Draft Genome Sequence of the First Isolate of Extensively Drug-Resistant Mycobacterium tuberculosis in Ireland.

Extensive drug resistance is an emerging threat to the control of tuberculosis (TB) worldwide, even in countries with low TB incidence. We report the draft whole-genome sequence of the first reported extensively drug-resistant TB (XDR-TB) strain isolated in Ireland (a low-incidence setting) and describe a number of single-nucleotide variations that correlate with its XDR phenotype.

GENOME ANNOUNCEMENT

Multidrug resistance (MDR) in tuberculosis (TB) threatens the global management of the disease, which is already a leading cause of infectious mortality worldwide, with an estimated 450,000 MDR-TB cases reported in 2012 (1). Approximately 10% of MDR-TB cases (those resistant to rifampin and isoniazid) are further defined as extensively drug resistant (XDR)-TB, due to their resistance to second-line drugs, fluoroquinolones and injectable aminoglycosides (2). Long turnaround times (2 to 4 weeks) for phenotypic drug susceptibility testing (DST) (due to the fastidious nature of the organism) can hamper the appropriate treatment of XDR-TB by delaying access to antibiotic susceptibility data (3). Next-generation sequencing (NGS) can highlight resistance in a timely manner in order to effectively manage treatment and minimize further transmission of resistant strains (4–6).

The first Irish XDR-TB strain was isolated in the Irish Mycobacteria Reference Laboratory (IMRL) in 2005 (IEXDR1) (7, 8). First-line DST was completed within 3 weeks (found to be streptomycin, isoniazid, rifampin, ethambutol, and pyrazinamide resistant), second-line DST within 5 weeks (found to be amikacin, clarithromycin, ciprofloxacin, and rifabutin resistant, as well as capreomycin, clofazimine, and prothionamide susceptible), and the remainder within 14 weeks (found to be para-aminosalicylate sodium [PAS] resistant and ethionamide and cycloserine susceptible).

In 2014, NGS was performed to provide further molecular characterization of IEXDR1 (lineage 2 or Beijing strain). Genomic DNA was sequenced using an Illumina MiSeq. Paired-end reads were mapped to the Mycobacterium tuberculosis H37Rv reference genome (accession no. AL123456.3) using the Burrows-Wheeler Aligner (9). This yielded a mapped-read depth of 196-fold, covering 97.6% of the H37Rv genome. A consensus sequence was called using the SAMtools mpileup command (10). The IMAGE algorithm was employed to extend contigs and close gaps in the assembly, producing a final draft assembly of 4,340,174 bp, consisting of 109 contigs (11). Single-nucleotide polymorphism (SNP) analysis was performed using Geneious R7 (version 7.1.5; Biomatters); 1,492 SNPs were detected in the assembled genome with respect to the genome of H37Rv, of which 810 were nonsynonymous (depth of coverage, ≥20-fold [average, 276]; variant frequency, ≥95%).

Nonsynonymous mutations were identified in genes Rv0667/rpoB (H526Y) and Rv1908c/katG (S315T). There is strong correlation between substitutions in rpoB (H526Y) and katG (S315T) and phenotypic resistance to rifampin and isoniazid, respectively (4, 12). High-confidence SNPs were also found for fluoroquinolone resistance in gene Rv0006 (gyrA) (D94A) and aminoglycoside resistance in MTB000019/rrs (a1401g) (12). This is consistent with the XDR phenotype of IEXDR1. Other high-confidence mutations found in IEXDR1 for ethambutol (Rv3795/embB [M306V]) and streptomycin (Rv0682/rpsL [K43R]) correlate with its drug resistance profile (13, 14). SNPs that may confer resistance to pyrazinamide (Rv2043c/pncA [G132C]) and PAS (Rv3764c/thyA [Q97R]) were also identified, although their specificities and sensitivities are not as well defined (http://www.broadinstitute.org/annotation/genome/mtb_drug_resistance.1/DirectedSequencingHome.html).

Previously described phylogenetically informative polymorphisms (Rv1908c/katG [R463L], Rv2629 [D64A], Rv3794/embA [C76C, TGC/TGT] and [Q38Q, CAA/CAG], Rv1630/rpsA [R212R, CGA/CGC], Rv3919c/gidB [E92D], and Rv0486/mshA [A187V]) confirm the presence of a Beijing strain (15).

In summary, using NGS, this isolate was confirmed to be XDR-TB in a considerably shorter turnaround time than that for conventional DST. This underlines the potential of NGS in the diagnostic laboratory, especially for MDR- and XDR-TB cases.

Nucleotide sequence accession number.

This whole-genome sequencing project has been deposited in the European Nucleotide Archive under the accession no. CCJS00000000.