Characterization of rifampin-resistance in pathogenic mycobacteria

Abstract

The emergence of rifampin-resistant strains of pathogenic mycobacteria has threatened the usefulness of this drug in treating mycobacterial diseases. Critical to the treatment of individuals infected with resistant strains is the rapid identification of these strains directly from clinical specimens. It Rifampin is an important component of effective multidrug therapies for tuberculosis and leprosy; however, widespread use has led to the emergence of rifampin-resistant (Rif) strains, threatening its usefulness in treating mycobacterial diseases (4-6, 8, 26, 27). Rapid information about drug susceptibility patterns is critical to the treatment of individuals with mycobacterial disease for which rifampin is indicated. Since conventional drug susceptibility testing can require 2 to 4 weeks after growth detection (Mycobacterium tuberculosis) or up to a year (Mycobacterium leprae) in mouse footpads, improvements are needed to yield accurate analysis in a shorter time. DNA diagnostic assays have the potential to provide rapid analysis of rifampin resistance in mycobacteria because of their high degree of sensitivity and specificity and the fact that they do not rely on in vitro growth for results. Shortening the time between diagnosis and the onset of effective therapy should improve patients' survival (tuberculosis) or decrease physical deformities and ocular manifestations resulting in disabilities and blindness (leprosy).Developing such assays requires knowledge of the molecular basis of Rif' in pathogenic mycobacteria. Mutations resulting in the Rif' phenotype in prokaryotes have been mapped to the gene encoding the 1-subunit of the DNA-dependent RNA polymerase (rpoB gene) (10, 11). Recently, the entire rpoB genes of M. leprae (7) resistance have been identified in both species (8,12,28,29). To further characterize mutations associated with the Rif phenotype in M. tuberculosis, M. leprae, and other pathogenic mycobacteria, we developed a rapid PCR-based, DNA sequencing protocol targeted to a 305-bp region of rpoB. By direct DNA sequencing of PCR products, the nucleic acid sequence within this region was determined in 4 rifampinsusceptible (Rifs) and 4 Rif' strains of M. leprae and in 12 Rif' and 110 Rif' strains of M. tuberculosis. In addition, mutations were identified in this region of Rif' strains of Mycobacterium africanum and Mycobacterium avium, the latter causing frequent opportunistic infections in immunocompromised hosts. On the basis of these results we have established conditions for a PCR-heteroduplex formation assay (PCR-HDF) for the rapid detection of the Rif' phenotype in pathogenic mycobacteria. MATERUILS AND METHODSMycobacterial strains. Rifampin-susceptible and -resistant strains of M. leprae were isolated initially from homogenates of skin biopsy samples from lepromatous leprosy patients not responding to antileprosy therapy, which included rifampin, and were subsequently defined as resistant to rifampin by the standard mouse footpad drug susceptibility assay (23). These strains were amplifie...

Multi-drug Resistant Mycobacterium leprae from Patients with Leprosy.

“…Mutations in the rpoB gene, encoding the ␤ subunit of RNA polymerase, were reported to result in resistance to rifampin in several mycobacterial species, including M. leprae (4,18). In the present study, 26 isolates showed the mutations which had already been observed in rifampinresistant bacteria (Fig.…”

Tuberculose resistente: revisão molecular

“…39 A caracterização do gene rpoB em Echerichia coli demonstrou que a rifampicina interage especificamente com a subunidade β da RNA polimerase e que mutações no locus rpoB conferem trocas conformacionais, impedindo uma ligação eficiente do fármaco e, conseqüentemente, a resistência. 16,41 Essas informações auxiliaram na compreensão das bases de resistência à RMP em M. tuberculosis. (Figura 1).…”

“…As posições dos aminoácidos substituídos ou ausentes devido a inserções ou mutações são mostradas nas caixas acima da seqüência. Extraida de Willians et al 41 (1994 …”

Nonconventional and new methods in the diagnosis of tuberculosis: feasibility and applicability in the field

“…It has been thoroughly used for characterising mutations in the rpoB gene in rifampicin-resistant strains and to detect mutations responsible for resistance to other anti-TB drugs [57,60,61]. Several other genotypic methods have been proposed to detect resistance to antibiotics, such as PCR-single strand conformation polymorphism [57], PCR-heteroduplex formation [62], and solid-phase hybridisation assays. Moreover, the Line Probe Assay (LiPA-Rif) based on the hybridisation of amplified DNA from cultured strains or clinical samples to ten probes encompassing the core region of the rpoB gene of M. tuberculosis, immobilised on a nitrocellulose strip [63], and the GenoType mycobacterium tuberculosis test (MTB) DR (Hain, Germany), a commercial system for the detection of the M. tuberculosis complex and its resistance to rifampicin and isoniazid from culture samples based on the detection of the most common mutations in the rpoB and katG genes, respectively.…”