In vivo Bioluminescence Imaging of Burkholderia mallei Respiratory Infection and Treatment in the Mouse Model

Bioluminescent imaging (BLI) technology is a powerful tool for monitoring infectious disease progression and treatment approaches. BLI is particularly useful for tracking fastidious intracellular pathogens that might be difficult to recover from certain organs. Burkholderia mallei, the causative agent of glanders, is a facultative intracellular pathogen and has been classified by the CDC as a Category B select agent due to its highly infectious nature and potential use as a biological weapon. Very little is known regarding pathogenesis or treatment of glanders. We investigated the use of bioluminescent reporter constructs to monitor the dynamics of infection as well as the efficacy of therapeutics for B. mallei in real-time. A stable luminescent reporter B. mallei strain was created using the pUTmini-Tn5::luxKm2 plasmid and used to monitor glanders in the BALB/c murine model. Mice were infected via the intranasal route with 5 × 103 bacteria and monitored by BLI at 24, 48, and 72 h. We verified that our reporter construct maintained similar virulence and growth kinetics compared to wild-type B. mallei and confirmed that it maintains luminescent stability in the presence or absence of antibiotic selection. The luminescent signal was initially seen in the lungs, and progressed to the liver and spleen over the course of infection. We demonstrated that antibiotic treatment 24 h post-infection resulted in reduction of bioluminescence that can be attributed to decreased bacterial burden in target organs. These findings suggest that BLI can be used to monitor disease progression and efficacy of therapeutics during glanders infections. Finally, we report an alternative method to mini-Tn5::luxKm2 transposon using mini-Tn7-lux elements that insert site-specifically at known genomic attachment sites and that can also be used to tag bacteria

Comparing in vitro and in vivo virulence phenotypes of Burkholderia pseudomallei type G strains.

Burkholderia pseudomallei (Bpm) is a saprophytic rod-shaped gram-negative bacterium and the causative agent of melioidosis. This disease has previously been described as endemic in areas such as northern Australia and Southeast Asia, but, more recently, a better understanding of the epidemiology of melioidosis indicated that the disease is distributed worldwide, including regions of the Americas and Africa. A 16S-23S rDNA internal transcribed spacer (ITS) typing system has been developed for Bpm and has revealed that ITS types C, E, and hybrid CE are mainly associated with Australia and Southeast Asia while type G strains are more associated with cases of melioidosis in the Western Hemisphere. The purpose of the current study was to determine the in vitro and in vivo virulence profiles of the understudied Bpm type G strains Ca2009, Ca2013a, Mx2013, and 724644 and compared such phenotypes to the commonly studied Bpm type C strain K96243. We evaluated virulence by measuring invasion/uptake and survival of these Bpm strains in murine respiratory epithelial LA-4 cells and alveolar macrophage MH-S cells using different multiplicity of infections (MOIs of 1 and 10). We also calculated the lethal dose 50 values (LD50) in BALB/c mice that were inoculated intranasally with either Ca2009, Ca2013a, or Mx2013. Overall, the virulence and lethality phenotypes of Bpm type G strains were similar to the Bpm type C strain K96243. Additional comparative analyses between the Bpm ITS types may lead to a better understanding of the contribution of the ITS type to the epidemiology and ecology of Bpm strains

Characterization of the <i>Burkholderia mallei tonB</i> Mutant and Its Potential as a Backbone Strain for Vaccine Development

