Detection of Multidrug Resistance in Mycobacterium tuberculosis

Abstract

We developed a DNA sequencing-based method to detect mutations in the genome of drug-resistant Mycobacterium tuberculosis. Drug resistance in M. tuberculosis is caused by mutations in restricted regions of the genome. Eight genome regions associated with drug resistance, including rpoB for rifampin (RIF), katG and the mabA (fabG1)-inhA promoter for isoniazid (INH), embB for ethambutol (EMB), pncA for pyrazinamide (PZA), rpsL and rrs for streptomycin (STR), and gyrA for levofloxacin, were amplified simultaneously by PCR, and the DNA sequences were determined. It took 6.5 h to complete all procedures. Among the 138 clinical isolates tested, 55 were resistant to at least one drug. Thirty-four of 38 INH-resistant isolates (89.5%), 28 of 28 RIF-resistant isolates (100%), 15 of 18 EMB-resistant isolates (83.3%), 18 of 30 STR-resistant isolates (60%), and 17 of 17 PZA-resistant isolates (100%) had mutations related to specific drug resistance. Eighteen of these mutations had not been reported previously. These novel mutations include one in rpoB, eight in katG, one in the mabA-inhA regulatory region, two in embB, five in pncA, and one in rrs. Escherichia coli isolates expressing individually five of the eight katG mutations showed loss of catalase and INH oxidation activities, and isolates carrying any of the five pncA mutations showed no pyrazinamidase activity, indicating that these mutations are associated with INH and PZA resistance, respectively. Our sequencing-based method was also useful for testing sputa from tuberculosis patients and for screening of mutations in Mycobacterium bovis. In conclusion, our new method is useful for rapid detection of multiple-drug-resistant M. tuberculosis and for identifying novel mutations in drug-resistant M. tuberculosis.The emergence and spread of drug-resistant strains of Mycobacterium tuberculosis, especially multidrug-resistant (MDR) strains, are serious threats to the control of tuberculosis and comprise an increasing public health problem (40). Patients infected with MDR strains, which are defined as strains resistant to both rifampin (RIF) and isoniazid (INH), are difficult to cure and are more likely to remain sources of infection for a longer period of time than are patients with drug-susceptible strains (40).It is essential that rapid drug susceptibility tests be developed to prevent the spread of MDR M. tuberculosis. The time necessary for culture of specimens was reduced by the radiometric BACTEC 460TB system (BD Biosciences, Sparks, MD), the nonradiometric ESP II system (Trek Diagnostics, Westlake, OH), and other rapid broth methods, such as BACTEC MGIT 960 SIRE (BD Biosciences) (20). These drug susceptibility tests, however, still require 1 to 2 weeks for final determination and reporting to the clinician (23). Additional reductions in the detection period are needed.Drug resistance in M. tuberculosis is caused by mutations in relatively restricted regions of the genome (17, 39). Mutations associated with drug resistance occur in rpoB for RIF, katG and the promote...

Detección de mutaciones asociadas a cepas multidrogo resistente de Mycobacterium tuberculosis en Chile

“…Las mutaciones en los codones 381 y 463 también han sido reportadas, sin embargo, se ha descrito que la mutación R463L (cGG→cTG) es un polimorfismo que no influencia la resistencia a INH 18,31,32,33 . En este estudio se encontraron 3 mutaciones en el gen katG que no han sido descritas en la literatura (T344P y las deleciones de 3 bases en los codones 450 y 485).…”

Clinical Laboratory Diagnostics for Mycobacterium tuberculosis

“…Several investigators have focused their efforts in developing assays similar to the MTBDRplus assay to include resistant marker to more than one class of antituberculosis drugs and for more than one mutation per gene target (Sekiguchi et al, 2007;Zhang et al, 2007;Ong et al, 2010;Pholwat et al, 2011).…”

In silico Analysis of Novel Mutation ala102pro Targeting pncA Gene of M. Tuberculosis

“…5 µl of supernatant was used as the template DNA. [13] with the thermal profile for 30 cycles of denaturation at 95°C for 1 min, annealing at 60°C for 30 sec and extension at 72°C for 1 min which yielded a 670 bp product. For each PCR run, negative control and positive control were included in the study.…”