Abstract
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.
Highlights
Burkholderia mallei are Gram negative, facultative intracellular bacteria, and the causative agent for glanders
Bacteria were grown in the presence or absence of antibiotic to ensure that selection was not necessary to maintain the transposon stability
The CSM001 strain sub-cultured in the presence of antibiotic exhibited the same level of luminescence as those sub-cultured in the absence of antibiotic
Summary
Burkholderia mallei are Gram negative, facultative intracellular bacteria, and the causative agent for glanders. B. mallei is highly infectious in aerosol form, requiring very few organisms to establish an infection (Lever et al, 2003). B. mallei is regarded as a potential biological threat due to their highly infectious nature and the incapacitating, often fatal disease progression in humans. Despite the history of B. mallei as a bio-weapon (Sharrer, 1995; Christopher et al, 1997), little data about the efficacy of vaccinations or antibiotic treatment methods is available. There are no vaccines available for either equine or human B. mallei infections. Due to the potential use as a biological weapon and the scarcity of in vivo treatment data, further evaluation of glanders progression in response to antibiotic challenge and/or vaccination is of utmost importance
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