Identification of Growth Promoting Effect of r BCG/BCG Culture Supernatant and Its Potential Applications

Abstract

Background: Although BCG is the most widely\radministered vaccine in the world, there have never been as many cases of TB as\rthere are now. Globally, more than 8.8 million people developed active TB and\r1.4 million—many\rof them—died in 2010. It\ris estimated that half of pulmonary TB cases arise from latent Mtb infection, making the study of\rlatency and reactivation of utmost importance. Methods: Widely administered BCG vaccines and a gene modified recombinant BCG (rBCG) strain, AERAS-422, were used as models to investigate the\rgrowth promoting function of resuscitation-promoting factors (Rpfs) in different bacilli culture phases.\rDifferent supernatant fractions were prepared by ultrafiltration, and the\rpromoting function of each fraction containing secreted Rpf(s) was evaluated by\rgrowth curve monitoring and colony counting on 7H10 agar plates. Results: The promoting effect of\rculture supernatants was mainly associated with the high molecular weight\rfraction (>30 kDa), which stimulated bacterial growth, but did not extend the exponential\rphase of stimulated culture. Anti-RpfB antibody showed significant growth\rrestriction of the tested cultures. When comparing rBCG cultures containing 7H9 medium, the 10 - 30 kDa fraction, or the >30 kDa\rfraction, only the >30 kDa fraction was displayed with down-regulation of\rthe secretion of RpfC, D and E. In colony counting tests, the plates containing\rthe >30 kDa fraction had total countable colony numbers 2 to 3 fold higher\rthan the plates with the 10 - 30 kDa fraction,\rand colonies appeared one to two weeks earlier than on the regular plates. The\rpotential applications of the prepared supernatant fractions containing RpfA\rand RpfB are discussed, which may include accelerating diagnosis of Mtb infection and future TB vaccine\rdevelopment.