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Thomas M Shinnick - One of the best experts on this subject based on the ideXlab platform.
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genome wide exploration of the drug action of Capreomycin on mycobacterium tuberculosis using affymetrix oligonucleotide genechips
Journal of Infection, 2007Co-Authors: Thomas M ShinnickAbstract:Summary Objective Multi-drug resistance and latent infection are two major issues in current tuberculosis (TB) control and management. Capreomycin is an important drug used for TB with multi-drug resistance. A recent study also indicates that this drug possesses unique bactericidal activity against non-replicating TB bacilli among known anti-TB drugs. Thus, there is an urgent need for investigating the full-spectrum action of Capreomycin. Methods Here we conduct the first microarray-based study on Capreomycin using the high-resolution Affymetrix oligonucleotide GeneChip system. Results The results indicate that Capreomycin primarily acts on the information pathways but it also significantly affects cell wall, cell processes, intermediate metabolism and respiration in Mycobacterium tuberculosis . Conclusions This study not only transcriptionally validates the specific molecular target, 16S rRNA, but also discovers potential new targets of Capreomycin, including genes operating at the DNA level, such as Rv0054 ( ssb ) and Rv3715c ( recR ), as well as genes involved in cell division like Rv3260c ( whiB2 ). In addition, the nuo gene cluster and the ATP synthase gene cluster are repressed.
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molecular analysis of cross resistance to Capreomycin kanamycin amikacin and viomycin in mycobacterium tuberculosis
Antimicrobial Agents and Chemotherapy, 2005Co-Authors: Courtney E Maus, Bonnie B Plikaytis, Thomas M ShinnickAbstract:Capreomycin, kanamycin, amikacin, and viomycin are drugs that are used to treat multidrug-resistant tuberculosis. Each inhibits translation, and cross-resistance to them is a concern during therapy. A recent study revealed that mutation of the tlyA gene, encoding a putative rRNA methyltransferase, confers Capreomycin and viomycin resistance in Mycobacterium tuberculosis bacteria. Mutations in the 16S rRNA gene (rrs) have been associated with resistance to each of the drugs; however, reports of cross-resistance to the drugs have been variable. We investigated the role of rrs mutations in Capreomycin resistance and examined the molecular basis of cross-resistance to the four drugs in M. tuberculosis laboratory-generated mutants and clinical isolates. Spontaneous mutants were generated to the drugs singularly and in combination by plating on medium containing one or two drugs. The frequencies of recovery of the mutants on single- and dual-drug plates were consistent with single-step mutations. The rrs genes of all mutants were sequenced, and the tlyA genes were sequenced for mutants selected on Capreomycin, viomycin, or both; MICs of all four drugs were determined. Three rrs mutations (A1401G, C1402T, and G1484T) were found, and each was associated with a particular cross-resistance pattern. Similar mutations and cross-resistance patterns were found in drug-resistant clinical isolates. Overall, the data implicate rrs mutations as a molecular basis for resistance to each of the four drugs. Furthermore, the genotypic and phenotypic differences seen in the development of cross-resistance when M. tuberculosis bacteria were exposed to one or two drugs have implications for selection of treatment regimens.
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mutation of tlya confers Capreomycin resistance in mycobacterium tuberculosis
Antimicrobial Agents and Chemotherapy, 2005Co-Authors: Courtney E Maus, Bonnie B Plikaytis, Thomas M ShinnickAbstract:Capreomycin, an important drug for the treatment of multidrug-resistant tuberculosis, is a macrocyclic peptide antibiotic produced by Saccharothrix mutabolis subspecies capreolus. The basis of resistance to this drug was investigated by isolating and characterizing Capreomycin-resistant strains of Mycobacterium smegmatis and Mycobacterium tuberculosis. Colonies resistant to Capreomycin were recovered from a library of transposon-mutagenized M. smegmatis. The transposon insertion site of one mutant was mapped to an open reading frame in the unfinished M. smegmatis genome corresponding to the tlyA gene (Rv1694) in the M. tuberculosis H37Rv genome. In M. smegmatis spontaneous Capreomycin-resistant mutants, the tlyA gene was disrupted by one of three different naturally occurring insertion elements. Genomic DNAs from pools of transposon mutants of M. tuberculosis H37Rv were screened by PCR by using primers to the tlyA gene and the transposon to detect mutants with an insertion in the tlyA gene. One Capreomycin-resistant mutant was recovered that contained the transposon inserted at base 644 of the tlyA gene. Complementation with the wild-type tlyA gene restored susceptibility to Capreomycin in the M. smegmatis and M. tuberculosis tlyA transposon mutants. Mutations were found in the tlyA genes of 28 spontaneous Capreomycin-resistant mutants generated from three different M. tuberculosis strains and in the tlyA genes of Capreomycin-resistant clinical isolates. In in vitro transcription-translation assays, ribosomes from tlyA mutant but not tlyA+ strains resist Capreomycin inhibition of transcription-translation. Therefore, TlyA appears to affect the ribosome, and mutation of tlyA confers Capreomycin resistance.
