In this article, the first isolation of Mycobacterium kyorinense specimens in Brazil is described. M. kyorinense is a recently identified species, with a few strains reported only in Japan. The Brazilian isolates were initially identified as Mycobacterium celatum by PCR restriction enzyme pattern analysis (PRA) with hsp65. However, biochemical tests indicated the same profile of M. kyorinense and distinguished them from M. celatum and Mycobacterium branderi. The sequencing of the hsp65, rpoB, and 16S rRNA genes allowed the accurate identification of isolates as M. kyorinense.
Mycobacterium kyorinense is a nonpigmented slowly growing mycobacterium that was first isolated from sputum of a patient with pneumonia. The species was described in 2009, and to date a few strains have been isolated, all from patients in Japan (5, 9). All patients were immunocompetent, and most of them had a history of pulmonary disease. M. kyorinense was considered a possible cause of clinically significant respiratory disease. The sequences of 16S rRNA genes, hsp65, and rpoB were identical in the strains tested but different from those of the two phylogenetically most related species, Mycobacterium celatum and Mycobacterium branderi (1, 4). Biochemical tests can also distinguish M. kyorinense from M. celatum and M. branderi (1,4,5,8).The aim of this work is to report the first isolation of M. kyorinense in Brazil and, to our knowledge, its first isolation outside Japan and to characterize its hsp65 restriction profile by PCR restriction enzyme pattern analysis (PRA).This study includes two pulmonary specimens (HF1629 and HF1836) that were initially identified as M. celatum in 2007 by PRA with hsp65 in the National Reference Laboratory for Tuberculosis of the Centro de Referência Professor Hélio Fraga/ENSP/ FIOCRUZ. These isolates were obtained from two separate expectorated sputum samples from a patient from Rio de Janeiro State, Brazil, according to microbiological diagnostic criteria for nontuberculous mycobacterial lung disease according to the American Thoracic Society guidelines (3).At admission to the health authority, the patient presented with complete destruction of the left lung by fibrotic lesions, which over the course of the disease extended to the right lung. Treatment with rifampin, isoniazid, pyrazinamide, and ethambutol temporarily improved the patient=s condition, but after less than 1 year, symptoms reappeared, leading to death.The DNA extraction protocol used in this work was developed in our laboratory and is being introduced here. Briefly, one loopful of bacteria grown on Lowenstein-Jensen medium was mixed in 300 l of 1% Triton X-100 and 100 l of acidwashed glass beads with a diameter of 106 m. The cells were shaken vigorously in a Vortex mixer for 1 min and incubated at room temperature for 8 min. After this incubation, 50 l of mixture was placed in a microtube with 100 l of lysis buffer (equal amounts of 15% Chelex 100, 0.5% Tween 20, and Tris-EDTA). The mixture was heated to 100°C for 20 min and shaken vigorously...
