Secretion of BCAA metabolism intermediates by infection induced colony morphotypes.

<p>Secreted amounts of α-keto-acids by K96243 isolates after <i>in vivo</i> (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004483#pntd.0004483.g001" target="_blank">Fig 1(iv/2)</a>) and <i>in vitro</i> (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004483#pntd.0004483.g001" target="_blank">Fig 1(iv/3)</a>) infection are shown. Data are shown as mean values ± SD of triplicate samples.</p

Secretion of BCAA metabolism intermediates.

<p>Absolute extracellular concentrations of secreted BCAA-pathway metabolites are displayed. The two most prominent morphotypes of K96243 and E8 after nutrient limitation are displayed. Data are shown as mean values ± SD of triplicate samples.</p

Consumption of extracellular amino acids in <i>B</i>. <i>pseudomallei</i> infection induced colony morphotypes.

<p><b>A)</b> Extracellular amino acid concentrations during the cultivation of K96243 morphotypes after <i>in vitro</i> (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004483#pntd.0004483.g001" target="_blank">Fig 1(iv/3)</a>) and <i>in vivo</i> (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004483#pntd.0004483.g001" target="_blank">Fig 1(iv/2)</a>) infection conditions are visualized in a color coded chart. The initial concentration in the culture medium was set to 100% and colored magenta whereas 10% is the lower limit and was colored light grey. Displayed are averages of triplicate growth experiments (SD in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004483#pntd.0004483.s006" target="_blank">S1 File</a>, Tables B and C). Arrows indicate the time line of cultivation from 5 h to 60 h for each morphotype. <b>B)</b> The combined value of optical densities (OD_650nm) of all K96243 morphotypes are displayed in red lines. Nutrition sets contain either glc+glcA (green), amino acids of set A (asp, asn, glu, gln, ser, pro, gly) (blue) or amino acids of set B (his, tyr, thr, phe, ile, val, leu, arg, lys, 5-oxoproline, 4-hydroxyproline) (brown). Percentage values of nutrition sets in K96243 morphotype cultures before or after <i>in vivo</i> and <i>in vitro</i> infection were calculated by summing up the average amount of the metabolites within a set and comparison to the initial value in the medium prior to cultivation (≙100%). Data are shown as mean values ± SD. <b>C)</b> The calculated amino acid concentrations of K96243 morphotypes after (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004483#pntd.0004483.g001" target="_blank">Fig 1(iv/1)</a>) nutrient limitation and (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004483#pntd.0004483.g001" target="_blank">Fig 1(iv/2)</a>) <i>ex vivo</i> isolates were mean centered and autoscaled and applied to principle component analysis. Clusters were generated by adding each sample, of all 6 morphotypes and 6 <i>ex vivo</i> isolates respectively, of the same time point to one group (t₁-t₆). The arrow above the plot indicates the time course. Groups are indicated by the same color and statistically clustered by an ellipse with a confidence interval of 90%. Displayed is component 1 versus component 3 with their corresponding proportion of variation.</p

Comparison of extracellular glucose and gluconate concentrations in starvation and infection induced colony morphotype cultures.

<p>Absolute extracellular concentrations of glucose (light grey) and gluconate (dark grey) in starvation and infection (<i>in vivo</i> and <i>in vitro</i>) induced K96243 morphotype cultures over time are displayed. Data are shown as mean values ± SD of triplicate samples.</p

Utilization of extracellular carbon sources and amino acids in <i>B</i>. <i>pseudomallei</i> starvation induced colony morphotypes.

<p>Concentrations (mM) of diverse carbon sources are shown for <b>A)</b> E8 and <b>B)</b> K96243 starvation induced morphotypes during the cultivation in RPMI medium. Data are shown as mean values ± SD of triplicate samples. <b>C)</b> Extracellular amino acid concentrations in starvation induced <i>B</i>. <i>pseudomallei</i> morphotype cultures during growth are visualized in a color coded chart. The initial concentration in the culture medium was set to 100% and colored magenta whereas 10% is the lower limit and was colored light grey. Displayed are averages of triplicate growth experiments (SD in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004483#pntd.0004483.s006" target="_blank">S1 File</a>, Table A). Arrows indicate the time line of cultivation from 5 h to 60 h for each morphotype.</p

Experimental setup and growth of diverse induced <i>B</i>. <i>pseudomallei</i> colony morphotypes.

