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Routinely used molecular diagnostic methods for mycobacterium identification are expensive and time-consuming. To tackle this problem, we develop a method to streamline identification of Mycobacterium tuberculosis complex (MTBC) in broth culture media by using detonation nanodiamonds (DNDs) as a platform to effectively capture the antigen secreted by MTBC which is cultured in BACTEC MGIT 960, followed by the analysis of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS). The 5 nm DNDs can capture the MTBC secretory antigen without albumin interference. With on diamond digestion, we confirm the DND captured antigen is cell filtrate protein 10 (CFP-10) because its Mascot analysis shows a score of 68. The dot blotting method further verifies a positive reaction with anti-CFP-10, indicating that CFP-10 is secreted in the medium of mycobacterium growth indicator tube (MGIT) and captured by DNDs. The minimal CFP-10 protein detection limit was 0.09 μg/mL. Furthermore, our approach can avoid the false-positive identification of MTBC by immunological methods due to cross-reactivity. Five hundred consecutive clinical specimens subjected to routine mycobacteria identification in hospital were used in this study, and the sensitivity of our method is 100% and the specificity is 98%. The analysis of each MTBC sample from culture solution can be finished within 1 h and thus shortens the turnaround time of MTBC identification of gold standard culture methods. In sum, DND MALDI-TOF MS for the detection of MTBC is rapid, specific, safe, reliable, and inexpensive.

Abstract:The high incidence of Mycobacterium infection, notably multidrug-resistant M. tuberculosis infection, has become a significant public health concern worldwide. In this study, we isolate and analyze a mycobacteriophage, BTCU-1, and a foundational study was performed to evaluate the antimycobacterial activity of BTCU-1 and its cloned lytic endolysins. Using Mycobacterium smegmatis as host, a mycobacteriophage, BTCU-1, was isolated from soil in eastern Taiwan. The electron microscopy images revealed that BTCU-1 displayed morphology resembling the Siphoviridae family. In the genome of BTCU-1, two putative lytic genes, BTCU-1_ORF7 and BTCU-1_ORF8 (termed lysA and lysB, respectively), were identified, and further subcloned and expressed in Escherichia coli. When applied exogenously, both LysA and LysB were active against M. smegmatis tested. Scanning electron microscopy revealed that LysA and LysB caused a remarkable modification of the cell shape of M. smegmatis. Intracellular bactericidal activity assay showed that treatment of M. smegmatis- OPEN ACCESSMolecules 2015, 20 19278 infected RAW 264.7 macrophages with LysA or LysB resulted in a significant reduction in the number of viable intracellular bacilli. These results indicate that the endolysins derived from BTCU-1 have antimycobacterial activity, and suggest that they are good candidates for therapeutic/disinfectant agents to control mycobacterial infections.