Courtney E Maus - One of the best experts on this subject based on the ideXlab platform.
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Capreomycin binds across the ribosomal subunit interface using tlya encoded 2 o methylations in 16s and 23s rrnas
Molecular Cell, 2006Co-Authors: Shanna K Johansen, Courtney E Maus, Bonnie B Plikaytis, Stephen DouthwaiteAbstract:The cyclic peptide antibiotics Capreomycin and viomycin are generally effective against the bacterial pathogen Mycobacterium tuberculosis. However, recent virulent isolates have become resistant by inactivation of their tlyA gene. We show here that tlyA encodes a 2'-O-methyltransferase that modifies nucleotide C1409 in helix 44 of 16S rRNA and nucleotide C1920 in helix 69 of 23S rRNA. Loss of these previously unidentified rRNA methylations confers resistance to Capreomycin and viomycin. Many bacterial genera including enterobacteria lack a tlyA gene and the ensuing methylations and are less susceptible than mycobacteria to Capreomycin and viomycin. We show that expression of recombinant tlyA in Escherichia coli markedly increases susceptibility to these drugs. When the ribosomal subunits associate during translation, the two tlyA-encoded methylations are brought into close proximity at interbridge B2a. The location of these methylations indicates the binding site and inhibitory mechanism of Capreomycin and viomycin at the ribosome subunit interface.
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molecular analysis of cross resistance to Capreomycin kanamycin amikacin and viomycin in mycobacterium tuberculosis
Antimicrobial Agents and Chemotherapy, 2005Co-Authors: Courtney E Maus, Bonnie B Plikaytis, Thomas M ShinnickAbstract:Capreomycin, kanamycin, amikacin, and viomycin are drugs that are used to treat multidrug-resistant tuberculosis. Each inhibits translation, and cross-resistance to them is a concern during therapy. A recent study revealed that mutation of the tlyA gene, encoding a putative rRNA methyltransferase, confers Capreomycin and viomycin resistance in Mycobacterium tuberculosis bacteria. Mutations in the 16S rRNA gene (rrs) have been associated with resistance to each of the drugs; however, reports of cross-resistance to the drugs have been variable. We investigated the role of rrs mutations in Capreomycin resistance and examined the molecular basis of cross-resistance to the four drugs in M. tuberculosis laboratory-generated mutants and clinical isolates. Spontaneous mutants were generated to the drugs singularly and in combination by plating on medium containing one or two drugs. The frequencies of recovery of the mutants on single- and dual-drug plates were consistent with single-step mutations. The rrs genes of all mutants were sequenced, and the tlyA genes were sequenced for mutants selected on Capreomycin, viomycin, or both; MICs of all four drugs were determined. Three rrs mutations (A1401G, C1402T, and G1484T) were found, and each was associated with a particular cross-resistance pattern. Similar mutations and cross-resistance patterns were found in drug-resistant clinical isolates. Overall, the data implicate rrs mutations as a molecular basis for resistance to each of the four drugs. Furthermore, the genotypic and phenotypic differences seen in the development of cross-resistance when M. tuberculosis bacteria were exposed to one or two drugs have implications for selection of treatment regimens.
