Generation of Metabolically Diverse Strains ofStreptococcus pyogenesduring Survival in Stationary Phase

Abstract

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.