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JAC Advance Access published online on September 13, 2006
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkl363
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org
Received May 12, 2006
Revised July 26, 2006
Accepted August 12, 2006

Original article

pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis clinical isolates from the southeast region of Brazil

P. Barco 1, R. F. Cardoso 2 *, R. D. C. Hirata 1, C. Q. F. Leite 3, J. R. Pandolfi 3, D. N. Sato 4, M. L. Shikama 5, F. Fiúza de Melo 6, E. M. Mamizuka 1, P. A. Z. Campanerut 7, and M. H. Hirata 1

1 Department of Clinical Analysis and Toxicology, University of São Paulo, São Paulo, Brazil
2 Department of Clinical Analysis, State University of Maringá, Paraná, Brazil; Present address. Laboratório de Bacteriologia Clínica, Departamento de Análises Clínicas, Universidade Estadual de Maringá, Avenida Colombo, 5790, Bloco J-90, sala 05, Jardim Universitário, CEP 87020-900, Maringá, PR, Brasil
3 Department of Biological Science, Paulista State University, Araraquara, São Paulo, Brazil
4 Institute Adolfo Lutz, Ribeirão Preto, São Paulo, Brazil
5 Institute Adolfo Lutz, Sorocaba, São Paulo, Brazil
6 Institute Clemente Ferreira, São Paulo, Brazil
7 Department of Clinical Analysis, State University of Maringá, Paraná, Brazil

* To whom correspondence should be addressed.
R. F. Cardoso, E-mail: rfcardoso{at}uem.br


  Abstract

Objectives: To investigate the presence of mutations in the pncA gene in 31 pyrazinamide-resistant Mycobacterium tuberculosis and 5 susceptible strains. MICs and pyrazinamidase (PZase) activity were also determined.

Methods: All 36 M. tuberculosis clinical isolates were genotyped by mycobacterial interspersed repetitive units (MIRUs) and most were also typed by spoligotyping. The MIC value necessary to inhibit 99% of the resistant mycobacterial isolates was determined by microplate Alamar Blue assay (MABA) and by Löwenstein-Jensen assay (LJA). The PZase activity was measured by pyrazinamide deamination to pyrazinoic acid and ammonia, and the entire pncA sequence including the 410 bp upstream from the start codon was determined by DNA sequencing of purified PCR products.

Results: Of the 31 isolates resistant to pyrazinamide, 26 (83.9%) showed at least one mutation in the pncA gene or in its putative regulatory region. Among the 22 different mutations detected in the pncA gene and in its regulatory region, 9 (40.9%) mutations (consisting of six substitutions, two insertions and one deletion) have not been described in previous studies. Three pyrazinamide-resistant isolates, confirmed by MIC varying from 800 to 1600 mg/L, carried the wild-type pncA sequence and retained PZase activity.

Conclusions: These results contribute to the knowledge of the molecular mechanism of pyrazinamide resistance in Brazil and also expand the profile of pncA mutations worldwide. The MABA was successfully used to determine the MICs of pyrazinamide.

Keywords: Alamar Blue; MIRU; pyrazinamidase.
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