Midori Kato-Maeda

Out-of-Africa migration and Neolithic coexpansion of Mycobacterium tuberculosis with modern humans

Tuberculosis caused 20% of all human deaths in the Western world between the seventeenth and nineteenth centuries and remains a cause of high mortality in developing countries. In analogy to other crowd diseases, the origin of human tuberculosis has been associated with the Neolithic Demographic Transition, but recent studies point to a much earlier origin. We analyzed the whole genomes of 259 M. tuberculosis complex (MTBC) strains and used this data set to characterize global diversity and to reconstruct the evolutionary history of this pathogen. Coalescent analyses indicate that MTBC emerged about 70,000 years ago, accompanied migrations of anatomically modern humans out of Africa and expanded as a consequence of increases in human population density during the Neolithic period. This long coevolutionary history is consistent with MTBC displaying characteristics indicative of adaptation to both low and high host densities.

Whole-genome sequencing of rifampicin-resistant Mycobacterium tuberculosis strains identifies compensatory mutations in RNA polymerase genes

Epidemics of drug-resistant bacteria emerge worldwide, even as resistant strains frequently have reduced fitness compared to their drug-susceptible counterparts. Data from model systems suggest that the fitness cost of antimicrobial resistance can be reduced by compensatory mutations; however, there is limited evidence that compensatory evolution has any significant role in the success of drug-resistant bacteria in human populations. Here we describe a set of compensatory mutations in the RNA polymerase genes of rifampicin-resistant M. tuberculosis, the etiologic agent of human tuberculosis (TB). M. tuberculosis strains harboring these compensatory mutations showed a high competitive fitness in vitro. Moreover, these mutations were associated with high fitness in vivo, as determined by examining their relative clinical frequency across patient populations. Of note, in countries with the worlds highest incidence of multidrug-resistant (MDR) TB, more than 30% of MDR clinical isolates had this form of mutation. Our findings support a role for compensatory evolution in the global epidemics of MDR TB.

Genomic analysis identifies targets of convergent positive selection in drug-resistant Mycobacterium tuberculosis.

M. tuberculosis is evolving antibiotic resistance, threatening attempts at tuberculosis epidemic control. Mechanisms of resistance, including genetic changes favored by selection in resistant isolates, are incompletely understood. Using 116 newly sequenced and 7 previously sequenced M. tuberculosis whole genomes, we identified genome-wide signatures of positive selection specific to the 47 drug-resistant strains. By searching for convergent evolution—the independent fixation of mutations in the same nucleotide position or gene—we recovered 100% of a set of known resistance markers. We also found evidence of positive selection in an additional 39 genomic regions in resistant isolates. These regions encode components in cell wall biosynthesis, transcriptional regulation and DNA repair pathways. Mutations in these regions could directly confer resistance or compensate for fitness costs associated with resistance. Functional genetic analysis of mutations in one gene, ponA1, demonstrated an in vitro growth advantage in the presence of the drug rifampicin.

Microarray analysis of pathogens and their interaction with hosts.

Microarrays are a promising technique for elucidating and interpreting the mechanistic roles of genes in the pathogenesis of infectious disease. Microarrays have been used to analyse the genetic polymorphisms of specific loci associated with resistance to antimicrobial agents, to explore the distribution of genes among isolates from the same and similar species, to understand the evolutionary relationship between closely related species and to integrate the clinical and genomic data. This technique has also been used to study host–pathogen interactions, mainly by identifying genes from pathogens that may be involved in pathogenicity and by surveying the scope of the host response to infection. The RNA expression profile of pathogens has been used to identify regulatory mechanisms that ensure gene expression in the appropriate environment, to hypothesize functions of hundreds of uncharacterized genes and to identify virulence genes that promote colonization or tissue damage. This information also has the potential to identify targets for drug design. Furthermore, microarrays have been used to investigate the mechanism of drug action and to delineate and predict adverse effects of new drugs. In this paper, we review the use of spotted and high‐density oligonucleotide arrays to study the genetic polymorphisms of pathogens, host–pathogen interactions and whole‐genome expression profiles of pathogens, as well as their use for drug discovery.

Genomic Analysis Distinguishes Mycobacterium africanum

ABSTRACT Mycobacterium africanum is thought to comprise a unique species within the Mycobacterium tuberculosis complex. M. africanum has traditionally been identified by phenotypic criteria, occupying an intermediate position between M. tuberculosis and M. bovis according to biochemical characteristics. Although M. africanum isolates present near-identical sequence homology to other species of the M. tuberculosis complex, several studies have uncovered large genomic regions variably deleted from certain M. africanum isolates. To further investigate the genomic characteristics of organisms characterized as M. africanum, the DNA content of 12 isolates was interrogated by using Affymetrix GeneChip. Analysis revealed genomic regions of M. tuberculosis deleted from all isolates of putative diagnostic and biological consequence. The distribution of deleted sequences suggests that M. africanum subtype II isolates are situated among strains of “modern” M. tuberculosis. In contrast, other M. africanum isolates (subtype I) constitute two distinct evolutionary branches within the M. tuberculosis complex. To test for an association between deleted sequences and biochemical attributes used for speciation, a phenotypically diverse panel of “M. africanum-like” isolates from Guinea-Bissau was tested for these deletions. These isolates clustered together within one of the M. africanum subtype I branches, irrespective of phenotype. These results indicate that convergent biochemical profiles can be independently obtained for M. tuberculosis complex members, challenging the traditional approach to M. tuberculosis complex speciation. Furthermore, the genomic results suggest a rational framework for defining M. africanum and provide tools to accurately assess its prevalence in clinical specimens.

