Abstract
BackgroundBacteria can be selectively imaged in experimentally-infected animals using exogenously administered 1-(2′deoxy-2′-fluoro-β-D-arabinofuranosyl)-5-[125I]-iodouracil ([125I]-FIAU), a nucleoside analog substrate for bacterial thymidine kinase (TK). Our goal was to use this reporter and develop non-invasive methods to detect and localize Mycobacterium tuberculosis.Methodology/Principal Findings We engineered a M. tuberculosis strain with chromosomally integrated bacterial TK under the control of hsp60 - a strong constitutive mycobacterial promoter. [125I]FIAU uptake, antimicrobial susceptibilities and in vivo growth characteristics were evaluated for this strain. Using single photon emission computed tomography (SPECT), M. tuberculosis Phsp60 TK strain was evaluated in experimentally-infected BALB/c and C3HeB/FeJ mice using the thigh inoculation or low-dose aerosol infection models. M. tuberculosis Phsp60 TK strain actively accumulated [125I]FIAU in vitro. Growth characteristics of the TK strain and susceptibility to common anti-tuberculous drugs were similar to the wild-type parent strain. M. tuberculosis Phsp60 TK strain was stable in vivo and SPECT imaging could detect and localize this strain in both animal models tested.ConclusionWe have developed a novel tool for non-invasive assessment of M. tuberculosis in live experimentally-infected animals. This tool will allow real-time pathogenesis studies in animal models of TB and has the potential to simplify preclinical studies and accelerate TB research.
Highlights
Current tools for monitoring tuberculosis (TB) in preclinical animal studies are costly and time consuming
We have developed a novel tool for non-invasive assessment of M. tuberculosis in live experimentally-infected animals
M. tuberculosis strains and media M. tuberculosis H37Rv and M. tuberculosis Phsp60 thymidine kinase (TK) strains were grown to mid-log phase in plastic roller bottles or as shaken cultures in plastic tubes at 37uC in Middlebrook 7H9 liquid broth (Difco Laboratories) supplemented with oleic acid albumin dextrose catalase (OADC) (Becton Dickinson), 0.5% glycerol, and 0.05% Tween 80
Summary
Current tools for monitoring tuberculosis (TB) in preclinical animal studies are costly and time consuming. While mouse chemotherapy is generally concordant with human studies, and animal-to-animal variability is low owing to the availability of inbred strains, a significant drawback of the standard mouse model is the lack of caseation – the hallmark of human disease. It may be essential to perform cross-species studies in larger animal models such as guinea pigs, rabbits, non-human primates (NHP) which develop microenvironments relevant to human TB [1]. Animal-to-animal variability is a more serious concern with these larger, more expensive species, which are generally not available as inbred strains. The need for non-invasive biomarkers that can monitor response in the same group of animals and significantly reduce the numbers of animals required per study will be essential to cost-effectively conduct these studies in the larger and more expensive animals. Our goal was to use this reporter and develop non-invasive methods to detect and localize Mycobacterium tuberculosis
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