Geographical distribution of isolates from this study.

<p>Circle size represents the number of isolates found at a specific sampling site and the color represents different species as determined by <i>recA</i> or 16s <i>rRNA</i> sequencing. Division of circles indicates that multiple species were found at a site. <i>B</i>. <i>ubonensis</i> is commonly found throughout Darwin and the surrounding areas. Several species were found in the same soil sample as <i>B</i>. <i>pseudomallei</i> including <i>B</i>. <i>ubonensis</i>, several <i>B</i>. <i>cepacia</i> complex species, proposed <i>B</i>. <i>humptydooensis</i>, <i>Pandoraea</i>, and <i>Cupriavidus</i>.</p

Colony morphology of MSMB isolates on Ashdown’s Agar (ASA) compared to <i>B</i>. <i>pseudomallei</i> MSHR305.

<p>ASA is considered a selective growth medium for <i>Burkholderia pseudomallei</i>. In this study, at least 20 different species demonstrated the ability to grow on ASA including multiple <i>Burkholderia</i> speices as well as <i>Ralstonia</i>, <i>Cupriavidus</i>, and <i>Panoraea</i>. Many different colony morphologies were observed during this study as evident in the pictures (a) proposed <i>B</i>. <i>humptydooensis</i>MSMB43, (b) unknown <i>Burkholderia spp</i>. MSMB 175, (c) <i>B</i>. <i>multivorans</i> MSMB105, (d) <i>B</i>. <i>thailandensis</i> MSMB60, (e) <i>B</i>. <i>ubonensis</i> MSMB153, (f) <i>B</i>. <i>pseudomallei</i> MSHR 305. Each strain was grown on ASA for 72 hours at 37°C aerobically.</p

Comparison of environmental species identified in the Darwin region of Australia.

<p>(-) indicates that no <i>B</i>. <i>pseudomallei</i> was found to occur with a species or that the molecular assay was negative for that target.</p><p>^a Site refers to the GPS location from which soil or water samples were taken.</p><p>^b<i>B</i>. <i>pseudomallei</i> were recovered from the same soil or water sample as the isolate.</p><p>Comparison of environmental species identified in the Darwin region of Australia.</p

Number and origin of <i>B. pseudomallei</i>, <i>B. mallei</i>, and genetic near-neighbor strains used in this study.

<p>Number and origin of <i>B. pseudomallei</i>, <i>B. mallei</i>, and genetic near-neighbor strains used in this study.</p

Amplification plots of BurkDiff.

<p>Quadruplicates of 10-fold serial dilutions of DNA from a crude heat lysis extraction of A. <i>B. mallei</i> strain 2002734303 and B. <i>B. pseudomallei</i> strain 2002721637 were screened on BurkDiff to determine the limit of detection of the assay. Two of 4 replicates at the 10¹ copies template amount did not amplify for both species.</p

Species and number of differential diagnostic and background flora strains screened across BurkDiff to validate the assay's specificity.

<p>Out of the 328 strains from approximately 80 species, none amplified.</p

BurkDiff allelic discrimination plot.

<p>Results from the assay across 45 <i>B. pseudomallei</i> and 23 <i>B. mallei</i> strains are shown, along with 2 no template controls (NTCs) and 26 near-neighbor and differential diagnostic species.</p

Colony morphologies of various <i>B. pseudomallei</i> near-neighbour species on Ashdown’s agar.

<p>Panels: A, <i>Burkholderia ubonensis</i> MSMB700; B, <i>B. ubonensis</i> MSMB704; C, <i>B. ubonensis</i> MSMB1138; D, <i>B. ubonensis</i> MSMB718; E, <i>B. ubonensis</i> MSMB1191; F, <i>B. ubonensis</i> MSMB1165; G, <i>B. ubonensis</i> MSMB1202; H, <i>Pandoraea</i> sp. MSMB824; I, <i>Herbaspirillum seropedicae</i> MSMB1000; J, <i>Burkholderia diffusa</i> MSMB1075; K, <i>Chryseobacterium</i> sp. MSMB1448; L, <i>Cupriavidus metallidurans</i> MSMB1495; M, <i>Burkholderia vietnamiensis</i> MSMB1224; N, <i>Burkholderia multivorans</i> MSMB1271; O, <i>Burkholderia pyrrocinia</i> MSMB1147; P, <i>Delftia</i> sp. MSMB943; Q, <i>Ralstonia mannitolilytica</i> MSMB1253; R, <i>Burkholderia thailandensis</i> MSMB1415; S, <i>Burkholderia cepacia</i> MSMB1456; T, <i>B. cepacia</i> MSMB1011; U, <i>Acidovorax caeni</i> MSMB1260. On this medium, <i>Burkholderia ubonensis</i> demonstrates similar morphological characteristics to its potentially deadly near-neighbour, <i>Burkholderia pseudomallei</i>, including uptake of crystal violet and neutral red, and wrinkling of colonies after ∼72 h growth <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071647#pone.0071647-Ashdown1" target="_blank">[13]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071647#pone.0071647-Chantratita1" target="_blank">[24]</a>. Molecular genotyping is therefore necessary for differentiation of <i>B. ubonensis</i> from other bacterial species that grow on Ashdown’s medium. Note the morphological differences among <i>B. ubonensis</i> strains; several morphotypes have also been observed in <i>B. pseudomallei </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071647#pone.0071647-Chantratita1" target="_blank">[24]</a>.</p

Bu550 <i>B. ubonensis-</i>specific real-time PCR.

<p>Bu550 differentiates <i>Burkholderia ubonensis</i> from other soil- and water-borne bacterial species that grow on Ashdown’s agar <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071647#pone.0071647-Ashdown1" target="_blank">[13]</a>. Only <i>B. ubonensis</i> (shown in blue) amplifies with this assay. Other species (shown in red) fail to amplify.</p

Bacterial strain panel used in this study.

a<p>Numbers in parentheses indicate Thai strains; all other strains were isolated in the Northern Territory, Australia.</p>b<p>Species assignment based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071647#pone.0071647-Gee1" target="_blank">[28]</a>.</p