<p><b>A)</b> i) Strains E8 and K96243 were taken from glycerol stocks and ii) different media (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004483#sec005" target="_blank">Materials and Methods</a>) were inoculated with them for 24 days. iii) Colony morphotypes were identified after plating on Ashdown agar, stored at -80°C and plated again on Ashdown agar to confirm colony morphotype stability for further experiments. iv/1) Glycerol stocks of 14 colony morphotypes from both strains were plated on Ashdown agar (v) and used for inoculation of modified RPMI medium (vi). Obtained morphotypes of strain K96243 were further used in (iv/2) <i>in vivo</i> infection experiments with BALB/c mice or (iv/3) for <i>in vitro</i> infection experiments in macrophages RAW267.4. v/1) Colony morphotypes after <i>in vivo</i> and <i>in vitro</i> experiments were identified after plating on Ashdown agar, stored at -80°C and used for inoculation of modified RPMI medium (vi). <b>B)</b> Bacteria were plated after (i) nutrient limitation for 28 days (ii) or after (iv/3) <i>in vitro</i> and (iv/2) <i>in vivo</i> infection, respectively, on Ashdown agar.</p

Characteristics of the <i>Burkholderia pseudomallei</i> antigens used in this study.

<p>Characteristics of the <i>Burkholderia pseudomallei</i> antigens used in this study.</p

Rapid and Sensitive Multiplex Detection of <i>Burkholderia pseudomallei</i>-Specific Antibodies in Melioidosis Patients Based on a Protein Microarray Approach

<div><p>Background</p><p>The environmental bacterium <i>Burkholderia pseudomallei</i> causes the infectious disease melioidosis with a high case-fatality rate in tropical and subtropical regions. Direct pathogen detection can be difficult, and therefore an indirect serological test which might aid early diagnosis is desirable. However, current tests for antibodies against <i>B</i>. <i>pseudomallei</i>, including the reference indirect haemagglutination assay (IHA), lack sensitivity, specificity and standardization. Consequently, serological tests currently do not play a role in the diagnosis of melioidosis in endemic areas. Recently, a number of promising diagnostic antigens have been identified, but a standardized, easy-to-perform clinical laboratory test for sensitive multiplex detection of antibodies against <i>B</i>. <i>pseudomallei</i> is still lacking.</p><p>Methods and Principal Findings</p><p>In this study, we developed and validated a protein microarray which can be used in a standard 96-well format. Our array contains 20 recombinant and purified <i>B</i>. <i>pseudomallei</i> proteins, previously identified as serodiagnostic candidates in melioidosis. In total, we analyzed 196 sera and plasmas from melioidosis patients from northeast Thailand and 210 negative controls from melioidosis-endemic and non-endemic regions. Our protein array clearly discriminated between sera from melioidosis patients and controls with a specificity of 97%. Importantly, the array showed a higher sensitivity than did the IHA in melioidosis patients upon admission (cut-off IHA titer ≥1:160: IHA 57.3%, protein array: 86.7%; <i>p</i> = 0.0001). Testing of sera from single patients at 0, 12 and 52 weeks post-admission revealed that protein antigens induce either a short- or long-term antibody response.</p><p>Conclusions</p><p>Our protein array provides a standardized, rapid, easy-to-perform test for the detection of <i>B</i>. <i>pseudomallei</i>-specific antibody patterns. Thus, this system has the potential to improve the serodiagnosis of melioidosis in clinical settings. Moreover, our high-throughput assay might be useful for the detection of anti-<i>B</i>. <i>pseudomallei</i> antibodies in epidemiological studies. Further studies are needed to elucidate the clinical and diagnostic significance of the different antibody kinetics observed during melioidosis.</p></div

Comparative calculation of IHA and protein array sensitivity.^#

<p>Comparative calculation of IHA and protein array sensitivity.<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004847#t003fn001" target="_blank">^#</a></p

Average signal intensities of IgG antibodies bound to <i>B</i>. <i>pseudomallei</i> proteins probed with melioidosis-positive and negative control samples.

<p>The diagram shows the average signal intensity of each antigen (spotted protein solution of 0.45 mg/ml) incubated with sera from melioidosis-positive groups (week 0, 12 and 52 <i>p</i>.<i>a</i>.), healthy control individuals from endemic and non-endemic areas, as well as samples from patients with other bacteremia or fungaemia obtained in the non-endemic area of Greifswald. Not shown are values for antigens with His-tag. Error bars indicate standard error of the mean (SEM).</p