FIG. 3.

Quantitation of M. catarrhalis and S. pyogenes coaggregation. (A) biotinylated (+bMc, gray bar) or nonbiotinylated (+M, dark bar) M. catarrhalis was incubated with S. pyogenes or S. aureus. Nonbiotinylated S. pyogenes (+Sp, dark bar) or biotinylated S. pyogenes (+bSp, gray bar) was incubated with M. catarrhalis. Avidin-conjugated magnetic beads were added, and biotinylated bacteria were purified by extensive washing on a magnetic separator. Purified aggregates were serially diluted and plated on appropriate media. The bars represent CFU of the nonbiotinylated bacteria recovered after purification. Error bars represent the standard deviation of three independent experiments. In each case, the difference between biotinylated and nonbiotinylated bacteria was significant (P < 0.001) as determined by Student's t test. More than 30-fold more S. pyogenes was recovered with the streptavidin-magnetic beads in the presence of biotinylated M. catarrhalis (gray bars) than in the presence of nonbiotinylated M. catarrhalis (dark bars). No enhancement of binding was observed with M. catarrhalis and S. aureus (data not shown). Biotinylated S. pyogenes (gray bar) was also able to enhance the recovery of nonbiotinylated M. catarrhalis (dark bar) by 100-fold. (B) S. pyogenes strains (gray bars) were biotinylated and incubated with M. catarrhalis O35E, and coaggregation was measured as described above. M. catarrhalis was also incubated with beads in the absence of S. pyogenes (dark bar). M. catarrhalis coaggregated with each of the S. pyogenes strains. (C) Wild-type (WT) and adhesin mutant M. catarrhalis O35E bacteria were incubated with biotinylated S. pyogenes 1881, and coaggregation was measured as described above. Wild-type M. catarrhalis O35E was also incubated with beads in the absence of S. pyogenes (dark bar). All of the M. catarrhalis adhesin mutants coaggregated with S. pyogenes, suggesting that these adhesin proteins do not mediate coaggregation of these bacteria.