Bacterial enumeration at moribund disease for respiratory melioidosis models.

<p>Groups of 5 female albino C57BL/6J mice were infected by either the i.n. (10^5.1 CFU) or IMIT (10^4.6 CFU) routes of infection and euthanized at moribund disease endpoints. Bacteria were enumerated from tissues homogenized in 1 ml PBS, from a 1 ml PBS BAL collection, or from cardiac-drawn blood. Bacterial burden was calculated as CFU/tissue (lung, liver, and spleen) or bacteria per ml of body fluid (BAL and blood). Significant differences between log transformed data were evaluated by 2-way ANOVA with Bonferroni multiple comparisons (n.s., not significant; *, p<0.05; **, p<0.01; ****, p<0.0001).</p

Bacterial enumeration of <i>B. pseudomallei</i> mutants in moribund respiratory disease.

<p>Groups of 5 female albino C57BL/6J mice were infected with wild type JW280 (10^4.2 CFU), Δ<i>wcb</i> capsule mutant (10^6.0 CFU), or Δ<i>sctU</i>Bp3 T3SS3 mutant (10^6.5 CFU), and euthanized at moribund disease endpoints. Bacteria were enumerated from tissues <i>ex vivo</i> by optical imaging (left Y-axis: cps/tissue) with presentation of the estimated tissue CFU burdens based on calculated tissue-specific cps:CFU correlation (right Y-axis: CFU/tissue est.) for lung (A), liver (B) and spleen (C). The 95% LOD was calculated as a technical background luminescence and indicated as a dotted horizontal line. Data points below the 95% LOD were set to the 95% LOD value. Significant differences (1-way ANOVA with Tukey posttest) between log transformed data sets are indicated with an adjoining line (*, p<0.05; **, p<0.01; ***, p<0.001).</p

Host response to lung-specific delivery of <i>B. pseudomallei</i>.

<p>Groups of 5 female albino C57BL/6J mice were challenged with increasing doses of luminescent <i>B. pseudomallei</i> strain JW280 by either the intranasal (A) or IMIT (B) routes of infection. Mice were monitored for 14 days (336 hr) for disease progression and euthanized at the onset of moribund disease presentation. Survival curves are used to present the dose-dependent response of the host to increasing bacterial challenges. The MTTD was calculated for groups with ≥50% mortality, as indicated.</p

Detection of pulmonary growth rates of <i>B. pseudomallei</i> mutants <i>in vivo</i>.

<p>Groups of 5 female albino C57BL/6J mice were infected with wild type JW280 (10^4.2 CFU), Δ<i>wcb</i> capsule mutant (10^6.0 CFU), or Δ<i>sctU</i>Bp3 T3SS3 mutant (10^6.5 CFU), and monitored by optical diagnostic imaging once to twice daily. ROIs from the dorsally-imaged thoracic cavity were plotted as a function of infection time for each mutant. The 95% LOD was calculated for the background luminescence of uninfected mice and indicated as a dotted horizontal line. The calculated doubling rate of bioluminescent signal of each strain is indicated.</p

Host response to capsule and T3SS3 mutants of <i>B. pseudomallei</i>.

<p>Groups of 5 female albino C57BL/6J mice were challenged by the IMIT route of infection with increasing doses of either Δ<i>wcb</i> capsule mutant (A) or Δ<i>sctU</i>_Bp3 T3SS3 mutant (B) in the luminescent <i>B. pseudomallei</i> strain JW280 background. Mice were monitored for 14 days (336 hr) for disease progression and euthanized at the onset of moribund disease presentation. Survival curves are used to present the dose-dependent response of the host to increasing bacterial challenges of <i>B. pseudomallei</i> mutants. The MTTD was calculated for groups with ≥50% mortality, as indicated.</p

Detection of dissemination of <i>B. pseudomallei</i> mutants by optical imaging.

<p>Groups of 5 female albino C57BL/6J mice were challenged by the IMIT route of infection with either wild type luminescent <i>B. pseudomallei</i> strain JW280, the Δ<i>wcb</i> capsule mutant, or the Δ<i>sctU</i>_Bp3 T3SS3 mutant. Representative images of disease endpoints are presented, with uniform image settings adjusted to a range of 2.5×10³ to 3×10⁴ p/s/cm2/sr on a logarithmic scale.</p

Male host response to lung-specific delivery of <i>B. pseudomallei</i>.

<p>Groups of 3 male albino C57BL/6J mice were challenged with increasing doses of luminescent <i>B. pseudomallei</i> strain JW280 by IMIT infection. Mice were monitored for 14 days (336 hr) for disease progression and euthanized at the onset of moribund disease presentation. Survival curves are used to present the dose-dependent response of the host to increasing bacterial challenges. The MTTD was calculated for groups with ≥50% mortality, as indicated.</p

Genetic organization of the ATCC 43816 K2 capsule locus.

<p>Scale representation of the capsular polysaccharide biosynthetic locus (<i>galF-uge</i>) and LPS locus (<i>wzm-yvet</i>). The central domain of the capsule locus (<i>orf7-orf13</i>) represents the antigenic diversity region unique to K2 serotype capsules, while the remainder of the locus is well conserved with other <i>K. pneumoniae</i> capsule loci.</p

Growth potential of <i>K. pneumoniae</i> strains in cultured murine macrophages.

<p><i>K. pneumoniae</i> strains ATCC 43816 (K2), NTUH-K2044 (K1) and MGH 78578 (K52) were incubated in the presence of cultured murine macrophage cell lines J774A.1 or RAW264.7 at an MOI of 10 in 96 well plates. At one hour post-infection, gentamicin was introduced to eradicate extracellular bacteria. At 3, 4.5, 6, 9, and 12 hr post-infection, a triplicate set of samples was processed for enumeration of intracellular bacteria. The data is representative of at least three independent trials.</p

Uptake of <i>K. pneumoniae</i> wild type and capsular polysaccharide mutants strains into cultured murine macrophages.

<p><i>K. pneumoniae</i> strains ATCC 43816 (K2), NTUH-K2044 (K1) and MGH 78578 (K52) or capsule mutants ATCC Δ<i>manC</i>, and NTUH Δ<i>wzc</i> were incubated in the presence of cultured murine macrophage cell lines J774A.1 or RAW264.7 at an MOI of 10 in 96 well plates. At one hour post-infection, gentamicin was introduced to eradicate extracellular bacteria, and uptake of <i>K. pneumoniae</i> strains into macrophages was assessed at 3 hr post-infection by plate counting. Triplicate samples were enumerated and data analyzed as a percentage of the inoculum, with the results representative of at least two independent trials.</p