Generation of metabolically diverse strains of Streptococcus pyogenes during survival in stationary phase.

Streptococcus pyogenes, in addition to causing fulminant disease, can be carried asymptomatically and may survive in the host without causing disease. Long-term stationary-phase cultures were used to characterize the metabolism of cultures surviving after glucose depletion. Survival of stationary-phase cultures in glucose-depleted rich medium was truncated by switching the cells to phosphate-buffered saline or by the addition of antibiotics, suggesting that survival depended on the presence of nutrients and metabolic activity. The metabolites of the pyruvate-to-acetate (PA) pathway (acetate and formate) and amino acid catabolic pathways (ammonia) accumulated throughout long-term stationary phase (12 weeks). Acid and ammonia production was balanced so that the culture pH was maintained above pH 5.6. Strains isolated from long-term stationary-phase cultures accumulated mutations that resulted in unique exponential-phase metabolisms, with some strains expressing the PA pathway, some strains producing ammonia, and some strains expressing both in the presence of glucose. Strains expressing high levels of PA pathway activity during exponential growth were unable to survive when regrown in pure culture due to the production of excess acid. These data suggest that S. pyogenes diversifies during survival in stationary phase into distinct strains with different metabolisms and that complementary metabolism is required to control the pH in stationary-phase cultures. One of three survivor strains isolated from tonsillar discard material from patients expressed high levels of the PA pathway during exponential growth. Sequencing of multiple group A streptococcus regulators revealed two different mutations in two different strains, suggesting that random mutation occurs during survival.

Evaluation of the MDR-MDCK cell line as a permeability screen for the blood-brain barrier.

The objectives of this study were to (1) characterize MDR-MDCK monolayers as an in vitro model to predict brain uptake potential; (2) examine the ability of MDR-MDCK monolayers to identify the brain uptake potential of compounds that interact with P-glycoprotein (P-gp). The study measured the bi-directional transport of 28 compounds across MDR-MDCK monolayers. The brain uptake of a subset of the compounds was determined in the rat brain perfusion model. Drug concentrations were analyzed by LC-MS-MS. CNS-positive drugs exhibited absorptive permeability coefficients (Papp, A-B) values ranging from 3.4 x 10(-6) to 20.2 x 10(-6) cm/s; whereas CNS-negative drugs showed Papp (A-B) ranging from 0.03 x 10(-6) to 0.83 x 10(-6) cm/s. Inhibition of P-gp by cyclosporin A (CsA) significantly reduced secretory flux of compounds known to be P-pg substrates, but only enhanced the absorptive flux of compounds with high efflux ratio (>100). In vitro results were confirmed by brain perfusion studies on selected compounds. MDR-MDCK monolayers can be used to classify compounds into CNS-positive or CNS-negative based on the permeability coefficients (Papp, A-B). Under our experimental conditions, compounds with Papp (A-B)>3 x 10(-6) cm/s have high brain uptake potential; compounds with Papp (A-B)<1 x 10(-6) cm/s are unable to penetrate the blood-brain barrier (BBB); the brain uptake of compounds with Papp (A-B)<1 x 10(-6) cm/s and a P-gp-mediated efflux ratio of >100 may be enhanced by inhibiting P-gp.