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mutation of tlya confers Capreomycin resistance in mycobacterium tuberculosis
Antimicrobial Agents and Chemotherapy, 2005Co-Authors: Courtney E Maus, Bonnie B Plikaytis, Thomas M ShinnickAbstract:Capreomycin, an important drug for the treatment of multidrug-resistant tuberculosis, is a macrocyclic peptide antibiotic produced by Saccharothrix mutabolis subspecies capreolus. The basis of resistance to this drug was investigated by isolating and characterizing Capreomycin-resistant strains of Mycobacterium smegmatis and Mycobacterium tuberculosis. Colonies resistant to Capreomycin were recovered from a library of transposon-mutagenized M. smegmatis. The transposon insertion site of one mutant was mapped to an open reading frame in the unfinished M. smegmatis genome corresponding to the tlyA gene (Rv1694) in the M. tuberculosis H37Rv genome. In M. smegmatis spontaneous Capreomycin-resistant mutants, the tlyA gene was disrupted by one of three different naturally occurring insertion elements. Genomic DNAs from pools of transposon mutants of M. tuberculosis H37Rv were screened by PCR by using primers to the tlyA gene and the transposon to detect mutants with an insertion in the tlyA gene. One Capreomycin-resistant mutant was recovered that contained the transposon inserted at base 644 of the tlyA gene. Complementation with the wild-type tlyA gene restored susceptibility to Capreomycin in the M. smegmatis and M. tuberculosis tlyA transposon mutants. Mutations were found in the tlyA genes of 28 spontaneous Capreomycin-resistant mutants generated from three different M. tuberculosis strains and in the tlyA genes of Capreomycin-resistant clinical isolates. In in vitro transcription-translation assays, ribosomes from tlyA mutant but not tlyA+ strains resist Capreomycin inhibition of transcription-translation. Therefore, TlyA appears to affect the ribosome, and mutation of tlyA confers Capreomycin resistance.
Michael G Thomas - One of the best experts on this subject based on the ideXlab platform.
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mechanistically distinct nonribosomal peptide synthetases assemble the structurally related antibiotics viomycin and Capreomycin
ChemBioChem, 2011Co-Authors: John J Barkei, Elizabeth A Felnagle, Angela M Podevels, Michael G ThomasAbstract:Tuberculosis (TB) is a worldwide burden for human health. More than 1.8 million people succumbed to TB infection in 2008 alone, and as many as two billion people may be passively infected by Mycobacterium tuberculosis, the causative agent of TB.[1] One of the challenges facing the successful treatment of M. tuberculosis infections is the development of strains that are resistant to many of the antibiotics used in the clinic. Cases of multidrug-resistant TB (MDR-TB), defined as TB caused by a M. tuberculosis strain that is resistant to both rifampin and isoniazid, have been identified in nearly every country surveyed.[1, 2] Extensively drug-resistant TB (XDR-TB), defined as MDR-TB that is also resistant to a flouroquinolone and at least one of three injectables (Capreomycin, streptomycin, or amikacin), has spread to more than 45 countries, including the United States.[1, 2] The development and spread of drug-resistant strains of M. tuberculosis has put a high priority on the development of new antituberculosis drugs and the generation of derivatives of known drugs that regain their antibiotic activity against resistant strains. The tuberactinomycin family of antituberculosis drugs are important components of our drug arsenal against drug-resistant M. tuberculosis. The most prominent member of this family is Capreomycin (CMN), which is a key drug in the treatment of MDR-TB based on its inclusion on the World Health Organization’s “Model List of Essential Medicines.”[3] Furthermore, if CMN is the injectable that XDR-TB infection is resistant to, it almost guarantees treatment failure.[4] Based on its level of importance in treating drug-resistant forms of TB, it is important that new CMN derivatives be developed that regain activity against resistant M. tuberculosis strains. Synthetic procedures to synthesize derivatives have been difficult.[5, 6] More success has been accomplished by semisynthetic approaches,[7–9] but the derivatives are limited by the functional groups present on the cyclic pentapeptide core. Complementing these approaches with metabolic engineering of the enzymology that produces the cyclic pentapeptide core has the potential of enabling further structural diversification. Our focus is on the metabolic engineering aspect of drug development. Harnessing the full potential of metabolic engineering to generate new drug derivatives requires a complete understanding of how the targeted drug is biosynthesized by the producing organism. To this end, we have focused on understanding tuberactinomycin biosynthesis at the molecular and biochemical level by using CMN and the structural analog viomycin (VIO) as model systems. Our focus on both CMN and VIO was based on our hypothesis that by studying the biosynthesis of two structurally related molecules (Scheme 1), we would gain considerable insights into how the tuberactinomycins are biosynthesized and structurally modified by the natural enzymology. To date, we have sequenced and annotated the CMN and VIO biosynthesis gene clusters,[10, 11] reconstituted CMN and VIO production in the heterologous host Streptomyces lividans,[12] biochemically characterized L-capreomycidine formation,[13] and identified the amino acids activated by each of the adenylation (A) domains of the CMN nonribosomal peptide synthetases (NRPS) and some of the A domains of the VIO NRPS (Scheme 2).[14] Zabriskie and colleagues have also contributed important genetic and biochemical information that enable more refined models of tuberactinomycin biosynthesis to be developed.[15–18] Open in a separate window Scheme 1 Chemical structures of viomycin (VIO), tuberactinamine A (TMN A; des-β-lysine VIO) and the four derivatives that make up Capreomycin (CMN). The numbering within the cyclic pentapeptide cores of the antibiotics is used to identify positions noted in the text.
