Search

ABSTRACTMoxifloxacin has shown excellent activity against drug-sensitive as well as drug-resistant tuberculosis (TB), thus confirming DNA gyrase as a clinically validated target for discovering novel anti-TB agents. We have identified novel inhibitors in the pyrrolamide class which kill Mycobacterium tuberculosisthrough inhibition of ATPase activity catalyzed by the GyrB domain of DNA gyrase. A homology model of the M. tuberculosisH37Rv GyrB domain was used for deciphering the structure-activity relationship and binding interactions of inhibitors with mycobacterial GyrB enzyme. Proposed binding interactions were later confirmed through cocrystal structure studies with the Mycobacterium smegmatisGyrB ATPase domain. The most potent compound in this series inhibited supercoiling activity of DNA gyrase with a 50% inhibitory concentration (IC50) of <5 nM, an MIC of 0.03 μg/ml against M. tuberculosisH37Rv, and an MIC90of <0.25 μg/ml against 99 drug-resistant clinical isolates of M. tuberculosis. The frequency of isolating spontaneous resistant mutants was ∼10−6to 10−8, and the point mutation mapped to the M. tuberculosisGyrB domain (Ser208 Ala), thus confirming its mode of action. The best compound tested for in vivoefficacy in the mouse model showed a 1.1-log reduction in lung CFU in the acute model and a 0.7-log reduction in the chronic model. This class of GyrB inhibitors could be developed as novel anti-TB agents.

ABSTRACTThis study aimed to investigate the prevalence of resistance to second-line antituberculosis (anti-TB) drugs and its association with resistance-related mutations in Mycobacterium tuberculosisisolated in China. In the present study, we collected 380 isolates from a population-based study in China and tested the drug susceptibility to first- and selected second-line drugs. These results were compared with polymorphisms in the DNA sequences of genes associated with drug resistance and MIC values of the studied second-line drugs. Of 43 multidrug-resistant M. tuberculosisisolates, 13 showed resistance to fluoroquinolones or injectable second-line drugs (preextensively drug-resistant TB [pre-XDR-TB]), and 4 were resistant to both and thus defined as extensively drug-resistant TB (XDR-TB). Age and previous TB therapy, including use of second-line drugs, were two independent factors associated with increased resistance to both first- and second-line drugs. Molecular analysis identified the most frequent mutations in the resistance-associated genes: D94G in gyrA(29.1%) and A1401G in rrs(30.8%). Meanwhile, all 4 XDR-TB isolates had a mutation in gyrA, and 3 of them carried the A1401G mutation in rrs. Mutations in gyrAand rrswere associated with high-level resistance to fluoroquinolones and the second-line injectable drugs. In addition to the identification of resistance-associated mutations and development of a rapid molecular test to diagnose the second-line drug resistance, it should be a priority to strictly regulate the administration of second-line drugs to maintain their efficacy to treat multidrug-resistant TB.

ABSTRACTThe antigen 85 (Ag85) protein family, consisting of Ag85A, -B, and -C, is vital for Mycobacterium tuberculosisdue to its role in cell envelope biogenesis. The mycoloyl transferase activity of these proteins generates trehalose dimycolate (TDM), an envelope lipid essential for M. tuberculosisvirulence, and cell wall arabinogalactan-linked mycolic acids. Inhibition of these enzymes through substrate analogs hinders growth of mycobacteria, but a link to mycolic acid synthesis has not been established. In this study, we characterized a novel inhibitor of Ag85C, 2-amino-6-propyl-4,5,6,7-tetrahydro-1-benzothiophene-3-carbonitrile (I3-AG85). I3-AG85 was isolated from a panel of four inhibitors that exhibited structure- and dose-dependent inhibition of M. tuberculosisdivision in broth culture. I3-AG85 also inhibited M. tuberculosissurvival in infected primary macrophages. Importantly, it displayed an identical MIC against the drug-susceptible H37Rv reference strain and a panel of extensively drug-resistant/multidrug-resistant M. tuberculosisstrains. Nuclear magnetic resonance analysis indicated binding of I3-AG85 to Ag85C, similar to its binding to the artificial substrate octylthioglucoside. Quantification of mycolic acid-linked lipids of the M. tuberculosisenvelope showed a specific blockade of TDM synthesis. This was accompanied by accumulation of trehalose monomycolate, while the overall mycolic acid abundance remained unchanged. Inhibition of Ag85C activity also disrupted the integrity of the M. tuberculosisenvelope. I3-AG85 inhibited the division of and reduced TDM synthesis in an M. tuberculosisstrain deficient in Ag85C. Our results indicate that Ag85 proteins are promising targets for novel antimycobacterial drug design.

