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.

Paths to selection on life history loci in different natural environments across the native range of Arabidopsis thaliana.

Selection on quantitative trait loci (QTL) may vary among natural environments due to differences in the genetic architecture of traits, environment-specific allelic effects or changes in the direction and magnitude of selection on specific traits. To dissect the environmental differences in selection on life history QTL across climatic regions, we grew a panel of interconnected recombinant inbred lines (RILs) of Arabidopsis thaliana in four field sites across its native European range. For each environment, we mapped QTL for growth, reproductive timing and development. Several QTL were pleiotropic across environments, three colocalizing with known functional polymorphisms in flowering time genes (CRY2, FRI and MAF2-5), but major QTL differed across field sites, showing conditional neutrality. We used structural equation models to trace selection paths from QTL to lifetime fitness in each environment. Only three QTL directly affected fruit number, measuring fitness. Most QTL had an indirect effect on fitness through their effect on bolting time or leaf length. Influence of life history traits on fitness differed dramatically across sites, resulting in different patterns of selection on reproductive timing and underlying QTL. In two oceanic field sites with high prereproductive mortality, QTL alleles contributing to early reproduction resulted in greater fruit production, conferring selective advantage, whereas alleles contributing to later reproduction resulted in larger size and higher fitness in a continental site. This demonstrates how environmental variation leads to change in both QTL effect sizes and direction of selection on traits, justifying the persistence of allelic polymorphism at life history QTL across the species range.

The role of speciation in positive Lowenstein-Jensen culture isolates from a high tuberculosis burden country.

OBJECTIVE: To determine the need for routine speciation of positive Lowenstein-Jensen mycobacterial cultures in HIV-infected patients suspected of having pulmonary tuberculosis at Mulago Hospital in Kampala, Uganda. METHODS: Sputum and bronchoalveolar lavage Lowenstein-Jensen mycobacterial culture isolates from consecutive, HIV-infected patients admitted to Mulago Hospital with 2 weeks or more of cough were subjected to IS6110 PCR and rpoB genetic analysis to determine the presence of Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM). RESULTS: Eighty (100%) mycobacterial cultures from 65 patients were confirmed to be members of MTBC. Subsequent analysis of the cultures from 54 patients by PCR and sequence analyses to identify co-infection with NTM confirmed the presence of MTBC as well as the presence of Micrococcus luteus (n = 4), Janibacter spp. (n = 1) and six cultures had organisms that could not be identified. CONCLUSIONS: Presumptive diagnosis of tuberculosis on the basis of a positive Lowenstein-Jensen culture is sufficient in HIV-infected Ugandans suspected of having tuberculosis. Routine molecular confirmation of positive Lowenstein-Jensen cultures is unnecessary in this low resource setting.

Major-effect alleles at relatively few loci underlie distinct vernalization and flowering variation in Arabidopsis accessions.

We have explored the genetic basis of variation in vernalization requirement and response in Arabidopsis accessions, selected on the basis of their phenotypic distinctiveness. Phenotyping of F2 populations in different environments, plus fine mapping, indicated possible causative genes. Our data support the identification of FRI and FLC as candidates for the major-effect QTL underlying variation in vernalization response, and identify a weak FLC allele, caused by a Mutator-like transposon, contributing to flowering time variation in two N. American accessions. They also reveal a number of additional QTL that contribute to flowering time variation after saturating vernalization. One of these was the result of expression variation at the FT locus. Overall, our data suggest that distinct phenotypic variation in the vernalization and flowering response of Arabidopsis accessions is accounted for by variation that has arisen independently at relatively few major-effect loci.

Effects of genetic perturbation on seasonal life history plasticity.

Like many species, the model plant Arabidopsis thaliana exhibits multiple different life histories in natural environments. We grew mutants impaired in different signaling pathways in field experiments across the species' native European range in order to dissect the mechanisms underlying this variation. Unexpectedly, mutational loss at loci implicated in the cold requirement for flowering had little effect on life history except in late-summer cohorts. A genetically informed photothermal model of progression toward flowering explained most of the observed variation and predicted an abrupt transition from autumn flowering to spring flowering in late-summer germinants. Environmental signals control the timing of this transition, creating a critical window of acute sensitivity to genetic and climatic change that may be common for seasonally regulated life history traits.