Mycobacterium bolletii Respiratory Infections

Contrary to other species in the Mycobacterium chelonae-abscessus complex, we reidentified M. bolletii strains isolated from 4 respiratory patients and found these strains to be uniformly resistant to clarithromycin. No mutations previously associated with macrolide resistance in bacteria were detected in either the 23S rDNA or the genes encoding riboproteins L4 and L22.

tive results of a direct microscopic examination; refi ned identifi cation performed 8 years later in 2003 found both isolates to be M. bolletii. The patient received rifampin, clarithromycin, isoniazid, and ciprofl oxacin for 16 months. After an initial improvement, the patient continued to have an episodic cough and hemoptysis, and M. bolletii was grown from 2 sputum specimens; direct microscopic examination yielded acid-fast bacilli. In November 1998, the patient still had symptoms. In 2003, he was admitted to an intensive care unit for acute respiratory distress syndrome. A broncho-alveolar lavage specimen yielded a mixed culture of multidrug-resistant M. bolletii and Klebsiella pneumoniae despite negative results of direct examination. The patient eventually died of K. pneumoniae septicemia within days of his admission to the ICU. Patient 3, a 77-year-old man who smoked 3 packs of cigarettes per month and who had a history of pulmonary TB, was admitted to Timone Hospital, Marseilles, in October 2000 with a diagnosis of bronchitis. Corticoid therapy was prescribed. In March 2001, the patient was admitted for respiratory insuffi ciency. A chest radiograph showed diffuse bullous emphysema in both lungs. Microscopic examination of 1 sputum specimen and 1 bronchial aspirate yielded the presence of acid-fast bacilli that were later identifi ed as M. bolletii. The patient left the hospital without treatment, and no further information on his condition is available.
Patient 4, a 90-year-old woman, sought treatment with a temperature of 38°C, hemoptysis, and bilateral micronodular infi ltrates of the upper lung lobes. She had a history of childhood pulmonary TB. Sputum specimens yielded mycobacteria that were identifi ed later as M. bolletii despite negative results of direct examination. The patient remained febrile after 1 month of treatment with intravenous imipenem and amikacin, her pulmonary condition worsened, and a sputum smear showed numerous acid-fast bacilli. The treatment regimen was changed to a combination of ciprofl oxacin, clarithromycin, and ethambutol, but the patient died 2 weeks after treatment began.
Twelve of 31 isolates were identifi ed by rpoB sequencing as M. abscessus, 11 as M. chelonae, 4 as M. massiliense, and 4 as M. bolletii. M. abscessus and M. bolletii isolates showed no intraspecifi c rpoB sequence variation. In contrast, 0.7% sequence divergence was observed in M. chelonae (6 sequevars) and M. massiliense (3 sequevars) isolates ( Figure). The 4 M. bolletii isolates were unique among these 31 MCAC isolates in that they were multidrug resistant (Table). The isolates exhibited clarithromycin MICs >256 μg/mL, whereas the other MCAC isolates had clarithromycin MICs <2 μg/mL. The E-test is not a validated method for MIC determination in rapidly growing mycobacteria, yet the results we obtained were similar to those previously reported for the reference broth microdilution method (9). In the M. bolletii isolates, we found no substitutions, deletions, or insertions in domain V (A2058, A2059, C2611 position, Escherichia coli numbering) of the 23S rDNA or in the L4 and L22 ribosomal protein genes.

Conclusions
M. bolletii is an emerging pathogen responsible for respiratory tract infections in patients with underlying com-promised respiratory function. In our study, M. bolletii was responsible for pulmonary infection in 3 of 4 patients (patients 2-4) (10). M. bolletii was repeatedly isolated from different samples from those 3 patients over a period of several weeks. A coexisting broncho-pulmonary disease was found in 3 of the 4 patients, and clinical features and radiograph patterns that suggested nontuberculous mycobacteria infection were observed in all 4 patients (10). Infected patients were >75 years of age. Three of 4 patients had hemoptysis and lung infi ltrates, M. bolletii was the sole organism isolated from respiratory tract specimens. In this study, 16S rDNA sequencing misidentifi ed 25% of  31 MCAC isolates that were eventually identifi ed as M. massiliense and M. bolletii. This statistic agrees with observations made during the recent description of M. bolletii infection after mesotherapy (11). Clarithromycin was administrated to 2 of the 4 patients, both of whom died within several weeks after treatment began. The 4 M. bolletii isolates were highly resistant to clarithromycin, yet they did not harbor the 23S rDNA mutations that have been previously found in clarithromycin-resistant M. abscessus strains (12). Additionally, no mutations in riboproteins L4 and L22, which are associated with macrolide resistance in Streptococcus pneumoniae (13) (14). Further investigations are therefore needed to clarify the mechanism of clarithromycin resistance in M. bolletii.
Clarithromycin has been recommended as the fi rstline antimicrobial drug for treating rapidly growing mycobacteria infections in patients with compromised respiratory function (1,10). Patient deaths (3) have been linked to clarithromycin resistance, with a risk of secondary clarithromycin resistance during monotherapy estimated to be <10% (12). Recent studies showed that 21%-36% of MCAC isolates were resistant to clarithromycin (15). The recommendation for treating M. abscessus infection with clarithromycin was made before discovering M. bolletii, a multidrug-resistant species that mimics M. abscessus. Our report illustrates that accurate species identifi cation and in vitro clarithromycin susceptibility testing should be recommended for MCAC isolates of clinical interest. GenBank accession nos. were as follows: