Gastrointestinal bacterial burden following oral <i>B. pseudomallei</i> challenge.

<p>BALB/c mice (n = 8–9) were inoculated orally with 5×10⁵ CFU <i>B. pseudomallei</i> strain 1026b. On day 3, 14 and 56 after inoculation, mice were euthanized and organs were processed for determination of bacterial burden as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037324#s4" target="_blank">Materials and Methods</a>. Data are presented as individual log₁₀ CFU/organ values with bars representing the mean titer for each organ. The limit of detection was 20 CFU/organ. Data were pooled from two independent experiments.</p

<i>B. pseudomallei</i> is persistently shed in feces following i.n. inoculation, but not after s.c. or i.p. inoculation.

<p>BALB/c mice were inoculated with <i>B. pseudomallei</i> using the following route and dose combinations: Oral inoculation (5×10⁵ CFU) (n = 10–18 animals), i.n. inoculation (∼500 CFU) (n = 9–17 animals), s.c. inoculation (5×10⁴ CFU–5×10⁷ CFU) (n = 11–24 animals), or i.p. inoculation (10⁶ CFU–10⁸ CFU) (n = 11–12 animals). On day 3, 14, 35 and 56 after infection, fecal pellets were collected and processed for determination of bacterial burden. Data are graphed as individual log₁₀ CFU/gram values with bars representing the mean value for each time point. Oral fecal shedding data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037324#pone-0037324-g002" target="_blank">figure 2</a> are reproduced in this figure for reference. Data were pooled from 2–6 experiments per infection route.</p

Gastric colonization following oral infection with different <i>B. pseudomallei</i> isolates.

<p>BALB/c mice were infected orally with <i>B. pseudomallei</i> strain Bp1026b (5×10⁵ CFU), Bp2671a (2.0×10⁴ CFU), Bp2685a (4.8×10⁴ CFU) or Bp2719a (2.8×10⁴ CFU). Stomach tissues were collected from Bp1026b mice 56 days after infection, Bp2671a mice 21 days after infection, Bp2685a mice 3 days after infection, and Bp2719a mice 4 days after infection. All tissues were fixed in 10% NBF and embedded in paraffin before sectioning. FISH was performed on stomach tissue sections from mice infected with Bp1026b (A), Bp2671a (B), Bp2685a (C) and Bp2719a (D) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037324#s4" target="_blank">materials and methods</a>. Tissues were counterstained with DAPI (blue), and observed at 1000× final magnification. Tissue sections were hybridized with a eubacterial probe (green) and two <i>B. pseudomallei</i> specific probes (red). (E) The location of 1000× fields positive for <i>B. pseudomallei</i> was determined for each <i>B. pseudomallei</i> isolate. Positive 1000× fields are indicated by red (Bp1026b), yellow (Bp2671a), green (Bp2685a) or blue (Bp2719a) shaded outlines. Outlines were overlaid onto a representative stomach image created by combining images from a hematoxylin and eosin stained section. The esophagus (esoph.), body (corpus) and pylorus of the stomach are labeled for reference. In (A–D) the scale bar represents 10 microns, and in (E) the scale bar represents 2 mm.</p

GI organs colonized more heavily and more frequently than extra-intestinal organs after i.n. challenge.

<p>BALB/c mice (n = 11 animals per group) were infected via the i.n. route with ∼500 CFU Bp1026b. After 21 days, blood, organs and feces were processed for determination of bacterial burden. (A) Bacterial burden in each organ following low-dose i.n. infection. Data are graphed as individual values with bars representing the mean log₁₀ titer. Organ titers are plotted as log₁₀ CFU/organ, blood as log₁₀ CFU/ml, and feces as log₁₀ CFU/gram. Statistical differences between organs were determined using a one-way ANOVA followed by a Tukey's multiple means test. (*** = <i>p</i><0.001). (B) The percentage of mice with positive <i>B. pseudomallei</i> cultures from each organ was determined. Statistical differences between percentages in the intestine versus other organs were determined using a two-tailed Fisher's exact test. The limit of detection was 1 CFU/organ, 10 CFU/ml for blood, and 10–60 CFU/gram for feces, depending on the number of fecal pellets collected from each mouse. Both figures were generated by pooling data from 3 independent experiments. ns = not significant.</p

<i>B. pseudomallei</i> is persistently shed in the feces following oral inoculation.

<p>BALB/c mice (n = 10–18 animals per group) were inoculated orally with 5×10⁵ CFU. On days 3, 14, 35 and 56 after inoculation, feces were collected and processed for determination of bacterial burden as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037324#s4" target="_blank">Materials and Methods</a>. Data are presented as individual log₁₀ CFU/gram values with bars representing the mean titer at each time point. The limit of detection was 10–60 CFU/gram, depending on the number of fecal pellets collected from each mouse. Data was pooled from two independent experiments.</p

Histology in gastrointestinal organs following oral <i>B. pseudomallei</i> infection.

