title: Reporter enzyme fluorescence (REF) imaging and quantification of tuberculosis in live animals.
abstract: The slow growth rate of Mycobacterium tuberculosis hinders research progress, since estimating the bacterial numbers present in all experiments normally relies on determination of colony forming units on agar plates. M. tuberculosis colonies can take as long as four to six weeks to become visible. Whole animal imaging is an emerging technology that has broad applications in all areas of biological sciences, including monitoring infections. Imaging allows bacterial numbers to be determined in real-time for each infected animal, individually, which allows inter-animal variability to be observed and controlled for. Reporter enzyme fluorescence (REF) utilizes custom substrates that allow production of a fluorescent product after cleavage by a bacterial enzyme. In our recently published studies, we demonstrate that the enzyme β-lactamase, a naturally occurring enzyme expressed by M. tuberculosis, can be used for REF. The resulting imaging system is the first that allows non-invasive detection of natural M. tuberculosis strains directly in pulmonary infected living animals. Use of REF for M. tuberculosis infected mice allows detection of ~10⁴ CFU in the lungs, which is very sensitive. This system also displays promise for allowing rapid evaluation of differences in virulence strains and efficacy of therapeutics and vaccines. This system could be developed into a diagnostic tool for tuberculosis through the use of REF to identify infected tissues or other diagnostic specimens.
authors.affiliation: Department of Microbial and Molecular Pathogenesis, Texas A&M Health Sciences Center, College Station, TX, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Kong
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Cirillo
mesh_terms: D000818:Animals; D003952:Diagnostic Imaging / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes; D008168:Lung / Q000382:microbiology; D051379:Mice; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000302:isolation & purification*; D014397:Tuberculosis, Pulmonary / Q000382:microbiology*; D001618:beta-Lactamases / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D001618:beta-Lactamases