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investigations into viomycin biosynthesis by using heterologous production in streptomyces lividans
ChemBioChem, 2009Co-Authors: John J Barkei, Brian M Kevany, Elizabeth A Felnagle, Michael G ThomasAbstract:Viomycin and Capreomycin are members of the tuberactinomycin family of antituberculosis drugs. As with many antibacterial drugs, resistance to the tuberactinomycins is problematic in treating tuberculosis; this makes the development of new derivatives of these antibiotics to combat this resistance of utmost importance. To take steps towards developing new derivatives of this family of antibiotics, we have focused our efforts on understanding how these antibiotics are biosynthesized by the producing bacteria so that metabolic engineering of these pathways can be used to generate desired derivatives. Here we present the heterologous production of viomycin in Streptomyces lividans 1326 and the use of targeted-gene deletion as a mechanism for investigating viomycin biosynthesis as well as the generation of viomycin derivatives. Deletion of vioQ resulted in nonhydroxylated derivatives of viomycin, while strains lacking vioP failed to acylate the cyclic pentapeptide core of viomycin with beta-lysine. Surprisingly, strains lacking vioL produced derivatives that had the carbamoyl group of viomycin replaced by an acetyl group. Additionally, the acetylated viomycin derivatives were produced at very low levels. These two observations suggested that the carbamoyl group of the cyclic pentapeptide core of viomycin was introduced at an earlier step in the biosynthetic pathway than previously proposed. We present biochemical evidence that the carbamoyl group is added to the beta-amino group of L-2,3-diaminopropionate prior to incorporation of this amino acid by the nonribosomal peptide synthetases that form the cyclic pentapeptide cores of both viomycin and Capreomycin.
Stephen Douthwaite - One of the best experts on this subject based on the ideXlab platform.
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Capreomycin susceptibility is increased by tlya directed 2 o methylation on both ribosomal subunits
Molecular Microbiology, 2012Co-Authors: Tanakarn Monshupanee, Shanna K Johansen, Albert E Dahlberg, Stephen DouthwaiteAbstract:The binding site of the cyclic peptide antibiotics Capreomycin and viomycin is located on the ribosomal subunit interface close to nucleotides C1409 in 16S rRNA and C1920 in 23S rRNA. In Mycobacterium tuberculosis, the 2'-hydroxyls of both nucleotides are methylated by the enzyme TlyA. Loss of these methylations through inactivation of TlyA confers resistance to Capreomycin and viomycin. We report here that TlyA orthologues occur in diverse bacteria and fall into two distinct groups. One group, now termed TlyA(I) , has shorter N- and C-termini and methylates only C1920; the second group (now TlyA(II) ) includes the mycobacterial enzyme, and these longer orthologues methylate at both C1409 and C1920. Ribosomal subunits are the preferred substrates for both groups of orthologues. Amino acid substitutions at the N-terminus of TlyA(II) reduce its ability to methylate these substrates. Growing pairs of recombinant TlyA(II) Escherichia coli strains in competition shows that even subtle changes in the level of rRNA methylation lead to significant differences in susceptibility to sub-inhibitory concentrations of Capreomycin. The findings reveal that 2'-O-methyls at both C1409 and C1920 play a role in facilitating the inhibitory effects of Capreomycin and viomycin on the bacterial ribosome.