The nature and consequence of genetic variability within Mycobacterium tuberculosis.

Viewed in terms of its historical and contemporary disease burden, Mycobacterium tuberculosis (MTB) is undeniably the most successful human pathogen. Molecular paleoarchaeologic evidence clearly identified MTB as the cause of lesions in 5,000-year-old mummies. Over recorded history, the burden of MTB has been staggering. In Europe the mortality was between 200 and 300 per 100,000 at the beginning of the 19th century. Today, tuberculosis still remains an important cause of morbidity and has been displaced from the position of leading infectious cause of death only by the accounting policies of the World Health Organization, which attribute tuberculosis deaths to human immunodeficiency virus in the increasing number of coinfected persons. The future is not brighter. The most optimistic scenarios predict in excess of 80 million new cases and 20 million deaths in the coming decade, 95% of which will occur in resource-poor countries. After decades of neglect, a resurgence of tuberculosis in industrialized countries reinvigorated research efforts, and tuberculosis is now back on the cutting edge of science. Molecular epidemiologic approaches have provided novel insights into the transmission dynamics of tuberculosis and have helped to refocus and refine control practices. These studies highlighted old evidence that there is significant variability in the clinical and epidemiologic consequences of infection with MTB. In the near future, mycobacteria are likely to be one of the most deeply sequenced pathogenic groups. The complete genomes of two MTB isolates are now available, and the sequencing of isolates of M. bovis, bacille Calmette-Guerin (BCG), M. smegmatis, M. paratuberculosis, M. leprae, M. marinum, M. ulcerans, and M. avium are somewhere between contemplated and completed. Although much is known about environmental and host factors that contribute to infection and disease variability, little is know about the bacteria’s role. Given recent advances, we are now poised to explore the nature and consequences of genetic variability in MTB. Here, we review the data supporting phenotypic and genotypic diversity of laboratory and natural strains of MTB and our understanding of the relationship between them.

Large Sequence Polymorphisms Classify Mycobacterium tuberculosis Strains with Ancestral Spoligotyping Patterns

ABSTRACT Genomic deletion analysis revealed that strains of Mycobacterium tuberculosis exhibiting spoligotyping patterns with almost all spacers present belong either to a strain lineage that includes the W-Beijing strain family or to the ancestral strain lineage of M. tuberculosis.

Mycobacterium tuberculosis lineage 4 comprises globally distributed and geographically restricted sublineages

Generalist and specialist species differ in the breadth of their ecological niches. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of Mycobacterium tuberculosis lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that, whereas the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration.

Beijing sublineages of Mycobacterium tuberculosis differ in pathogenicity in the guinea pig.

ABSTRACT The Beijing family of Mycobacterium tuberculosis strains is part of lineage 2 (also known as the East Asian lineage). In clinical studies, we have observed that isolates from the sublineage RD207 of lineage 2 were more readily transmitted among humans. To investigate the basis for this difference, we tested representative strains with the characteristic Beijing spoligotype from four of the five sublineages of lineage 2 in the guinea pig model and subjected these strains to comparative whole-genome sequencing. The results of these studies showed that all of the clinical strains were capable of growing and causing lung pathology in guinea pigs after low-dose aerosol exposure. Differences between the abilities of the four sublineages to grow in the lungs of these animals were not overt, but members of RD207 were significantly more pathogenic, resulting in severe lung damage. The RD207 strains also induced much higher levels of markers associated with regulatory T cells and showed a significant loss of activated T cells in the lungs over the course of the infections. Whole-genome sequencing of the strains revealed mutations specific for RD207 which may explain this difference. Based on these data, we hypothesize that the sublineages of M. tuberculosis are associated with distinct pathological and clinical phenotypes and that these differences influence the transmissibility of particular M. tuberculosis strains in human populations.

Use of Whole Genome Sequencing to Determine the Microevolution of Mycobacterium tuberculosis during an Outbreak

Rationale Current tools available to study the molecular epidemiology of tuberculosis do not provide information about the directionality and sequence of transmission for tuberculosis cases occurring over a short period of time, such as during an outbreak. Recently, whole genome sequencing has been used to study molecular epidemiology of Mycobacterium tuberculosis over short time periods. Objective To describe the microevolution of M. tuberculosis during an outbreak caused by one drug-susceptible strain. Method and Measurements We included 9 patients with tuberculosis diagnosed during a period of 22 months, from a population-based study of the molecular epidemiology in San Francisco. Whole genome sequencing was performed using Illumina’s sequencing by synthesis technology. A custom program written in Python was used to determine single nucleotide polymorphisms which were confirmed by PCR product Sanger sequencing. Main results We obtained an average of 95.7% (94.1–96.9%) coverage for each isolate and an average fold read depth of 73 (1 to 250). We found 7 single nucleotide polymorphisms among the 9 isolates. The single nucleotide polymorphisms data confirmed all except one known epidemiological link. The outbreak strain resulted in 5 bacterial variants originating from the index case A1 with 0–2 mutations per transmission event that resulted in a secondary case. Conclusions Whole genome sequencing analysis from a recent outbreak of tuberculosis enabled us to identify microevolutionary events observable during transmission, to determine 0–2 single nucleotide polymorphisms per transmission event that resulted in a secondary case, and to identify new epidemiologic links in the chain of transmission.