FIG 1.

Illustration of the reporter enzyme fluorescence (REF) assay used to evaluate clinical specimens. The M. tuberculosis β-lactamase, BlaC, is surface localized (∼90%) and secreted (∼10%) when the bacteria are grown under laboratory conditions in liquid medium (28), making it easily accessible to fluorogenic substrates for REF. The fluorogenic substrates CDG-1, CDG-OMe, and CDG-3 produce the fluorescent product Tokyo green after cleavage by BlaC. The catalytic activity of BlaC can be measured by detecting the presence of fluorescence using a plate reader or similar system that allows excitation at 492 nm and fluorescence emission at 515 or 535 nm. Although a 515-nm emission was used for all in vitro studies due to the maximal emission of Tokyo green at this wavelength, 535 nm was chosen for use with clinical specimens because this wavelength displays a lower autofluorescent background due to components of clinical sputum samples.