<div><p>Background</p><p>In this study, a <i>Burkholderia mallei tonB</i> mutant (TMM001) deficient in iron acquisition was constructed, characterized, and evaluated for its protective properties in acute inhalational infection models of murine glanders and melioidosis.</p><p>Methodology/Principal Findings</p><p>Compared to the wild-type, TMM001 exhibits slower growth kinetics, siderophore hyper-secretion and the inability to utilize heme-containing proteins as iron sources. A series of animal challenge studies showed an inverse correlation between the percentage of survival in BALB/c mice and iron-dependent TMM001 growth. Upon evaluation of TMM001 as a potential protective strain against infection, we found 100% survival following <i>B</i>. <i>mallei</i> CSM001 challenge of mice previously receiving 1.5 x 10⁴ CFU of TMM001. At 21 days post-immunization, TMM001-treated animals showed significantly higher levels of <i>B</i>. <i>mallei</i>-specific IgG1, IgG2a and IgM when compared to PBS-treated controls. At 48 h post-challenge, PBS-treated controls exhibited higher levels of serum inflammatory cytokines and more severe pathological damage to target organs compared to animals receiving TMM001. In a cross-protection study of acute inhalational melioidosis with <i>B</i>. <i>pseudomallei</i>, TMM001-treated mice were significantly protected. While wild type was cleared in all <i>B</i>. <i>mallei</i> challenge studies, mice failed to clear TMM001.</p><p>Conclusions/Significance</p><p>Although further work is needed to prevent chronic infection by TMM001 while maintaining immunogenicity, our attenuated strain demonstrates great potential as a backbone strain for future vaccine development against both glanders and melioidosis.</p></div

Attenuated virulence of TMM001 is partially rescued by iron supplementation.

<p>Mice (n = 8) were challenged i.n. with 1.5 x 10⁵ CFU (solid circle/open circle), 1.5 x 10⁶ CFU (solid square/open square) or 1.5 x 10⁷ CFU (solid triangle/open triangle) of TMM001 grown in LBG with (open) or without (closed) 200 μM FeSO₄. The statistical significance of differences in survival times was determined by plotting Kaplan-Meier curves, followed by a log rank test. ^★★★★ p ≤ 0.0001.</p

<i>B</i>. <i>mallei-</i>specific immunoglobulin levels in TMM001- vs PBS-treated mice before challenge.

<p>Murine serum samples were taken 21 days post-immunization, diluted 1:10,000 and analyzed for <i>B</i>. <i>mallei-</i>specific IgG1 (A), IgG2a (B) and IgM (C). Mean ± SEM of three representative animals is plotted. Statistical significance was determined by the unpaired <i>t</i> test with equal SD. ^★ p ≤ 0.05 ^★★★ p ≤ 0.001, ^★★★★ p ≤ 0.0001.</p

TMM001 (1.5x10⁴ CFU) provides 100% protection against CSM001 challenge.

<p>Mice were immunized i.n. with PBS (solid triangle), 1.5 x 10⁴ CFU (solid circle), 1.5 x 10³ CFU (solid inverted triangle), or 1.5 x 10² CFU (solid square) of TMM001. Three weeks later, mice were challenged with 1.5 x 10⁵ CFU of CSM001. The statistical significance of differences in survival times was determined by plotting Kaplan-Meier curves, followed by a log rank test. ^★★★ p ≤ 0.001, ^★★ p ≤ 0.01.</p

Treatment with TMM001 reduces the pro-inflammatory cytokine and chemokine response to <i>B</i>. <i>mallei</i> challenge.

<p>Serum cytokine/chemokine profile of PBS- or TMM001-treated mice following exposure to <i>B</i>. <i>mallei</i> CSM001 (1.5x10⁵ CFU) at 0 h (A) and 48 h (B) post challenge. Mean ± SEM plotted are representative of three animals. Statistical significance was determined by one-way ANOVA followed by the Dunnett's test. ^★ p ≤ 0.05.</p

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</p

Colonization of target organs by TMM001 is partially rescued by iron supplementation.

<p>Bacterial burden in the lungs (A) and spleen (B) of mice infected with CSM001 and TMM001 grown ± 200 μM FeSO₄ at 24, 48 and 72 h post infection. Bars plotted with their SD represent the mean of three independent experiments. Significant differences in colonization at 24 and 48 h were individually ascertained via one-way ANOVA followed by Tukey’s multiple comparisons test. Significant difference in colonization at 72 h was extrapolated by using an unpaired <i>t</i> test with equal SD. ^★ p ≤ 0.05, ^★★★p ≤ 0.001, ^★★★★p ≤ 0.0001, ns = no significance.</p