Competition between cryptic species explains variations in rates of lineage evolution.
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ABSTRACT:
SUBMITTER: Alizon S
PROVIDER: S-EPMC2527920 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:RATIONALE:Minority drug-resistant Mycobacterium tuberculosis subpopulations can be associated with phenotypic resistance but are poorly detected by Sanger sequencing or commercial molecular diagnostic assays. OBJECTIVES:To determine the role of targeted next-generation sequencing in resolving these minor variant subpopulations. METHODS:We used single molecule overlapping reads (SMOR), a targeted next-generation sequencing approach that dramatically reduces sequencing error, to analyze primary cultured isolates phenotypically resistant to rifampin, fluoroquinolones, or aminoglycosides, but for which Sanger sequencing found no resistance-associated variants (RAVs) within respective resistance-determining regions (study group). Isolates also underwent single-colony selection on antibiotic-containing agar, blinded to sequencing results. As a positive control, isolates with multiple colocalizing chromatogram peaks were also analyzed (control group). MEASUREMENTS AND MAIN RESULTS:Among 61 primary culture isolates (25 study group and 36 control group), SMOR described 66 (49%) and 45 (33%) of 135 total heteroresistant RAVs at frequencies less than 5% and less than 1% of the total mycobacterial population, respectively. In the study group, SMOR detected minor resistant variant subpopulations in 80% (n?=?20/25) of isolates with no Sanger-identified RAVs (median subpopulation size, 1.0%; interquartile range, 0.2-3.9%). Single-colony selection on drug-containing media corroborated SMOR results for 90% (n?=?18/20) of RAV-containing specimens, and the absence of RAVs in 60% (n?=?3/5) of isolates. Overall, Sanger sequencing was concordant with SMOR for 77% (n?=?53/69) of macroheteroresistant (5-95% total population), but only 5% of microheteroresistant (<5%) subpopulations (n?=?3/66) across both groups. CONCLUSIONS:Cryptic minor variant mycobacterial subpopulations exist below the resolving capability of current drug susceptibility testing methodologies, and may explain an important proportion of false-negative resistance determinations.
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2024-03-11 | GSE255232 | GEO
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