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mutations in conserved helix 69 of 23s rrna of thermus thermophilus that affect Capreomycin resistance but not posttranscriptional modifications
Journal of Bacteriology, 2008Co-Authors: Tanakarn Monshupanee, Stephen Douthwaite, Steven T Gregory, Wipa Chungjatupornchai, Albert E DahlbergAbstract:Translocation during the elongation phase of protein synthesis involves the relative movement of the 30S and 50S ribosomal subunits. This movement is the target of tuberactinomycin antibiotics. Here, we describe the isolation and characterization of mutants of Thermus thermophilus selected for resistance to the tuberactinomycin antibiotic Capreomycin. Two base substitutions, A1913U and mU1915G, and a single base deletion, ΔmU1915, were identified in helix 69 of 23S rRNA, a structural element that forms part of an interribosomal subunit bridge with the decoding center of 16S rRNA, the site of previously reported Capreomycin resistance base substitutions. Capreomycin resistance in other bacteria has been shown to result from inactivation of the TlyA methyltransferase which 2′-O methylates C1920 of 23S rRNA. Inactivation of the tlyA gene in T. thermophilus does not affect its sensitivity to Capreomycin. Finally, none of the mutations in helix 69 interferes with methylation at C1920 or with pseudouridylation at positions 1911 and 1917. We conclude that the resistance phenotype is a consequence of structural changes introduced by the mutations.
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Capreomycin binds across the ribosomal subunit interface using tlya encoded 2 o methylations in 16s and 23s rrnas
Molecular Cell, 2006Co-Authors: Shanna K Johansen, Courtney E Maus, Bonnie B Plikaytis, Stephen DouthwaiteAbstract:The cyclic peptide antibiotics Capreomycin and viomycin are generally effective against the bacterial pathogen Mycobacterium tuberculosis. However, recent virulent isolates have become resistant by inactivation of their tlyA gene. We show here that tlyA encodes a 2'-O-methyltransferase that modifies nucleotide C1409 in helix 44 of 16S rRNA and nucleotide C1920 in helix 69 of 23S rRNA. Loss of these previously unidentified rRNA methylations confers resistance to Capreomycin and viomycin. Many bacterial genera including enterobacteria lack a tlyA gene and the ensuing methylations and are less susceptible than mycobacteria to Capreomycin and viomycin. We show that expression of recombinant tlyA in Escherichia coli markedly increases susceptibility to these drugs. When the ribosomal subunits associate during translation, the two tlyA-encoded methylations are brought into close proximity at interbridge B2a. The location of these methylations indicates the binding site and inhibitory mechanism of Capreomycin and viomycin at the ribosome subunit interface.
Sabine Ruschgerdes - One of the best experts on this subject based on the ideXlab platform.
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first evaluation after implementation of a quality control system for the second line drug susceptibility testing of mycobacterium tuberculosis joint efforts in low and high incidence countries
PLOS ONE, 2013Co-Authors: Doris Hillemann, Sabine Ruschgerdes, Sven Hoffner, Daniela M Cirillo, Francis Drobniewski, Elvira Richter, Tb Pannet, Ecdc Erlntb NetworksAbstract:Three networks/projects involving 27 European countries were established to investigate the quality of second-line drug (SLD) susceptibility testing with conventional and molecular methods. 1. The “Baltic-Nordic TB-Laboratory Network” comprised 11 reference laboratories in the Baltic-Nordic States. They performed SLD testing in the first phase with a panel of 20 Mycobacterium tuberculosis strains. After several laboratories made technical changes a second panel of 10 strains with a higher proportion of resistant strains were tested. Although the concordance for Ofloxacin, Kanamycin, and Capreomycin was consistently high, the largest improvements in performance were achieved for the analysis of Ofloxacin resistant (from 88.9 to 95.0%), and Capreomycin resistant (from 71.0 to 88.9%) strains. 2. Within the FP7 TB PAN-NET project (EU Grant agreement 223681) a quality control panel to standardize the EQA (External Quality Assurance) for first-line drugs (FLD) and SLD testing for phenotypic and molecular methods was established. The strains were characterized by their robustness, unambiguous results when tested, and low proportion of secondary drug resistances. 3. The (European Reference Laboratory Network-TB) ERLN-TB network analyzed four different panels for drug resistance testing using phenotypic and molecular methods; in two rounds in 2010 the 31 participating laboratories began with 5 strains, followed by 10 strains and 6 additional crude DNA extracts in 2011 and 2012 were examined by conventional DST and molecular methods. Overall, we demonstrated the importance of developing inter-laboratory networks to establish quality assurance and improvement of SLD testing of M. tuberculosis.
