<i>emb</i>B mutations and Bactec MGIT MIC values for study isolates.

<p><i>emb</i>B mutations and Bactec MGIT MIC values for study isolates.</p

Time-to-Detection of Inducible Macrolide Resistance in <i>Mycobacterium abscessus</i> Subspecies and Its Association with the <i>Erm</i>(41) Sequevar

<div><p>Mutations in the <i>erm</i>(41) gene of <i>M</i>.<i>abscessus</i> group organisms are associated with differences in inducible macrolide resistance, with current recommendations being to hold rapidly growing isolates for up to 14 days in order to ensure that resistance which develops more slowly can be detected. This study aimed to determine the ideal incubation time for accurate identification of inducible macrolide resistance as well as to determine if there was an association between the time taken to detect inducible resistance in <i>M</i>.<i>abscessus</i> group organisms and their <i>erm</i>(41) sequevar. We amplified and sequenced the <i>erm</i>(41) genes of a total of 104 <i>M</i>.<i>abscessus</i> group isolates and determined their sequevars. The isolates were tested for phenotypic clarithromycin resistance at days 7, 10, 14 and 21, using Trek Diagnostics Sensititre RAPMYCO microbroth dilution plates. Associations between <i>erm</i>(41) gene sequevars and time to detection of resistance were evaluated using Fisher’s exact test in R. The samples included in this study fell into 14 sequevars, with the majority of samples falling into Sequevar02 (16), Sequevar06 (15), Sequevar08 (7) and Sequvar 15 (31), and several isolates that were in small clusters, or unique. The majority (82.7%) of samples exhibiting inducible macrolide resistance were interpreted as resistant by day 7. Two isolates in Sequevar02, which has a T28C mutation that is associated with sensitivity, showed intermediate resistance at day 14, though the majority (13) were sensitive at day 14. The majority of isolates with inducible macrolide resistance fell into Sequevars 06,08 and 15, none of which contain the T28C mutation. These sequevars were analyzed to determine if there was any correlation between sequevar and time to detection of resistance. None was found. Based on these findings, we recommend the addition of a day 7 read to the CLSI guidelines to improve turn-around-times for these isolates. It is also recommended that <i>erm</i>(41) gene sequencing be added to routine phenotypic testing for the resolution of cases with difficult-to-interpret phenotypic results.</p></div

Subspeciation, days to detection of inducible macrolide resistance, <i>erm</i>(41) sequence and Sequevar assignment for study strains¹.

<p>Subspeciation, days to detection of inducible macrolide resistance, <i>erm</i>(41) sequence and Sequevar assignment for study strains<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158723#t001fn001" target="_blank">¹</a>.</p

Evaluation of the sequencing depth of coverage across the three library preparation methods explored in this experiment.

<p>Coverage across H37Rv MTB reference genome (NC_018143.2) using A) the NX-500 method B) the NX-600 method C) the TS method (600 cycle). D) Illustrates the mean values across the genome via each of the methods. Isolates prepared using TS had significantly higher depth of coverage across the genome than both NX-500 and NX-600 (<i>p</i><0.05).</p

GC content vs. genome size adjusted motif frequency.

<p>A) GC-rich motif (CGSCNGSCGKYGCCGSCGSYG) identified that is commonly found in regions of ultra-low sequencing depth of coverage in our MTB isolate analysis. B) AGNTYWRANCT Motif described by Adey <i>et al (</i>[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148676#pone.0148676.ref023" target="_blank">23</a>]).</p

Scatter plot depicting the relationship between A_260/230 (A, C & E) and A_260/280 (B, D & F) ratios and mean depth of read coverage.

<p>Blue lines illustrate the line of best fit. Isolates deemed outliers, with mean sequencing depth of coverage <25x or >195x, have been excluded. Pearson correlation values and corresponding linear regression <i>p</i>-values are expressed.</p

Pairwise intra- and inter-cluster SNV variability in four MIRU groups.

<p>Pairwise intra- and inter-cluster SNV variability in four MIRU groups.</p

Application of whole genome sequence analysis to the study of <i>Mycobacterium tuberculosis</i> in Nunavut, Canada

<div><p>Canada has one of the lowest rates of tuberculosis (TB) in the world, however, among certain sub-populations, disease incidence rates approach those observed in sub-Saharan Africa, and other high incidence regions. In this study, we applied mycobacterial interspersed repetitive unit (MIRU) variable number of tandem repeat (VNTR) and whole genome sequencing (WGS) to the analysis of <i>Mycobacterium tuberculosis</i> isolates obtained from Northern communities in the territory of Nunavut. WGS was carried out using the Illumina MiSeq, with identified variants used to infer phylogenetic relationships and annotated to infer functional implications. Additionally, the sequencing data from these isolates were augmented with publically available WGS to evaluate data from the Nunavut outbreak in the broader Canadian context. In this study, isolates could be classified into four major clusters by MIRU-VNTR analysis. These could be further resolved into sub-clusters using WGS. No evidence for antimicrobial resistance, either genetic or phenotypic, was observed in this cohort. Among most subjects with multiple samples, reactivation/incomplete treatment likely contributed to recurrence. However, isolates from two subjects appeared more likely to have occurred via reinfection, based on the large number of genomic single nucleotide variants detected. Finally, although quite distinct from previously reported Canadian MTB strains, isolates obtained from Nunavut clustered most closely with a cohort of samples originating in the Nunavik region of Northern Quebec. This study demonstrates the benefit of using WGS for discriminatory analysis of MTB in Canada, especially in high incidence regions. It further emphasizes the importance of focusing epidemiological intervention efforts on interrupting transmission chains of endemic TB throughout Northern communities, rather than relying on strategies applied in regions where the majority of TB cases result from importation of foreign strains.</p></div

Nonsense single nucleotide variants (resulting in premature stop, or abrogation of start).

<p>Loci based on genomic position and numbering in the H37Rv reference genome (NC_00962.3). All described alternate alleles at specified loci are in relation to the reference sequence at that position. Included loci are those with at least 5 isolates possessing the variant genotype. All were significantly associated with a MIRU cluster (p_FDR <0.05).</p

Repeat sampling of individuals from whom multiple isolates were obtained.

<p>Repeat sampling of individuals from whom multiple isolates were obtained.</p