and Mycobacterium abscessus subsp. bolletii and designation of Mycobacterium abscessus subsp. massiliense comb. nov. Int J Syst Evol Microbiol 66:4471–4479. [DOI] [PubMed] [Google Scholar]
- 7.Koh WJ, Stout JE, Yew WW. 2014. Advances in the management of pulmonary disease due to Mycobacterium abscessus complex. Int J Tuberc Lung Dis 18:1141–1148. doi: 10.5588/ijtld.14.0134. [DOI] [PubMed] [Google Scholar]
- 8.Nessar R, Cambau E, Reyrat JM, Murray A, Gicquel B. 2012. Mycobacterium abscessus: a new antibiotic nightmare. J Antimicrob Chemother 67:810–818. doi: 10.1093/jac/dkr578. [DOI] [PubMed] [Google Scholar]
- 9.Koh WJ, Jeon K, Lee NY, Kim BJ, Kook YH, Lee SH, Park YK, Kim CK, Shin SJ, Huitt GA, Daley CL, Kwon OJ. 2011. Clinical significance of differentiation of Mycobacterium massiliense from Mycobacterium abscessus. Am J Respir Crit Care Med 183:405–410. doi: 10.1164/rccm.201003-0395OC. [DOI] [PubMed] [Google Scholar]
- 10.Lyu J, Kim BJ, Kim BJ, Song JW, Choi CM, Oh YM, Lee SD, Kim WS, Kim DS, Shim TS. 2014. A shorter treatment duration may be sufficient for patients with Mycobacterium massiliense lung disease than with Mycobacterium abscessus lung disease. Respir Med 108:1706–1712. doi: 10.1016/j.rmed.2014.09.002. [DOI] [PubMed] [Google Scholar]
- 11.Park J, Cho J, Lee CH, Han SK, Yim JJ. 2017. Progression and treatment outcomes of lung disease caused by Mycobacterium abscessus and Mycobacterium massiliense. Clin Infect Dis 64:301–308. doi: 10.1093/cid/ciw723. [DOI] [PubMed] [Google Scholar]
- 12.Koh WJ, Jeong BH, Kim SY, Jeon K, Park KU, Jhun BW, Lee H, Park HY, Kim DH, Huh HJ, Ki CS, Lee NY, Kim HK, Choi YS, Kim J, Lee SH, Kim CK, Shin SJ, Daley CL, Kim H, Kwon OJ. 2017. Mycobacterial characteristics and treatment outcomes in Mycobacterium abscessus lung disease. Clin Infect Dis 64:309–316. doi: 10.1093/cid/ciw724. [DOI] [PubMed] [Google Scholar]
- 13.Diel R, Ringshausen F, Richter E, Welker L, Schmitz J, Nienhaus A. 2017. Microbiological and clinical outcomes of treating non-Mycobacterium avium complex nontuberculous mycobacterial pulmonary disease: a systematic review and meta-analysis. Chest 152:120–142. doi: 10.1016/j.chest.2017.04.166. [DOI] [PubMed] [Google Scholar]
- 14.Nash KA, Brown-Elliott BA, Wallace RJ Jr. 2009. A novel gene, erm(41), confers inducible macrolide resistance to clinical isolates of Mycobacterium abscessus but is absent from Mycobacterium chelonae. Antimicrob Agents Chemother 53:1367–1376. doi: 10.1128/AAC.01275-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Bastian S, Veziris N, Roux AL, Brossier F, Gaillard JL, Jarlier V, Cambau E. 2011. Assessment of clarithromycin susceptibility in strains belonging to the Mycobacterium abscessus group by erm(41) and rrl sequencing. Antimicrob Agents Chemother 55:775–781. doi: 10.1128/AAC.00861-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Shallom SJ, Moura NS, Olivier KN, Sampaio EP, Holland SM, Zelazny AM. 2015. New real-time PCR assays for detection of inducible and acquired clarithromycin resistance in the Mycobacterium abscessus group. J Clin Microbiol 53:3430–3437. doi: 10.1128/JCM.01714-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Wallace RJ Jr, Meier A, Brown BA, Zhang Y, Sander P, Onyi GO, Bottger EC. 1996. Genetic basis for clarithromycin resistance among isolates of Mycobacterium chelonae and Mycobacterium abscessus. Antimicrob Agents Chemother 40:1676–1681. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Maurer FP, Ruegger V, Ritter C, Bloemberg GV, Bottger EC. 2012. Acquisition of clarithromycin resistance mutations in the 23S rRNA gene of Mycobacterium abscessus in the presence of inducible erm(41). J Antimicrob Chemother 67:2606–2611. doi: 10.1093/jac/dks279. [DOI] [PubMed] [Google Scholar]
- 19.Rubio M, March F, Garrigo M, Moreno C, Espanol M, Coll P. 2015. Inducible and acquired clarithromycin resistance in the Mycobacterium abscessus complex. PLoS One 10:e0140166. doi: 10.1371/journal.pone.0140166. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Yang B, Jhun BW, Moon SM, Lee H, Park HY, Jeon K, Kim DH, Kim SY, Shin SJ, Daley CL, Koh WJ. 2017. A clofazimine-containing regimen for the treatment of Mycobacterium abscessus lung disease. Antimicrob Agents Chemother 61:e02052-16. doi: 10.1128/AAC.02052-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Clinical and Laboratory Standards Institute. 2011. Susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes; approved standard, 2nd ed CLSI document M24-A2. Clinical and Laboratory Standards Institute, Wayne, PA. [PubMed] [Google Scholar]
