Rapid identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry.

PURPOSE: The purpose of this study was to evaluate the identification of Mycobacterium tuberculosis which is often plagued with ambiguity. It is a time consuming process requiring 4-8 weeks after culture positivity, thereby delaying therapeutic intervention. For a successful treatment and disease management, timely diagnosis is imperative. We evaluated a rapid, proteomic based technique for identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). MATERIALS AND METHODS: Freshly grown mycobacterial isolates were used. Acetonitrile/trifluoroacetic acid extraction procedure was carried out, following which cinnamic acid charged plates were subjected to identification by MALDI-TOF MS. RESULTS: A comparative analysis of 42 clinical mycobacterial isolates using the MALDI-TOF MS and conventional techniques was carried out. Among these, 97.61% were found to corroborate with the standard methods at genus level and 85.36% were accurate till the species level. One out of 42 was not in accord with the conventional assays because MALDI-TOF MS established it as Mycobacterium tuberculosis (log (score)>2.0) and conventional methods established it to be non-tuberculous Mycobacterium. CONCLUSIONS: MALDI-TOF MS was found to be an accurate, rapid, cost effective and robust system for identification of mycobacterial species. This innovative approach holds promise for early therapeutic intervention leading to better patient care.

Expression of a functional single-chain antibody via Corynebacterium pseudodiphtheriticum.

Antibody-based therapeutics are effective against conditions ranging from acute infections to malignancy. They may prove crucial in combating bioterrorism and responding to drug-resistant and emerging pathogens. At present the cost of producing therapeutic monoclonal antibodies is between $1,000 to $6,000 per gram. The need to administer antibodies parenterally at frequent intervals further drives the cost of this treatment. Here we present an antibody delivery system, termed paratransgenesis, with the potential to overcome these limitations. The paratransgenic approach involves genetically transforming a commensal or symbiont bacterium to express foreign molecules that target pathogens. We describe transformation of Corynebacterium pseudodiptheriticum, a commensal bacterium found in the human respiratory tract, to express a murine single-chain antibody binding progesterone. The antibody was functional and bound specifically to progesterone in a concentration-dependent manner. This marker antibody system is the precursor to development of expression systems producing recombinant humanized single-chain antibodies. Studies are in progress evaluating fitness, transgene stablility, and pathogenecity of the genetically engineered C. pseudodiptheriticum. We anticipate developing a repertoire of expressed molecules targeting infectious agents and surface epitopes of pulmonary mass lesions. If expression systems for anti-pathogen molecules in C. pseudodiptheriticum and other respiratory commensal bacteria can be optimized, these bacteria have the potential for a range of therapeutic and prophylactic applications.