Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with rifampin (RIF) resistance in the rpoB gene of Mycobacterium tuberculosis. DGGE scans for mutations across large regions of DNA and is comparable to DNA sequencing in detecting DNA alterations. Specific mutations are often recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint. Five DGGE primer sets that scanned for DNA alterations across 775 bp of rpoB were developed. These primer sets were used to scan rpoB for DNA alterations in 296 M. tuberculosis patient isolates from the United States-Mexico border states of Texas and Tamaulipas. The most useful primer set scanned for mutations in the rifampin resistance-determining region (RRDR) and detected mutations in 95% of the RIF-resistant isolates compared to 2% of RIF-susceptible isolates. Thirty-four different alterations were observed within the RRDR by DGGE. In addition, isolates harboring mixtures of DNA within rpoB were readily detected by DGGE. A second PCR primer set was used to detect the V146A mutation in 5 to 7% of RIF-resistant isolates. A third primer set was used to detect mutations in 3% of RIF-resistant isolates, some of which also harbored mutations in the RRDR. Only 1 of 153 RIF-resistant isolates did not have a detectable rpoB mutation as determined by DGGE and DNA sequencing. These results demonstrate the power and usefulness of DGGE in detecting mutations associated with drug resistance in M. tuberculosis.Drug resistance is a major concern in the global tuberculosis epidemic. Multidrug resistance, defined as resistance to at least isoniazid and rifampin (RIF), is greater than 10% in many countries and appears to be more widespread than previously documented (25,26). Resistance to RIF is almost exclusively associated with mutations in the rpoB gene that encodes the -subunit of RNA polymerase (17, 28). Over 70 distinct rpoB mutations have been reported for RIF-resistant Mycobacterium tuberculosis isolates worldwide (7,17,20,28). Approximately 95% of RIF-resistant isolates harbor mutations in the rifampin resistance-determining region (RRDR), an 81-bp region within rpoB that spans codons 507 to 533. Mutations at the serine 531, histidine 526, and aspartate 516 codons have been observed in approximately 86% of RIF-resistant isolates and therefore represent hot spots within the RRDR (17, 28). Alterations outside of the RRDR have also been reported for the N-terminal, CII, and CIII regions of rpoB (6, 28) (Fig. 1).Molecular techniques are receiving increased scrutiny as alternatives to traditional drug susceptibility testing for M. tuberculosis (22). Molecular techniques can detect DNA alterations in hours or days, while assaying for drug resistance by culture methods takes weeks. Although the clustering of most mutations in the RRDR simplifies molecular analysis, the heterogeneity of mutations represents a challenge in detecting DNA alterations as a predictor of RIF resistance. These genetic considerations affect b...
To examine the transmission of drug-resistant (DR) tuberculosis between Texas and Mexico, Mycobacterium tuberculosis isolates resistant to one or more of the first-line antimycobacterial drugs were obtained from 606 patients who resided in Texas and 313 patients who resided in Mexico, primarily within the state of Tamaulipas. The isolates were genotyped by IS6110-based restriction fragment length polymorphism (RFLP) analysis and spoligotyping. Of the 919 isolates genotyped, 413 (45%) grouped into 105 clusters containing 2 or more isolates with identical genotypes. In addition to having identical genotypes, identical drug resistance patterns were identified in 250 isolates in 78 clusters (DR clusters). Twenty DR clusters, containing isolates from 32% of the total number of patients infected with DR strains, were geographically distributed across Mexico and Texas. Within this population of 919 patients infected with DR isolates, the probability of being in a DR cluster was the same for residents of Mexico and Texas. In Texas, the significant independent predictors of clustering within DR clusters as opposed to genotype clusters were found to be race, age, country of birth, human immunodeficiency virus (HIV) infection status, and resistance to more than one drug. Specifically, isolates from African Americans, individuals under age 65, individuals born in the United States, and HIV-positive individuals were each more likely to be associated with a DR cluster. By contrast, no significant independent predictors of clustering in a DR cluster were identified in Mexico. Although some DR M. tuberculosis strains are geographically restricted, this study suggests that a number of strains are transmitted between Mexico and the United States.
Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with pyrazinamide (PZA) resistance in the pncA gene of Mycobacterium tuberculosis. DGGE scans for mutations across large regions of DNA and rivals sequencing in its ability to detect DNA alterations. Specific mutations can often be recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint. Five PCR target fragments were designed to scan for DNA alterations across 600 bp of pncA in 181 M. tuberculosis isolates from patients residing in the U.S-Mexico border states of Texas and Tamaulipas, respectively. A region of pncA was observed with a high GC content and a melting temperature approaching 90°C that was initially refractory to denaturation, and a DGGE target fragment was specifically designed to detect mutations in this region. DGGE detected pncA mutations in 82 of 83 PZA-resistant isolates. By contrast, only 1 of 98 PZA-susceptible isolates harbored a detectable DNA alteration. The pncA gene was sequenced from 41 isolates, and 32 DNA alterations in 32 PZA-resistant isolates were identified, including 11 new mutations. DGGE also detected nine isolates whose susceptibility to PZA appeared to be incorrect, and DNA sequencing confirmed these apparent errors in drug susceptibility testing. These results demonstrate the power and usefulness of DGGE in detecting mutations associated with PZA resistance in M. tuberculosis.Pyrazinamide (PZA) is a front-line drug used in the treatment of tuberculosis (TB). In a typical short-course (6-month) therapy, PZA is administered with rifampin (RIF), isoniazid (INH), and ethambutol (EMB) for the first 2 months of treatment, followed by 4 months of treatment with INH and RIF (1). During the initial acute phase, PZA and RIF are responsible for much of the killing of persisting Mycobacterium tuberculosis bacteria (23, 48). The mode of action of PZA is complex and not fully understood. It requires activation to pyrazinoic acid by a pyrazinamidase/nicotinamidase (PZase) encoded by the pncA gene (48). The activated acid form is excreted and then reabsorbed in a protonated form. It is thought that the acidification of the M. tuberculosis cells by the protonated form represents the primary mechanism by which PZA kills tubercle bacilli (48, 49). PZase inactivation is the primary mechanism for developing resistance to PZA (31,35,47,48). Any mutation in pncA that inactivates the encoded enzyme appears to be sufficient to confer resistance. Consequently, PZA resistance mutations are highly diverse and are found throughout pncA. Cumulative reports indicate that between 72 and 98% of PZAresistant isolates harbor pncA mutations (33,46,49). More recent reports, however, tend to support the upper end of this range (48). Earlier reports may have been subject to inaccurate susceptibility testing using culture methods. Susceptibility testing media must have a pH of 6 for PZA to be active against susceptible cells, and this pH is near the limit for growth of tubercle bacill...
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