Fluorogenic Probes with Substitutions at the 2 and 7 Positions of Cephalosporin are Highly BlaC‐Specific for Rapid Mycobacterium tuberculosis Detection

Abstract

Current methods for the detection of Mycobacterium tuberculosis (Mtb) are either time consuming or require expensive instruments and are thus are not suitable for point-of-care diagnosis. The design, synthesis, and evaluation of fluorogenic probes with high specificity for BlaC, a biomarker expressed by Mtb, are described. The fluorogenic probe CDG-3 is based on cephalosporin with substitutions at the 2 and 7 positions and it demonstrates over 120 000-fold selectivity for BlaC over TEM-1 Bla, the most common β-lactamase. CDG-3 can detect 10 colony-forming units of the attenuated Mycobacterium bovis strain BCG in human sputum in the presence of high levels of contaminating β-lactamases expressed by other clinically prevalent bacterial strains. In a trial with 50 clinical samples, CDG-3 detected tuberculosis with 90 % sensitivity and 73 % specificity relative to Mtb culture within one hour, thus demonstrating its potential as a low-cost point-of-care test for use in resource-limited areas.

New directions using reporter enzyme fluorescence (REF) as a tuberculosis diagnostic platform

“…In our own laboratory, we have targeted diagnostic strategies, since they have the potential to change the TB management landscape due to the fact that current diagnostic methods often cannot make a diagnosis prior to transmission [1,15]. Recent optical imaging technological developments have created more sensitive ways to visualize and quantify mycobacteria within cells and in vivo [2,3,6–9,12,21]. Despite these advances, reduction in signal due to tissue depth make application to diagnosis in humans extremely difficult.…”

“…REF is very sensitive because it uses the robust catalytic beta-lactamase enzyme produced by M. tuberculosis called BlaC to cleave custom fluorogenic substrates designed to be specific for this unique enzyme [3,21]. REF technology should produce complementary results as compared to existing diagnostic technologies because it requires viability of the bacteria due to the need for ATP in twin-arginine translocation (Tat) secretion of BlaC, but does not require complete bacterial replication as would be required for culture-based diagnosis.…”

“…In order to determine the best wavelengths for our probe, designated CDG-3 [3], we scanned the excitation and emission of the substrate before and after exhaustive cleavage by BlaC (Figure 1) in 2-(N-morpholino)ethanesulfonic acid (MES) buffer as described previously [3]. Using the emission wavelength of 532 nm, we found that the best excitation wavelength was around 490 nm, giving a two- to three-fold difference in signal between the fully cleaved and un-cleaved substrate.…”

“…Once we had determined optimal excitation and emission conditions for CDG-3, we compared the sensitivity of this new substrate [3] to our first generation substrate CDG-OMe [21]. M. tuberculosis var.…”

“…M. tuberculosis var. bovis strain bacillus Calmette-Guérin (BCG) was used in REF assays [3]. BCG was cultured in 7H9 broth (Difco, Detroit, MI) supplemented with 0.5% glycerol, 10% oleic acid albumin dextrose complex (OADC) and 0.05% Tween 80.…”