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Use of whole genome sequencing to estimate the mutation rate of Mycobacterium tuberculosis during latent infection.

Ford CB, Lin PL, Chase MR, Shah RR, Iartchouk O, Galagan J, Mohaideen N, Ioerger TR, Sacchettini JC, Lipsitch M, Flynn JL, Fortune SM - Nat. Genet. (2011)

Bottom Line: Based on the distribution of SNPs observed, we calculated the mutation rates for these disease states.The pattern of polymorphisms suggests that the mutational burden in vivo is because of oxidative DNA damage.We show that Mtb continues to acquire mutations during disease latency, which may explain why isoniazid monotherapy for latent tuberculosis is a risk factor for the emergence of isoniazid resistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, USA.

ABSTRACT
Tuberculosis poses a global health emergency, which has been compounded by the emergence of drug-resistant Mycobacterium tuberculosis (Mtb) strains. We used whole-genome sequencing to compare the accumulation of mutations in Mtb isolated from cynomolgus macaques with active, latent or reactivated disease. We sequenced 33 Mtb isolates from nine macaques with an average genome coverage of 93% and an average read depth of 117×. Based on the distribution of SNPs observed, we calculated the mutation rates for these disease states. We found a similar mutation rate during latency as during active disease or in a logarithmically growing culture over the same period of time. The pattern of polymorphisms suggests that the mutational burden in vivo is because of oxidative DNA damage. We show that Mtb continues to acquire mutations during disease latency, which may explain why isoniazid monotherapy for latent tuberculosis is a risk factor for the emergence of isoniazid resistance.

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Experimental protocol for assessing mutational capacity in different disease states1) Cynomolgus macaques were infected with ~25CFU of Mtb Erdman via bronchoscopy. 2) Animals were euthanized in the indicated stages of disease for strain isolation. 3) 18 pathologic lesions were plated for bacterial colonies. 33 strains were isolated for WGS. 4) Genomic DNA was isolated from these strains and then analyzed via Illumina sequencing. 5) Reads were assembled using both de novo and scaffolded approaches. 15 SNPs were predicted by both methodologies. Insertions and deletions were not detected using either methodology. 6) Sanger sequencing confirmed 14 of the 15 putative SNPs identified by both scaffolded and de novo analysis.
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Figure 1: Experimental protocol for assessing mutational capacity in different disease states1) Cynomolgus macaques were infected with ~25CFU of Mtb Erdman via bronchoscopy. 2) Animals were euthanized in the indicated stages of disease for strain isolation. 3) 18 pathologic lesions were plated for bacterial colonies. 33 strains were isolated for WGS. 4) Genomic DNA was isolated from these strains and then analyzed via Illumina sequencing. 5) Reads were assembled using both de novo and scaffolded approaches. 15 SNPs were predicted by both methodologies. Insertions and deletions were not detected using either methodology. 6) Sanger sequencing confirmed 14 of the 15 putative SNPs identified by both scaffolded and de novo analysis.

Mentions: Conventional approaches to measuring bacterial mutation rates cannot be applied to Mtb in vivo. However, high-density whole genome sequencing (WGS) allows us to assess the capacity of Mtb for mutation over the course of infection with minimal bias and maximum sensitivity3–5. As the nonhuman primate is the only animal model that mimics the broad range of disease seen in human tuberculosis6,7, we performed WGS on the infecting strain of Mtb, Erdman, and 33 isolates from nine cynomolgus macaques that represented the three major clinical outcomes of infection (active disease, persistently latent infection and spontaneously reactivated disease after prolonged latency7) (Fig. 1). Genome coverage averaged 93% across these isolates, and average read depth was 117× across the genomes (Supplementary Table 1). Putative polymorphisms were identified using both a scaffolded approach8,9 and a de novo assembly method10, and polymorphic sites were validated by Sanger sequencing or through independent identification by WGS. Through these analyses, we identified 14 unique single nucleotide polymorphisms (SNPs) (Fig. 2). There was no evidence that these SNPs were present in the inoculum either from repeated deep sequencing and PCR resequencing of the inoculum, or from shared polymorphisms between bacteria from different lesions. While we have used WGS previously to detect insertions and deletions9, we did not detect either in the 33 genomes analyzed. Within lesions, we identified both shared and independent polymorphisms as would be expected if the SNPs accrued within lesions over the course of the infection.