<p>BALB/c mice were in inoculated with 5×10⁵ CFU <i>B. pseudomallei</i> 1026b. Mice were euthanized 56 days after infection and tissues were processed and stained with hematoxylin and eosin as described in methods. Tissues were also collected from uninfected BALB/c mice as control tissues. Stomach (A), small intestine (B), cecum (C) and colon (D) images from uninfected BALB/c mice are shown in the left column. Stomach (E), small intestine (F), cecum (G) and colon (H) images from <i>B. pseudomallei</i> infected mice are shown in the right hand column. For stomach images (A, E) the location of each H+E image within the stomach is indicated by the star on the stomach outline shown in the bottom left corner of each image. All small intestine images are from the ileum, and the colon images are from the proximal colon. Images were captured at 400× final magnification, and the scale bar on all images represents 25 microns.</p

Bacterial dissemination to systemic organs following oral inoculation with <i>B. pseudomallei</i>.

<p>BALB/c mice (n = 8–10 animals per time point) were inoculated orally with 5×10⁵ CFU Bp1026b. On day 3, 14 and 56 after inoculation mice were euthanized and organs were processed for determination of bacterial burden. Data are presented as mean ± SEM log₁₀ values. Lung, liver and spleen titers are graphed as CFU/organ, and blood is graphed as CFU/ml. The limit of detection was 20 CFU/organ, and 10 CFU/ml for blood. Data were pooled from two independent experiments.</p

Persistent gastrointestinal colonization following oral infection.

a<p>ID₅₀ values calculated according to the Reed-Muench method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037324#pone.0037324-Reed1" target="_blank">[101]</a>.</p

<i>B. pseudomallei</i> 1710b malleobactin mutants cannot use lactoferrin as an iron source A.–F.

<p>Microtiter plates containing 200 µl of M9-glucose minimal medium with 200 µM 2,2′-dipyridyl (open white symbols), or 200 µM 2,2′-dipyridyl and 1 µM lactoferrin (purple symbols) were inoculated with strain 1710b (<b>A.</b>) and its siderophore synthesis and hemin uptake mutants (<b>B.</b> ΔPCH, <b>C.</b> ΔMBA, <b>D.</b> Δ141-kb, <b>E.</b> Δ141-kb ΔPCH and <b>F.</b> Δ141-kb ΔPCH ΔHMU ΔHEM). The optical density at 600 nm (OD₆₀₀) was measured hourly. OD₆₀₀ means and standard deviation of three cultures from a single experiment are shown for all strains.</p

<i>B. pseudomallei</i> 1710b malleobactin mutant strains mimic the siderophore phenotypes of the <i>B. pseudomallei</i> 708a clinical isolate.

<p><b>A</b>. <i>Burkholderia</i> GBrowse map of the <i>B. pseudomallei</i> 1710b genomic region corresponding to the extent of the deletion found in the <i>B. pseudomallei</i> 708a clinical isolate <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001715#pntd.0001715-Trunck1" target="_blank">[32]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001715#pntd.0001715-Winsor1" target="_blank">[62]</a>. The extents of the <i>amrRAB-oprA</i> genes ecoding the AmrAB-OprA efflux pump and AmrR repressor and the malleobactin synthesis gene cluster (<i>mbaS-mbaF</i>) are noted with horizontal green lines. Vertical black lines and gene locus numbers indicate the borders of 1710b genomic region deletions constructed in this study. The shorthand nomenclatures for strains indicating the genomic region deletions contained in them are bolded. <b>B.</b> Quantitative CAS siderophore assays indicate similar amounts of secondary siderophore production by 1710b malleobactin minus strains and <i>B. pseudomallei</i> 708a. Supernatants from overnight cultures grown in low-iron TSBFC medium were tested by quantitative CAS assays for siderophore production adjusted for cell density by OD₆₀₀ of a 1∶10 dilution. Means and standard deviations of two measurements each from three independent experiments are shown. <b>C.</b> Bacterial colony and CAS halo diameters were measured daily for 4 days on CAS agar plates spotted and incubated as described above. Red bars indicate colony diameter and blue bars halo diameter. Means and standard deviations of two measurements each from three independent experiments are shown. <b>D.</b> CAS plate assays indicate similar secondary siderophore production by 1710b malleobactin deficient strains and <i>B. pseudomallei</i> 708a. Five µl samples of overnight cultures grown in low-iron TSBFC medium were spotted onto CAS agar plates and incubated at 37°C for 4 days prior to photographing.</p