ABSTRACTRifampin is a major drug used in the treatment of tuberculosis infections, and increasing rifampin resistance represents a worldwide clinical problem. Resistance to rifampin is caused by mutations in the rpoBgene, encoding the β-subunit of RNA polymerase. We examined the effect of three different rpoBmutations on the fitness of Mycobacterium tuberculosis. Rifampin-resistant mutants were isolated from a virulent clinical isolate of M. tuberculosis(strain Harlingen) in vitro at a mutation frequency of 2.3 × 10−8. Mutations in the rpoBgene were identified, and the growth rates of three defined mutants were measured by competition with the susceptible parent strain in laboratory medium and by single cultures in a macrophage cell line and in laboratory medium. All of the mutants showed a decreased growth rate in the three assays. The relative fitness of the mutants varied between 0.29 and 0.96 (that of the susceptible strain was set to 1.0) depending on the specific mutant and assay system. Unexpectedly, the relative fitness ranking of the mutants differed between the different assays. In conclusion, rifampin resistance is associated with a cost that is conditional.

ABSTRACTThis study evaluated cross-resistance of Mycobacterium tuberculosisstrains to ofloxacin, moxifloxacin, and gatifloxacin and investigated the presence of mutations in gyrAand gyrB. Fluoroquinolone susceptibilities were determined for 41 M. tuberculosisstrains by the proportion method on 7H11, and MICs were determined by the resazurin microtiter assay. Forty strains shared the same resistance results for the three fluoroquinolones. However, one strain, with an Asn-533 → Thr mutation in gyrB, was susceptible to ofloxacin but resistant to moxifloxacin and gatifloxacin.

ABSTRACTThirty-four pyrazinamide-resistant and 37 pyrazinamide-susceptible Mycobacterium tuberculosiscomplex strains were analyzed for pncAgene mutations. None of the sensitive strains had any mutations, apart from silent mutations, whereas all but one resistant strain showed pncAmutations. By using sequencing as a means of early resistance detection, the inconsistency of phenotypic pyrazinamide assays can be circumvented.

ABSTRACTThe distribution and resistance levels of 189 in vitro-selected rifampin-resistant Mycobacterium tuberculosismutants of Beijing and other genotypes were determined. Apart from a higher amount of codon 522 point mutations and large deletions, a spread of mutations similar to that reported for clinical isolates was seen. Most mutations were correlated with high-level resistance; a lower level, or a MIC of <16 mg/liter, was associated with codon 522 mutations. Multiple mutations were detected in two Beijing mutants.

ABSTRACTPyrazinamide (PZA) is a standard component of first-line treatment regimens for Mycobacterium tuberculosisand is included in treatment regimens for drug-resistant M. tuberculosiswhenever possible. Therefore, it is imperative that susceptibility to PZA be assessed reliably prior to the initiation of therapy. Currently available growth-based PZA susceptibility tests are time-consuming, and results can be inconsistent. Molecular tests have been developed for most first-line antituberculosis drugs; however, a commercial molecular test is not yet available for rapid detection of PZA resistance. Recently, a line probe assay, the Nipro Genoscholar PZA-TB II assay, was developed for the detection of mutations within the pncAgene, including the promoter region, that are likely to lead to PZA resistance. The sensitivity and specificity of this assay were evaluated by two independent laboratories, using a combined total of 249 strains with mutations in pncAor its promoter and 21 strains with wild-type pncA. Overall, the assay showed good sensitivity (93.2% [95% confidence interval, 89.3 to 95.8%]) and moderate specificity (91.2% [95% confidence interval, 77.0 to 97.0%]) for the identification of M. tuberculosisstrains predicted to be resistant to PZA on the basis of the presence of mutations (excluding known PZA-susceptible mutations) in the pncAcoding region or promoter. The assay shows promise for the molecular prediction of PZA resistance.