Integrated analysis of ethionamide resistance loci in Mycobacterium tuberculosis clinical isolates.

Tuberculosis patients taking second line drugs such as ethionamide (ETH) have often experienced previous treatment failure and usually have a complex history of disease and treatment that can span decades. Mutations in the ETH activating enzyme, EthA, confer resistance through undescribed mechanisms. To explore the impact of EthA mutations on ETH resistance, data from a total of 160 ETHR isolates was analysed. The most frequently mutated positions are within regions that display sequence conservation with the active site of OTEMO, another FAD-containing NADH-binding Baeyer-Villiger monooxygenase (BVMO), or with the sugar binding site of galectin-4N. Additionally, to look at a possible role of EthR on ETH resistance we purified an EthR mutant identified in a clinical isolate, F110L, and found it to bind the ethA-ethR intergenic region with higher affinity than the wild type regulator in gel shift assays. The ability of cyclic di-GMP to enhance DNA binding is maintained in the EthR mutant. To our knowledge, this is the first ETH resistance study that combines sequence and resistance data of clinical isolates with functional and structural information.

Description of a new Mycobacterium intracellulare pattern of PCR restriction enzyme analysis of hsp65 gene.

This work comprises 9 pulmonary nontuberculous mycobateria isolates obtained from sputum of 4 different patients from Brazil. The sequencing and phylogenetic analysis allowed their accurate identification as Mycobacterium intracellulare. We report a mutation at position 453 creating a new HaeIII cutting site and, therefore, a new PRA-hsp65 M. intracellulare profile.