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feasibility of the genotype mtbdrsl assay for fluoroquinolone amikacin Capreomycin and ethambutol resistance testing of mycobacterium tuberculosis strains and clinical specimens
Journal of Clinical Microbiology, 2009Co-Authors: Doris Hillema, Sabine Ruschgerdes, Elvira RichteAbstract:The new GenoType Mycobacterium tuberculosis drug resistance second line (MTBDRsl) assay (Hain Lifescience, Nehren, Germany) was tested on 106 clinical isolates and directly on 64 sputum specimens for the ability to detect resistance to fluoroquinolones, injectable drugs (amikacin or Capreomycin), and ethambutol in Mycobacterium tuberculosis strains. A total of 63 strains harboring fluoroquinolone, amikacin/Capreomycin, or ethambutol resistance and 43 fully susceptible strains were comparatively analyzed with the new MTBDRsl assay, by DNA sequencing, and by conventional drug susceptibility testing in liquid and solid media. No discrepancies were obtained in comparison with the DNA sequencing results. Fluoroquinolone resistance was detected in 29 (90.6%) of 32, amikacin/Capreomycin resistance was detected in 39/39 (84.8%/86.7%) of 46/45, and ethambutol resistance was detected in 36 (69.2%) of 52 resistant strains. A total of 64 sputum specimens (42 smear positive, 12 scanty, and 10 smear negative) were tested with the new MTBDRsl assay, and the results were compared with those of conventional drug susceptibility testing. Fluoroquinolone resistance was detected in 8 (88.9%) of 9, amikacin/Capreomycin resistance was detected in 6/7 (75.0%/87.5%) of 8, and ethambutol resistance was detected in 10 (38.5%) of 26 resistant strains. No mutation was detected in susceptible strains. The new GenoType MTBDRsl assay represents a reliable tool for the detection of fluoroquinolone and amikacin/Capreomycin resistance and to a lesser extent also ethambutol resistance. In combination with a molecular test for detection of rifampin and isoniazid resistance, the potential for the detection of extensively resistant tuberculosis within 1 to 2 days can be postulated.
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multicenter laboratory validation of the bactec mgit 960 technique for testing susceptibilities of mycobacterium tuberculosis to classical second line drugs and newer antimicrobials
Journal of Clinical Microbiology, 2006Co-Authors: Sabine Ruschgerdes, Gaby E Pfyffer, M Casal, M V Chadwick, Salman H SiddiqiAbstract:The BACTEC MGIT 960 system, a fully automated, nonradiometric, noninvasive system for detection and drug susceptibility testing of mycobacteria, was evaluated for the ability to test susceptibilities to second-line drugs. In this study, which was carried out in three phases (phase I, mostly susceptible strains; phase II, mostly resistant strains; phase III, final testing of the optimal drug concentrations found in phases I and II), we established the critical concentrations for seven drugs to be tested in the BACTEC MGIT 960 system compared to the BACTEC 460TB system. The critical concentrations for the seven drugs used in the MGIT 960 system are as follows: amikacin, 1.0 μg/ml; Capreomycin, 2.5 μg/ml; ethionamide, 5.0 μg/ml; protionamide, 2.5 μg/ml; ofloxacin, 2.0 μg/ml; rifabutin, 0.5 μg/ml; linezolid, 1.0 μg/ml. Our results demonstrate that the BACTEC MGIT 960 system is an accurate method for rapid testing of the susceptibilities of Mycobacterium tuberculosis to second-line drugs.
