Steps in the evolution of the seventh pandemic <i>Vibrio cholerae</i>.

<p>Environmental <i>V. cholerae</i> indigenous in coastal waters can harbor genomic islands (GIs) by horizontal gene transfer, rendering it pathogenic. Pathogenesis of toxigenic (toxin-producing) <i>V. cholerae</i> critically depends on the production of the cholera toxin, which is responsible for the cholera symptoms, and the toxin-coregulated pilus (TCP). The genes for the cholera toxin (<i>ctx</i>) are from the filamentous bacteriophage, CTXφ, that has been incorporated into the genome. The genes in the TCP island encode factors necessary for the colonization of the small intestine in the human host after ingestion of contaminated water. Additionally, seventh pandemic strains are distinguishable from pre-seventh pandemic strains due to the acquisition of additional GIs, the <i>Vibrio</i> seventh pandemic (VSP) islands.</p

How the Haiti cholera outbreak started.

<p>(A) MINUSTAH troops from Nepal were stationed in Haiti starting on October 8, 2010, and set up camp in Meille (red circle). Improper disposal of sewage led to the contamination of the Meille tributary, which connects downstream to the Latem River (red arrow). The first case of cholera occurred on October 12 along the Latem River in Mirebalais (orange circle), 2 km north of Meille. Water from the Latem River enters the Artibonite River (orange arrow), the major river that spans across Haiti, which flows downstream to St. Marc (blue arrow). The Artibonite River played a significant role in the rapid spread of cholera. During the early onset of the epidemic, reported cases were linked to proximity with the river. (B) A chronological timeline of events involving the Haiti cholera outbreak from July to December 2010.</p

Sulyok et al. BMCVetRes-alignment

Coxiella burnetii multispacer sequence typing alignmen

<i>PmeI</i> pulsed-field gel electrophoresis (PFGE) patterns for <i>F. tularensis</i> subsp. <i>holarectica</i>.

<p>Polymorphic band position 1 consists of two fragments in PFGE type 2. Polymorphic band position 2 and 3 consist of two fragments at the same position, both missing in PFGE type 2. Polymorphic band position 6 consists of two fragments in PFGE type 3.</p

VNTR markers.

<p>*Data obtained in this study.</p>†<p>The individual marker diversity (D) was calculated as D = [1-∑(allele frequency)²].</p>††<p>Location within an open reading frame.</p><p>VNTR markers.</p

Sulyok et al. BMCVetRes-Figure2_revised

Coxiella burnetii multispacer sequence typing pylogenetic tre

MRSA_phylogenetic_trees_data_Luxembourg

This data file contains SNP matrices used to build phylogenetic trees. The matrices are provided in both .fasta format (the input that MEGA accepts) and Excel file format. The phylogenetic trees are provided in MEGA tree session format (MEGAsoftware.net)

TaqMan Real-Time PCR Assays for Single-Nucleotide Polymorphisms Which Identify <i>Francisella tularensis</i> and Its Subspecies and Subpopulations

<div><p><i>Francisella tularensis</i>, the etiologic agent of tularemia and a Class A Select Agent, is divided into three subspecies and multiple subpopulations that differ in virulence and geographic distribution. Given these differences, there is a need to rapidly and accurately determine if a strain is <i>F. tularensis</i> and, if it is, assign it to subspecies and subpopulation. We designed TaqMan real-time PCR genotyping assays using eleven single nucleotide polymorphisms (SNPs) that were potentially specific to closely related groups within the genus <i>Francisella</i>, including numerous subpopulations within <i>F. tularensis</i> species. We performed extensive validation studies to test the specificity of these SNPs to particular populations by screening the assays across a set of 565 genetically and geographically diverse <i>F. tularensis</i> isolates and an additional 21 genetic near-neighbor (outgroup) isolates. All eleven assays correctly determined the genetic groups of all 565 <i>F. tularensis</i> isolates. One assay differentiates <i>F. tularensis</i>, <i>F. novicida</i>, and <i>F. hispaniensis</i> from the more genetically distant <i>F. philomiragia</i> and <i>Francisella-</i>like endosymbionts. Another assay differentiates <i>F. tularensis</i> isolates from near neighbors. The remaining nine assays classify <i>F. tularensis</i>-confirmed isolates into <i>F. tularensis</i> subspecies and subpopulations. The genotyping accuracy of these nine assays diminished when tested on outgroup isolates (i.e. non <i>F. tularensis</i>), therefore a hierarchical approach of assay usage is recommended wherein the <i>F. tularensis</i>-specific assay is used before the nine downstream assays. Among <i>F. tularensis</i> isolates, all eleven assays were highly sensitive, consistently amplifying very low concentrations of DNA. Altogether, these eleven TaqMan real-time PCR assays represent a highly accurate, rapid, and sensitive means of identifying the species, subspecies, and subpopulation of any <i>F. tularensis</i> isolate if used in a step-wise hierarchical scheme. These assays would be very useful in clinical, epidemiological, and/or forensic investigations involving <i>F. tularensis</i>.</p></div

Flow chart providing the hierarchical organization of 18 SNP-genotyping assays.

<p>The hierarchy provides the sequential order to allow for stepwise identification of the genotype of an unknown isolate. Genetic subgroups are represented by colored circles labeled with each genetic subgroup. Individual assays are represented by the black bar labeled with each assay ID. The derived and ancestral allele states for each assay are indicated immediately above and below the black bar.</p

Primers and TaqMan-MGB probes for <i>F. tularensis</i> canSNP assays.

<p>Primers and TaqMan-MGB probes for <i>F. tularensis</i> canSNP assays.</p