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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.

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