Abstract
BackgroundRepeated culture reduces within-sample Mycobacterium tuberculosis genetic diversity due to selection of clones suited to growth in culture and/or random loss of lineages, but it is not known to what extent omitting the culture step altogether alters genetic diversity. We compared M. tuberculosis whole genome sequences generated from 33 paired clinical samples using two methods. In one method DNA was extracted directly from sputum then enriched with custom-designed SureSelect (Agilent) oligonucleotide baits and in the other it was extracted from mycobacterial growth indicator tube (MGIT) culture.ResultsDNA directly sequenced from sputum showed significantly more within-sample diversity than that from MGIT culture (median 5.0 vs 4.5 heterozygous alleles per sample, p = 0.04). Resistance associated variants present as HAs occurred in four patients, and in two cases may provide a genotypic explanation for phenotypic resistance.ConclusionsCulture-free M. tuberculosis whole genome sequencing detects more within-sample diversity than a leading culture-based method and may allow detection of mycobacteria that are not actively replicating.
Highlights
Repeated culture reduces within-sample Mycobacterium tuberculosis genetic diversity due to selection of clones suited to growth in culture and/or random loss of lineages, but it is not known to what extent omitting the culture step altogether alters genetic diversity
We have previously reported the use of oligonucleotide enrichment technology SureSelect (Agilent, CA, USA) to sequence M. tuberculosis DNA directly from sputum [32] and demonstrated its utility in determining a rapid genetic drug resistance profile [33, 34]
Patient characteristics and drug susceptibility testing Whole genome sequences were obtained for 33 patients from both mycobacterial growth indicator tube (MGIT) culture and direct sputum sequencing
Summary
Repeated culture reduces within-sample Mycobacterium tuberculosis genetic diversity due to selection of clones suited to growth in culture and/or random loss of lineages, but it is not known to what extent omitting the culture step altogether alters genetic diversity. M. tuberculosis has a conserved genome with little genetic diversity between strains and no evidence of horizontal gene transfer [10], but more detailed analysis of individual patient samples with WGS has identified genetically separate bacterial subpopulations in sequential sputum samples [11,12,13,14,15,16] and across different anatomical sites [17]. This within-patient diversity can occur as a result of mixed infection with genetically distinct strains or within-host evolution of a single infecting strain [18]
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