European Society of Mycobacteriology - Instituto Nacional de Saúde ...

OP-12
THE CHARACTERIZATION OF MYCOBACTERIA FROM AN
OUTBREAK SUGGESTS A REVISION OF THE TAXONOMIC STATUS OF
MEMBERS OF THE Mycobacterium chelonae-abscessus GROUP
Sylvia Cardoso Leão 1 , Enrico Tortoli 2 , Cristina Viana-Niero 1 , Suely Yoko Mizuka Ueki 3 , Karla Valeria Batista Lima 4 , Maria
Luiza Lopes 4 , Jesus Yubero 5 María Carmen Menendez 5 , Maria Jesus Garcia 5
1 - Universidade Federal de São Paulo, São Paulo, Brazil
2 - Centro Regionale di Riferimento per la Diagnostica dei Micobatteri, Ospedale di Careggi, Firenze, Italy
3 - Instituto Adolfo Lutz, São Paulo, Brazil
4 - Instituto Evandro Chagas, Belém, Brazil
5 - Universidad Autonoma de Madrid, Madrid, Spain
An outbreak of infections by rapidly growing mycobacteria (RGM) related to invasive procedures has been ongoing in
Brazil since 2004. Isolates from patients submitted to laparoscopic or plastic surgery and to mesotherapy, a cosmetic
procedure, were previously identified by molecular methods as Mycobacterium massiliense and M. bolletii, respectively. The
similarity of rpoB and hsp65 sequences from the clinical isolates and the corresponding sequences from both M. massiliense
and M. bolletii type strains were above the accepted limit for interspecies variability, leading to conflicting results.
Therefore, an extensive characterization was carried out with six Brazilian clinical isolates from this outbreak study and
type strains from the members of the M. abscessus-M. chelonae group – M. abscessus, M. chelonae, M. immunogenum, M.
massiliense and M. bolletii. Phenotypic identification was performed by biochemical tests, high performance liquid chromatography
(HPLC) and drug susceptibility testing. Molecular identification included PCR-restriction enzyme pattern
analysis (PRA) of the hsp65 gene, as well as rpoB and hsp65 gene sequencing and analysis of the corresponding phylogenetic
trees. DNA-DNA hybridization (DDH) and restriction fragment length polymorphism (RFLP) of the 16S rRNA
gene were used as the gold standards for RGM speciation. The clinical isolates and the M. abscessus, M. massiliense and
M. bolletii type strains could not be separated by phenotypic tests and were grouped in the phylogenetic trees obtained.
Also, DDH results confirmed >70% relatedness, and indistinguishable RFLP 16S rRNA patterns were obtained. On the
contrary, separation from M. chelonae and M. immunogenum was supported by results from PRA-hsp65, rpoB and hsp65
phylogenetic trees, DDH and RFLP-16S. Taken together, these results led to the proposition that M. abscesus, M. massiliense
and M. bolletii represent a single species, that of M. abscessus. Two subspecies are also proposed, M. abscessus subsp.
abscessus and M. abscessus subsp. massiliense which can be distinguished by two different PRA-hsp65 patterns, differing in
a single Hae III band, and by differences in rpoB (3.4%) and hsp65 (1.3%) sequences.
54 ESM 2009