As the prevalence of multidrug-resistant and extensively drug-resistant tuberculosis strains continues to rise, so does the need to develop accurate and rapid molecular tests to complement time consuming growth-based drug susceptibility testing. Performance of molecular methods relies on the association of specific mutations with phenotypic drug resistance and while considerable progress has been made for resistance detection of first-line antituberculosis drugs, rapid detection of resistance for second-line drugs lags behind. The rrs A1401G allele is considered a strong predictor of cross-resistance between the three second-line injectable drugs, capreomycin (CAP), kanamycin, and amikacin. However, discordance is often observed between the rrs A1401G mutation and CAP resistance, with up to 40% of rrs A1401G mutants being classified as CAP susceptible. We measured the minimal inhibitory concentrations (MICs) to CAP in 53 clinical isolates harboring the A1401G mutation and found that the CAP MICs ranged from 8 mug/ml to 40 mug/ml. These results were drastically different from engineered A1401G mutants generated in isogenic Mycobacterium tuberculosis, which exclusively exhibited high-level CAP MICs of 40 mug/ml. These data support prior studies suggesting the critical concentration of CAP (10 mug/ml) used to determine resistance by indirect agar proportion may be too high to detect all CAP resistant strains and suggests that a larger percentage of resistant isolates could be identified by lowering the critical concentration. These data also suggest that differences in resistance levels among clinical isolates are possibly due to second site or compensatory mutations located elsewhere in the genome.

Since the discovery of streptomycin's bactericidal activity against Mycobacterium tuberculosis, aminoglycosides have been utilized to treat tuberculosis (TB). Today, the aminoglycosides kanamycin and amikacin are used to treat multidrug-resistant (MDR) TB, and resistance to any of the second-line injectable antibiotics, including kanamycin, amikacin, or capreomycin, is a defining characteristic of extensively drug-resistant (XDR) TB. Resistance to kanamycin and streptomycin is thought to be due to the acquisition of unlinked chromosomal mutations. However, we identified eight independent mutations in the 5' untranslated region of the transcriptional activator whiB7 that confer low-level resistance to both aminoglycosides. The mutations lead to 23- to 145-fold increases in whiB7 transcripts and subsequent increased expression of both eis (Rv2416c) and tap (Rv1258c). Increased expression of eis confers kanamycin resistance in these mutants, while increased expression of tap, which encodes an efflux pump, is a previously uncharacterized mechanism of low-level streptomycin resistance. Additionally, high-level resistance to streptomycin arose at a much higher frequency in whiB7 mutants than in a wild-type (WT) strain. Although whiB7 is typically associated with intrinsic antibiotic resistance in M. tuberculosis, these data suggest that mutations in an uncharacterized regulatory region of whiB7 contribute to cross-resistance against clinically used second-line antibiotics. As drug resistance continues to develop and spread, understanding the mechanisms and molecular basis of antibiotic resistance is critical for the development of rapid molecular tests to diagnose drug-resistant TB strains and ultimately for designing regimens to treat drug-resistant cases of TB.

The emergence of multi and extensively drug-resistant tuberculosis is a significant impediment to the control of this disease because treatment becomes more complex and costly. Reliable and timely drug susceptibility testing is critical to ensure patients receive effective treatment and become non-infectious. Molecular methods can provide accurate and rapid drug susceptibility results. We used DNA sequencing to detect resistance to the first-line antituberculosis drugs, isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB), and the second-line drugs, amikacin (AMK), capreomycin (CAP), kanamycin (KAN), ciprofloxacin, (CIP) and ofloxacin (OFX). Nine loci were sequenced: rpoB for resistance to RIF, katG and inhA (INH), pncA (PZA), embB (EMB), gyrA (CIP and OFX), rrs, eis, and tlyA (KAN, AMK, and CAP). A total of 314 clinical M. tuberculosis complex isolates, representing a variety of antibiotic resistance patterns, genotypes and geographical origins were analyzed. The molecular data were compared to the phenotypic data and the accuracy values were calculated. Sensitivity and specificity values (as percentages) for the first-line drug loci were rpoB (97.1, 93.6), katG (85.4, 100), inhA (16.5, 100), katG and inhA together (90.6, 100) pncA (84.6, 85.8), and embB (78.6, 93.1). The values for the second-line drugs were also calculated. The size and scope of this study, in numbers of loci and isolates examined, and the phenotypic diversity of those isolates, support the use of DNA sequencing to detect drug resistance in the M. tuberculosis complex. Further, the results can be used to design diagnostic tests utilizing other mutation detection technologies.

The current study describes the development of a unique real-time PCR assay for the detection of mutations conferring drug resistance in Mycobacterium tuberculosis (Mtb). The Rifampicin Resistance Determinant Region (RRDR) of rpoB and specific regions of katG and the inhA promoter were targeted for the detection of rifampicin (RIF) and isoniazid (INH) resistance, respectively. Additionally, this assay was multiplexed to discriminate Mycobacterium tuberculosis complex (MTBC) strains from Nontuberculous Mycobacteria (NTM) strains by targeting the IS6110 insertion element. High resolution melting (HRM) analysis following real-time PCR was used to identify Mtb strains containing mutations at the targeted loci, and locked nucleic acid (LNA) probes were used to enhance the detection of strains containing specific SNP transversion mutations. This method was used to screen 252 Mtb clinical isolates including 154 RIF resistant strains and 174 INH resistant strains based on the agar proportion method of drug susceptibility testing (DST). Of the 154 RIF resistant strains, 148 were also resistant to INH and therefore classified as multidrug resistant (MDR). The assay demonstrated a sensitivity and specificity of 91% and 98%, respectively, for the detection of RIF resistance, and 87% and 100% for the detection of INH resistance. Overall, this assay showed a sensitivity of 85% and a specificity of 98% for the detection of MDR strains. This method provides a rapid, robust, and inexpensive way to detect the dominant mutations known to confer MDR in Mtb strains and offers several advantages over current molecular and culture-based techniques.