Whole-Genome Sequencing and Mutation Analysis of Two Extensively Drug-Resistant Sputum Isolates of Mycobacterium tuberculosis (VRFCWCF XDRTB 232 and VRFCWCF XDRTB 1028) from Chennai, India.

We announce the draft genome sequence of two extensively drug-resistant Mycobacterium tuberculosis strains, VRFCWCF XDRTB 232 and VRFCWCF XDRTB 1028, isolated from the sputum samples of a patient clinically suspected to have tuberculosis, and we also report novel mutations that confer drug resistance.

GENOME ANNOUNCEMENT

The global threat of drug-resistant tuberculosis (multidrug-resistant tuberculosis [MDR-TB] and extensively drug-resistant tuberculosis [XDR-TB] has become of great significance in the field of public health. MDR-TB cases threaten the effectiveness of chemotherapy for both the treatment and control of TB and require the use of second-line drugs that are more expensive, toxic, and less effective than first-line anti-TB drugs. Recent reports state that 84 countries have reported at least one XDR-TB case (1). The situation has turned into a pressing demand for rapid genotypic drug susceptibility testing (DST) for the second-line drugs in order to develop efficient regimens for the appropriate treatment of individual cases (2). Therefore, the whole-genome sequencing of XDR-TB strains will aid in a better understanding of the mutation patterns conferring drug resistance, and it provides insights into the exploration of the molecular epidemiology of XDR-TB in a particular geographical location and in the development of newer drug targets to treat tuberculosis.

We announce here the draft genome sequence of two XDR-TB isolates (VRFCWCF XDRTB 232 and VRFCWCF XDRTB 1028) from the sputum samples of two clinically suspected tuberculosis patients. Whole-genome sequencing of two XDR-TB strains was performed using the Ion Torrent PGM platform, similar to that in our previous work (3). The generated sequence reads were adapter trimmed and subjected to reference-based assembly (Mycobacterium tuberculosis H37Rv [accession no. NC_000962.3]) using the BWA software 0.6.1-r104. The isolates VRFCWCF XDRTB 232 and VRFCWCF XDRTB 1028 resulted in 156 contigs with a sequence length of 4,369,955 bp and 188 contigs with a sequence length of 4,329,965, respectively, with 63.8× coverage. The assembled sequences were annotated by the NCBI PGAAP (http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html). The two strains were further studied for single-nucleotide polymorphism analysis using the SNPsFinder software (http://snpsfinder.lanl.gov/). The single-nucleotide polymorphism (SNP) analysis of VRFCWCF XDRTB 232 revealed novel mutations in the iniA (His140Gln) and rpoB (Leu1075Pro) genes coding for isoniazid (INH) and rifampin (RIF) drug resistance, respectively. In addition, a novel mutation was observed in the rpsl gene (Ala140Val) coding for streptomycin resistance. Regarding second-line antituberculous drugs, novel mutations were observed in the gyrA gene (Phe614Ser, Ala668Thr, and Gln384Stop) coding for fluoroquinolone (FQ) resistance, as well as in the eis gene (Arg155Stop) coding for aminoglycoside (AMI) resistance. VRFCWCF XDRTB 1028 revealed the presence of novel mutations in the iniC (Ala285Val) and rpoB (Leu1075Pro) genes coding for INH and RIF drug resistance, respectively. In case of second-line antituberculous drugs, novel mutations were observed in the gyrA gene (Phe614Ser, Ala668Thr, and Gln384Stop) coding for FQ resistance, as well as a novel mutation in the eis gene (Arg155Stop) coding for AMI resistance. This is the first report of whole-genome sequencing of XDR-TB circulating in the Chennai population from a private research institution in Chennai.

Nucleotide sequence accession numbers.

The whole-genome shotgun sequences of VRFCWCF XDRTB 232 and VRFCWCF XDRTB 1028 have been deposited at DDBJ/EMBL/GenBank under the accession numbers JNVI00000000 and JQGH00000000, respectively.