Complete genome sequence of Mycobacterium tuberculosis K from a Korean high school outbreak, belonging to the Beijing family.

Mycobacterium tuberculosis K, a member of the Beijing family, was first identified in 1999 as the most prevalent genotype in South Korea among clinical isolates of M. tuberculosis from high school outbreaks. M. tuberculosis K is an aerobic, non-motile, Gram-positive, and non-spore-forming rod-shaped bacillus. A transmission electron microscopy analysis displayed an abundance of lipid bodies in the cytosol. The genome of the M. tuberculosis K strain was sequenced using two independent sequencing methods (Sanger and Illumina). Here, we present the genomic features of the 4,385,518-bp-long complete genome sequence of M. tuberculosis K (one chromosome, no plasmid, and 65.59 % G + C content) and its annotation, which consists of 4194 genes (3447 genes with predicted functions), 48 RNA genes (3 rRNA and 45 tRNA) and 261 genes with peptide signals.

Microplate hybridization assay for detection of isoniazid resistance in Mycobacterium tuberculosis.

Early and accurate detection of drug resistant Mycobacterium tuberculosis can improve both the treatment outcome and public health control of tuberculosis. A number of molecular-based techniques have been developed including ones using probe molecules that target drug resistance-related mutations. Although these techniques are highly specific and sensitive, mixed signals can be obtained when the drug resistant isolates are mixed with drug susceptible isolates. In order to overcome this problem, we developed a new drug susceptibility test (DST) for one of the most effective anti-tuberculosis drug, isoniazid. This technique employed a microplate hybridization assay that quantified signals from each probe molecule, and was evaluated using clinical isolates. The evaluation analysis clearly showed that the microplate hybridization assay was an accurate and rapid method that overcame the limitations of DST based on conventional molecular techniques.

Novel polymorphic region of the rpoB gene containing Mycobacterium species-specific sequences and its use in identification of mycobacteria.

Sequence analysis of a specific region of the mycobacterium rpoB gene in 35 mycobacterial strains representing 26 different mycobacterial species of clinical importance showed that there exists a highly polymorphic region. Based on the sequences of the polymorphic region, the oligonucleotide probes of 14 mycobacterial species with relatively high clinical importance were designed and shown to be specific to their corresponding mycobacterial species by dot blot hybridization. The results showed that the probes designed in this study are highly specific to each mycobacterial species, which suggests that these sequences may be useful for the species identification of mycobacteria.

Mutations in the embB locus among Korean clinical isolates of Mycobacterium tuberculosis resistant to ethambutol.

Resistance of Mycobacterium tuberculosis to ethambutol (EMB) has been assigned to an operon, embCAB, which has been proposed to be a structural gene for mycobacterial arabinosyl transferases. Recently, genetic events resulting in structural mutations at embB have been proposed as major contributors to the EMB-resistance of isolates whose minimum inhibitory concentration (MIC) level is higher than 20 microgram/ml. On the contrary, isolates with a MIC level lower than 20 microgram/ml do not seem to contain any sequence alterations. In this study, in an effort to understand the role of embB mutations at a low-level of EMB resistance, we investigated the sequence polymorphisms of clinical isolates whose MIC levels are lower than 10 microgram/ml. Accordingly, the sequence alterations of a 312-bp region of the embB gene containing the 306th codon, which has been assigned as a hot-spot for EMB-resistance related mutations, were determined for 21 EMB-resistant and 5 EMB-susceptible clinical isolates. In brief, among 21 EMB- resistant isolates examined, 12 (57.1%) contained mutations in embB (10 at the 306th codon and 2 at other sites), and the remaining isolates 9 contained no mutations in any region of embB. The observed mutations included M306V, M306I, and M306L substitutions that have been reported previously. However, 3 were novel types, which included M306T, A313G and Y319C, D328Y [corrected] double substitutions. On the other hand, all of the EMB-susceptible isolates were found to be free of mutations. In conclusion, our findings suggest that sequence polymorphism of embB may play a pivotal role in the EMB- resistance of M. tuberculosis.