Effects of Host, Sample, and in vitro Culture on Genomic Diversity of Pathogenic Mycobacteria

Abstract

Mycobacterium tuberculosis ( M. tb ), an obligate human pathogen and the etiological agent of tuberculosis (TB), remains a major threat to global public health. Comparative genomics has been invaluable for monitoring the emergence and spread of TB and for gaining insight into adaptation of M. tb . Most genomic studies of M. tb are based on single bacterial isolates that have been cultured for several weeks in vitro . However, in its natural human host, M. tb comprises complex, in some cases massive bacterial populations that diversify over the course of infection and cannot be wholly represented by a single genome. Recently, enrichment via hybridization capture has been used as a rapid diagnostic tool for TB, circumventing culturing protocols and enabling the recovery of M. tb genomes directly from sputum. This method has further applicability to the study of M. tb adaptation, as it enables a higher resolution and more direct analysis of M. tb genetic diversity within hosts with TB. Here we analyzed genomic material from M. tb and Mycobacterium bovis populations captured directly from sputum and from cultured samples using metagenomic and Pool-Seq approaches. We identified effects of sampling, patient, and sample type on bacterial genetic diversity. Bacterial genetic diversity was more variable and on average higher in sputum than in culture samples, suggesting that manipulation in the laboratory reshapes the bacterial population. Using outlier analyses, we identified candidate bacterial genetic loci mediating adaptation to these distinct environments. The study of M. tb in its natural human host is a powerful tool for illuminating host pathogen interactions and understanding the bacterial genetic underpinnings of virulence.

Dynamics of within-host Mycobacterium tuberculosis diversity and heteroresistance during treatment

“…However, when reanalysing data collected by other groups, it may be difficult to account for the role that pathogen culture and subsequent subculturing steps may have on genetic diversity through random loss or selection of culture-adapted subpopulations. We and others have shown that culture-independent sequencing of Mtb directly from sputum identifies more genetic diversity than sequencing from culture [15,16], while other studies have shown subculture can lead to loss of heterozygous resistance-associated variants (RAVs) [17]. Furthermore, detailed analysis of low-frequency variants can be affected by errors induced during PCR steps of genomic library preparation [18] and incorrect mapping of contaminant DNA [19].…”

Previously undetected superspreading ofMycobacterium tuberculosisrevealed by deep sequencing

“…Due to methodological challenges of sequencing directly from sputum, 26-28 few studies have examined the effect of culture on genome diversity. A recent study by Shockey et al 29 . using cSNPs suggests that variation may be lost during the culturing process, however authors did not examine the impact on hSNPs or transmission, and several other studies 27,28,30 previously found congruent results between cSNP analyses from culture versus raw samples.…”

Direct transmission of within-host Mycobacterium tuberculosis diversity to secondary cases can lead to variable between-host heterogeneity without de novo mutation: A genomic investigation

“…Indeed sampling effects whereby subclonal populations are differentially captured would also present as purifying selection. These data raise major concerns that even when multiple samples are analysed, we do not capture the underlying within-host heterogeneity in clinical practice [11, 48]. Our study, however, has several strengths in that it provides an in-depth look at the variability within and between a cluster of patients who are pan-susceptible to first-line therapy and thus avoids variability introduced due to differing history of acquired drug resistance.…”