Cost-effective interrogation of single nucleotide polymorphisms using the mismatch amplification mutation assay and capillary electrophoresis.

The ability to characterize SNPs is an important aspect of many clinical diagnostic, genetic and evolutionary studies. Here, we designed a multiplexed SNP genotyping method to survey a large number of phylogenetically informative SNPs within the genome of the bacterium Bacillus anthracis. This novel method, CE universal tail mismatch amplification mutation assay (CUMA), allows for PCR multiplexing and automatic scoring of SNP genotypes, thus providing a rapid, economical and higher throughput alternative to more expensive SNP genotyping techniques.

Selective isolation of Yersinia pestis from plague-infected fleas.

We evaluated Yersinia CIN agar for the isolation of Yersinia pestis from infected fleas. CIN media is effective for the differentiation of Y. pestis from flea commensal flora and is sufficiently inhibitory to other bacteria that typically outcompete Y.

Within-host evolution of Burkholderia pseudomallei in four cases of acute melioidosis.

Little is currently known about bacterial pathogen evolution and adaptation within the host during acute infection. Previous studies of Burkholderia pseudomallei, the etiologic agent of melioidosis, have shown that this opportunistic pathogen mutates rapidly both in vitro and in vivo at tandemly repeated loci, making this organism a relevant model for studying short-term evolution. In the current study, B.

Single nucleotide polymorphisms for assessing genetic diversity in castor bean (Ricinus communis).

Background: Castor bean (Ricinus communis) is an agricultural crop and garden ornamental that is widely cultivated and has been introduced worldwide. Understanding population structure and the distribution of castor bean cultivars has been challenging because of limited genetic variability. We analyzed the population genetics of R.

Phylogeographic reconstruction of a bacterial species with high levels of lateral gene transfer.

Background: Phylogeographic reconstruction of some bacterial populations is hindered by low diversity coupled with high levels of lateral gene transfer. A comparison of recombination levels and diversity at seven housekeeping genes for eleven bacterial species, most of which are commonly cited as having high levels of lateral gene transfer shows that the relative contributions of homologous recombination versus mutation for Burkholderia pseudomallei is over two times higher than for Streptococcus pneumoniae and is thus the highest value yet reported in bacteria. Despite the potential for homologous recombination to increase diversity, B.

Francisella tularensis subsp. novicida isolated from a human in Arizona.

Background: Francisella tularensis is the etiologic agent of tularemia and is classified as a select agent by the Centers for Disease Control and Prevention. Currently four known subspecies of F. tularensis that differ in virulence and geographical distribution are recognized:tularensis (type A), holarctica (type B), mediasiatica, and novicida.

Humans and evolutionary and ecological forces shaped the phylogeography of recently emerged diseases.

The development of human civilizations and global commerce has led to the emergence and worldwide circulation of many infectious diseases. Anthrax, plague and tularaemia are three zoonotic diseases that have been intensely studied through genome characterization of the causative species and phylogeographical analyses. A few highly fit genotypes in each species represent the causative agents for most of the observed disease cases.

Phylogenetic understanding of clonal populations in an era of whole genome sequencing.

Phylogenetic hypotheses using whole genome sequences have the potential for unprecedented accuracy, yet a failure to understand issues associated with discovery bias, character sampling, and strain sampling can lead to highly erroneous conclusions. For microbial pathogens, phylogenies derived from whole genome sequences are becoming more common, as large numbers of characters distributed across entire genomes can yield extremely accurate phylogenies, particularly for strictly clonal populations. The availability of whole genomes is increasing as new sequencing technologies reduce the cost and time required for genome sequencing.

Phylogeography of Francisella tularensis: global expansion of a highly fit clone.

Francisella tularensis contains several highly pathogenic subspecies, including Francisella tularensis subsp. holarctica, whose distribution is circumpolar in the northern hemisphere. The phylogeography of these subspecies and their subclades was examined using whole-genome single nucleotide polymorphism (SNP) analysis, high-density microarray SNP genotyping, and real-time-PCR-based canonical SNP (canSNP) assays.

Whole-genome-based phylogeny and divergence of the genus Brucella.

Brucellae are worldwide bacterial pathogens of livestock and wildlife, but phylogenetic reconstructions have been challenging due to limited genetic diversity. We assessed the taxonomic and evolutionary relationships of five Brucella species-Brucella abortus, B. melitensis, B.

A high resolution four-locus multiplex single nucleotide repeat (SNR) genotyping system in Bacillus anthracis.

The allelic identities of Single Nucleotide Repeat (SNR) markers in Bacillus anthracis are typically ascertained by DNA sequencing through the direct repeat. Here we describe a reproducible method for genotyping closely related isolates by using four SNR loci in a multiplex-PCR capillary electrophoresis system amenable to high-throughput analysis.

Real-time PCR assays of single-nucleotide polymorphisms defining the major Brucella clades.

Members of the genus Brucella are known worldwide as pathogens of wildlife and livestock and are the most common organisms of zoonotic infection in humans. In general, brucellae exhibit a range of host specificity in animals that has led to the identification of at least seven Brucella species. The genomes of the various Brucella species are highly conserved, which makes the differentiation of species highly challenging.

Tandem repeat regions within the Burkholderia pseudomallei genome and their application for high resolution genotyping.

Background: The facultative, intracellular bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals. We identified and categorized tandem repeat arrays and their distribution throughout the genome of B. pseudomallei strain K96243 in order to develop a genetic typing method for B.

VNTR analysis of selected outbreaks of Burkholderia pseudomallei in Australia.

Molecular typing methods for Burkholderia pseudomallei have been successful at assigning isolates into epidemiologically related groups, but have not been able to detect differences and define evolutionary patterns within groups. Our variable number tandem repeat (VNTR) analysis of a set of 121 Australian B. pseudomallei isolates, 104 of which were associated with nine epidemiological groups, provides fine scale differentiation even among very closely related isolates.