Use of whole genome sequencing to estimate the mutation rate of Mycobacterium tuberculosis during latent infection.

Ford CB, Lin PL, Chase MR, Shah RR, Iartchouk O, Galagan J, Mohaideen N, Ioerger TR, Sacchettini JC, Lipsitch M, Flynn JL, Fortune SM - Nat. Genet. (2011)

Experimental protocol for assessing mutational capacity in different disease states1) Cynomolgus macaques were infected with ~25CFU of Mtb Erdman via bronchoscopy. 2) Animals were euthanized in the indicated stages of disease for strain isolation. 3) 18 pathologic lesions were plated for bacterial colonies. 33 strains were isolated for WGS. 4) Genomic DNA was isolated from these strains and then analyzed via Illumina sequencing. 5) Reads were assembled using both de novo and scaffolded approaches. 15 SNPs were predicted by both methodologies. Insertions and deletions were not detected using either methodology. 6) Sanger sequencing confirmed 14 of the 15 putative SNPs identified by both scaffolded and de novo analysis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3101871&req=5

Figure 1: Experimental protocol for assessing mutational capacity in different disease states1) Cynomolgus macaques were infected with ~25CFU of Mtb Erdman via bronchoscopy. 2) Animals were euthanized in the indicated stages of disease for strain isolation. 3) 18 pathologic lesions were plated for bacterial colonies. 33 strains were isolated for WGS. 4) Genomic DNA was isolated from these strains and then analyzed via Illumina sequencing. 5) Reads were assembled using both de novo and scaffolded approaches. 15 SNPs were predicted by both methodologies. Insertions and deletions were not detected using either methodology. 6) Sanger sequencing confirmed 14 of the 15 putative SNPs identified by both scaffolded and de novo analysis.
Mentions: Conventional approaches to measuring bacterial mutation rates cannot be applied to Mtb in vivo. However, high-density whole genome sequencing (WGS) allows us to assess the capacity of Mtb for mutation over the course of infection with minimal bias and maximum sensitivity3–5. As the nonhuman primate is the only animal model that mimics the broad range of disease seen in human tuberculosis6,7, we performed WGS on the infecting strain of Mtb, Erdman, and 33 isolates from nine cynomolgus macaques that represented the three major clinical outcomes of infection (active disease, persistently latent infection and spontaneously reactivated disease after prolonged latency7) (Fig. 1). Genome coverage averaged 93% across these isolates, and average read depth was 117× across the genomes (Supplementary Table 1). Putative polymorphisms were identified using both a scaffolded approach8,9 and a de novo assembly method10, and polymorphic sites were validated by Sanger sequencing or through independent identification by WGS. Through these analyses, we identified 14 unique single nucleotide polymorphisms (SNPs) (Fig. 2). There was no evidence that these SNPs were present in the inoculum either from repeated deep sequencing and PCR resequencing of the inoculum, or from shared polymorphisms between bacteria from different lesions. While we have used WGS previously to detect insertions and deletions9, we did not detect either in the 33 genomes analyzed. Within lesions, we identified both shared and independent polymorphisms as would be expected if the SNPs accrued within lesions over the course of the infection.

Bottom Line: Based on the distribution of SNPs observed, we calculated the mutation rates for these disease states.The pattern of polymorphisms suggests that the mutational burden in vivo is because of oxidative DNA damage.We show that Mtb continues to acquire mutations during disease latency, which may explain why isoniazid monotherapy for latent tuberculosis is a risk factor for the emergence of isoniazid resistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, USA.

ABSTRACT
Tuberculosis poses a global health emergency, which has been compounded by the emergence of drug-resistant Mycobacterium tuberculosis (Mtb) strains. We used whole-genome sequencing to compare the accumulation of mutations in Mtb isolated from cynomolgus macaques with active, latent or reactivated disease. We sequenced 33 Mtb isolates from nine macaques with an average genome coverage of 93% and an average read depth of 117×. Based on the distribution of SNPs observed, we calculated the mutation rates for these disease states. We found a similar mutation rate during latency as during active disease or in a logarithmically growing culture over the same period of time. The pattern of polymorphisms suggests that the mutational burden in vivo is because of oxidative DNA damage. We show that Mtb continues to acquire mutations during disease latency, which may explain why isoniazid monotherapy for latent tuberculosis is a risk factor for the emergence of isoniazid resistance.

Show MeSH
Related in: MedlinePlus