Map of Australasia.

<p><b>a</b>) 21,500 years ago during the last glacial maximum. The shaded regions represent what was dry land during the period. Note that Australia and PNG comprised a single continent (Sahul) and that most of Southeast Asia (Sunda) was linked by land bridges <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018343#pone.0018343-OConnell1" target="_blank">[19]</a>. <b>b</b>) The Balimo region of the Western Province of Papua New Guinea on the Aramia River floodplain.</p

Neighbor joining tree constructed from <i>B. pseudomallei</i> MLVA data.

<p>The isolates in box A represent those with very short branch lengths and resistance to chloramphenicol, suggesting a high level of relatedness.</p

Phylogeny of the major groups of <i>B. anthracis</i> after Pearson et al. (2004) and VanErt et al. (2007).

<p>The new branch, “A.Br.011,” is flanked by branches A.Br.008 and A.Br.009. Thus, the group A.Br.008/009 is now subdivided into two groups: A.Br.008/011 and A.Br.011/009. The canSNP signature and assay that defines this new branch is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028274#pone-0028274-t003" target="_blank">Table 3</a>.</p

Dendrogram of MLVA-8 analysis of <i>B. anthracis</i> isolates collected from the 1976 California, 2006 New York, 2007 Connecticut anthrax cases, and 2009 New Hampshire case.

<p>All other genotypes are reference genotypes from Keim et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028274#pone.0028274-Keim1" target="_blank">[8]</a>. For the California case, the clinical isolates were GT 76 (n = 4), while environmental isolates were GT 71 (n = 2), GT 72 (n = 2), GT 92 (n = 1), and GT 105 (n = 1). All clinical and environmental isolates from the New York (one clinical, 35 environmental) and Connecticut cases (one clinical specimen, 15 environmental isolates) were GT 1. All clinical (n = 2) and environmental (n = 9) isolates from the NH case were GT 149. Scale bar indicates amount of evolutionary change <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028274#pone.0028274-McDonald1" target="_blank">[12]</a>.</p

MLVA-8 results of clinical and environmental <i>B. anthracis</i> isolates associated with the California, New York, Connecticut, and New Hampshire anthrax cases.

<p>—, plasmid loci not detected.</p><p>NA, not applicable.</p><p>*, new allele size not previously described by Keim et al.</p

<i>B. pseudomallei</i> isolates used in this study.

<p>Multiple identifiers indicate isolates that were isolated concurrently from a single patient. Isolates C5 & C6 and C7 & C8 were recovered from siblings.</p

Novel <i>B. anthracis</i> canSNP assays developed in this report.

a<p>Using ‘Ames ancestor’ genome (GenBank ref: AE017334).</p>b<p>Underlined nucleotides indicate the position of the SNP; bolded nucleotides indicate an introduced GC clamp that increases the melt temperature of the primer, thus enhancing allelic discrimination <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028274#pone.0028274-Germer1" target="_blank">[13]</a>; small-case nucleotides represent deliberate mismatches incorporated into the allele-specific primers.</p>c<p>All assays were optimized on an Applied Biosystems ABI PRISM 7900HT Sequence Detection System using default thermocycling parameters, with the addition of the dissociation curve <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028274#pone.0028274-Germer1" target="_blank">[13]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028274#pone.0028274-Germer2" target="_blank">[14]</a>.</p

Variable Virulence Factors in <i>Burkholderia pseudomallei</i> (Melioidosis) Associated with Human Disease

<div><p><i>Burkholderia pseudomallei</i> is a Gram-negative environmental bacterium that causes melioidosis, a potentially life-threatening infectious disease affecting mammals, including humans. Melioidosis symptoms are both protean and diverse, ranging from mild, localized skin infections to more severe and often fatal presentations including pneumonia, septic shock with multiple internal abscesses and occasionally neurological involvement. Several ubiquitous virulence determinants in <i>B. pseudomallei</i> have already been discovered. However, the molecular basis for differential pathogenesis has, until now, remained elusive. Using clinical data from 556 Australian melioidosis cases spanning more than 20 years, we identified a <i>Burkholderia mallei-</i>like actin polymerization <i>bimA</i>_Bm gene that is strongly associated with neurological disease. We also report that a filamentous hemagglutinin gene, <i>fhaB</i>3, is associated with positive blood cultures but is negatively correlated with localized skin lesions without sepsis. We show, for the first time, that variably present virulence factors play an important role in the pathogenesis of melioidosis. Collectively, our study provides a framework for assessing other non-ubiquitous bacterial virulence factors and their association with disease, such as candidate loci identified from large-scale microbial genome-wide association studies.</p></div

Two-Locus (duplexed) Melt-MAMA development.

<p>(A) A phylogenetic topology of the three subspecies of <i>F. tularensis</i> rooted with <i>F. novicida</i>. The SNP-signatures specific for the two pathogenic subspecies of <i>F. tularensis</i> (indicated by black bars) were incorporated into Melt-MAMAs. The table (right) indicates expected allele states (derived and ancestral) for strains from each <i>F. tularensis</i> subspecies represented on the topology; <i>F. novicida</i> would have the same allelic states as <i>F. tularensis</i> subsp. <i>mediasiatica</i>. (Bi–iv) Temperature-dissociation (melt) curves (derivative) of allele-specific PCR products from <i>F. tularensis</i> strains amplified in the duplexed assay (Type A and Type B). Each profile show two melt-curve peaks generated from a single <i>F. tularensis</i> strain. Each peak corresponds to the allele-specific PCR product for a single SNP-locus in the duplexed assay.</p

Real-time PCR amplification and dissociation (melt) curve plots.

<p><i>B. anthracis</i> Melt-MAMA SYBR® Green assay targeting the A.Br.004 genetic clade. (A & C) The amplification of two alleles are illustrated for haploid template (<i>Bacillus anthracis</i>) possessing an ‘A’ polymorphic SNP-state or ‘G’ state. Each amplification plot represents a single PCR reaction containing a reverse “common” primer and two allele-specific MAMA primers. The AS-MAMA primers anneal to the same template target and then compete for extension across the SNP position. The polymerase-mediated extension rate of the 3′match AS-MAMA primer (perfect primer-template complex) exceeds that of the 3′mismatched MAMA primer (mismatched primer-template complex), thus the perfect match primer-template complex outcompetes the mismatched primer-template complex and dominates the PCR amplification. (B & D) Plots of the temperature-dissociation (melt) curve of the final PCR products for the two allele templates are shown next to their respective amplification plots (green arrows). Allele-specific PCR products are easily differentiated through temperature-dissociation (melt) curve analysis, which is conferred by the GC-clamp engineered on one of the AS-MAMA primer.</p