- 22.Jeon K, Kwon OJ, Lee NY, Kim BJ, Kook YH, Lee SH, Park YK, Kim CK, Koh WJ. 2009. Antibiotic treatment of Mycobacterium abscessus lung disease: a retrospective analysis of 65 patients. Am J Respir Crit Care Med 180:896–902. doi: 10.1164/rccm.200905-0704OC. [DOI] [PubMed] [Google Scholar]
- 23.Martiniano SL, Wagner BD, Levin A, Nick JA, Sagel SD, Daley CL. 5 May 2017. Safety and effectiveness of clofazimine for primary and refractory nontuberculous mycobacterial infection. Chest doi: 10.1016/j.chest.2017.04.175. [DOI] [PubMed] [Google Scholar]
- 24.Olivier KN, Shaw PA, Glaser TS, Bhattacharyya D, Fleshner M, Brewer CC, Zalewski CK, Folio LR, Siegelman JR, Shallom S, Park IK, Sampaio EP, Zelazny AM, Holland SM, Prevots DR. 2014. Inhaled amikacin for treatment of refractory pulmonary nontuberculous mycobacterial disease. Ann Am Thorac Soc 11:30–35. doi: 10.1513/AnnalsATS.201307-231OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Griffith DE, Philley JV, Brown-Elliott BA, Benwill JL, Shepherd S, York D, Wallace RJ Jr. 2015. The significance of Mycobacterium abscessus subspecies abscessus isolation during Mycobacterium avium complex lung disease therapy. Chest 147:1369–1375. doi: 10.1378/chest.14-1297. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Kang YA, Koh WJ. 2016. Antibiotic treatment for nontuberculous mycobacterial lung disease. Expert Rev Respir Med 10:557–568. doi: 10.1586/17476348.2016.1165611. [DOI] [PubMed] [Google Scholar]
- 27.Ryu YJ, Koh WJ, Daley CL. 2016. Diagnosis and treatment of nontuberculous mycobacterial lung disease: clinicians' perspectives. Tuberc Respir Dis (Seoul) 79:74–84. doi: 10.4046/trd.2016.79.2.74. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Koh WJ, Jeong BH, Jeon K, Kim SY, Park KU, Park HY, Huh HJ, Ki CS, Lee NY, Lee SH, Kim CK, Daley CL, Shin SJ, Kim H, Kwon OJ. 2016. Oral macrolide therapy following short-term combination antibiotic treatment for Mycobacterium massiliense lung disease. Chest 150:1211–1221. doi: 10.1016/j.chest.2016.05.003. [DOI] [PubMed] [Google Scholar]
- 29.Mougari F, Bouziane F, Crockett F, Nessar R, Chau F, Veziris N, Sapriel G, Raskine L, Cambau E. 2017. Selection of resistance to clarithromycin in Mycobacterium abscessus subspecies. Antimicrob Agents Chemother 61:e00943-16. doi: 10.1128/AAC.00943-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Mougari F, Amarsy R, Veziris N, Bastian S, Brossier F, Bercot B, Raskine L, Cambau E. 2016. Standardized interpretation of antibiotic susceptibility testing and resistance genotyping for Mycobacterium abscessus with regard to subspecies and erm41 sequevar. J Antimicrob Chemother 71:2208–2212. doi: 10.1093/jac/dkw130. [DOI] [PubMed] [Google Scholar]
- 31.Lee SH, Yoo HK, Kim SH, Koh WJ, Kim CK, Park YK, Kim HJ. 2014. Detection and assessment of clarithromycin inducible resistant strains among Korean Mycobacterium abscessus clinical strains: PCR methods. J Clin Lab Anal 28:409–414. doi: 10.1002/jcla.21702. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Sander P, Springer B, Prammananan T, Sturmfels A, Kappler M, Pletschette M, Böttger EC. 2002. Fitness cost of chromosomal drug resistance-conferring mutations. Antimicrob Agents Chemother 46:1204–1211. doi: 10.1128/AAC.46.5.1204-1211.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Kim HS, Lee KS, Koh WJ, Jeon K, Lee EJ, Kang H, Ahn J. 2012. Serial CT findings of Mycobacterium massiliense pulmonary disease compared with Mycobacterium abscessus disease after treatment with antibiotic therapy. Radiology 263:260–270. doi: 10.1148/radiol.12111374. [DOI] [PubMed] [Google Scholar]
- 34.American Thoracic Society. 2000. Diagnostic standards and classification of tuberculosis in adults and children. Am J Respir Crit Care Med 161:1376–1395. doi: 10.1164/ajrccm.161.4.16141. [DOI] [PubMed] [Google Scholar]
- 35.Kim SY, Shin SJ, Jeong BH, Koh WJ. 2016. Successful antibiotic treatment of pulmonary disease caused by Mycobacterium abscessus subsp. abscessus with C-to-T mutation at position 19 in erm(41) gene: case report. BMC Infect Dis 16:207. doi: 10.1186/s12879-016-1554-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Jamal MA, Maeda S, Nakata N, Kai M, Fukuchi K, Kashiwabara Y. 2000. Molecular basis of clarithromycin-resistance in Mycobacterium avium-intracellulare complex. Tuber Lung Dis 80:1–4. doi: 10.1054/tuld.1999.0227. [DOI] [PubMed] [Google Scholar]