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The emergence of the global threat of extensively drug-resistant (XDR) Mycobacterium tuberculosis reveals weaknesses in tuberculosis management and diagnostic services. We report the draft genome sequence of the first extensively drug-resistant Mycobacterium tuberculosis strain isolated in Malaysia. The sequence was also compared against a reference strain to elucidate the polymorphism that is related to their extensive resistance.
The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis is a major public health problem that threatens tuberculosis (TB) care and control in many countries (1). XDR Mycobacterium tuberculosis strain UM 1072388579 was isolated from a 57-year-old man. The isolate was resistant to amikacin, capreomycin, ciprofloxacin, ethionamide, isoniazid, kanamycin, ofloxacin, rifampin, and streptomycin but sensitive to ethambutol and para-salicylic acid.
The genomic DNA of UM 1072388579 was sequenced to 240× coverage with a 500-bp insert size library strategy, which consisted of 5,861,112 paired-end reads, using the Illumina HiSeq 2000 sequencer machine. After preprocessing, 2,800,000 high-quality paired-end reads (100-fold depth) were subsampled and assembled using Velvet v1.1.07 (2), producing 213 contigs (≥200 bp). The contigs were then scaffolded and gap filled with SSPACE v2.0 (3) and GapFiller v1.10 (4) programs by utilizing all paired-end information available. Final assembly consisted of 89 scaffolds (≥1,000 bp) with 158 contigs comprising a total length of 4.3 Mb. The assembly has an N50 scaffold size of 108,779 bp and G+C content of 65.4%. Protein coding sequences, rRNAs, and tRNAs of the XDR M. tuberculosis genome were predicted using GeneMarkS (5), RNAmmer 1.2 server (6), and tRNAscan-SE v1.23 (7), respectively. Our pipeline predicted 4,099 coding DNA sequences (CDS) with length ≥33 amino acids (aa), 45 tRNAs, and 3 rRNAs (5S, 16S, and 23S). Annotation of the coding sequences predicted was performed by a BLAST search against the NCBI nonredundant database with 4,069 (99.3%) genes annotated with function. All annotated genes were subjected to Gene Ontology classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis.
The genome sequence is about 93% complete compared to the F11 reference genome, which has a complete genome size of 4.42 Mb. A polymorphism study of the XDR M. tuberculosis genome was carried out by comparative analysis against the F11 genome (8) to identify the difference between intergenic and coding regions of drug-susceptible versus extensively drug-resistant strains. A total of 1,455 polymorphisms were observed in our comparative study, with 1,196 of these located in the coding regions of the genome. An insertion at the tlyA gene (a determinant for extensively drug-resistant M. tuberculosis) conferring capreomycin resistance to M. tuberculosis was observed. Mutations of other drug resistance sites such as ethA (ethionamide), gidB (streptomycin), gyrA and gyrB (fluoroquinolones), pncA (pyrazinamide), katG and ndh (isoniazid), and rpoB (rifampin) were also observed. The draft sequence of the XDR M. tuberculosis UM 1072388579 genome provides a genome sequence that may lead to better understanding of the genetic molecular differences in acquired drug resistance of XDR M. tuberculosis isolates locally and in other parts of the world.
The nucleotide sequences of the M. tuberculosis UM 1072388579 genome have been deposited in DDBJ/EMBL/GenBank under accession number AMXW00000000.
This study was supported by research grant UM.C/625/1/HIR/003/1 from the University of Malaya.
K.P.N., Y.F.N., and H.H. conceived the project and contributed to the writing and editing of the manuscript. K.P.N., J.C., S.N.T., T.S.S.H., C.L.C., and S.L.N. were responsible for the isolation, identification, and DNA extraction. S.M.Y., C.C.H., K.W.L., and W.Y.Y. performed the genome sequencing and bioinformatics analysis.
Citation Ng KP, Yew SM, Chan CL, Chong J, Tang SN, Soo-Hoo TS, Na SL, Hassan H, Ngeow YF, Hoh CC, Lee KW, Yee WY. 2013. Draft genome sequence of the first isolate of extensively drug-resistant (XDR) Mycobacterium tuberculosis in Malaysia. Genome Announc. 1(1):e00056-12. doi:10.1128/genomeA.00056-12.