FIG 4.

Role of capsule in biofilm structure, biomass, and antibiotic resistance in strains lacking or expressing different capsule levels. (A and B) Biofilms of M3 strains expressing various capsule amounts, i.e., high (AM3 and SS-90), medium (SS-1271 and 94421), or low (950802 and 87-136), were formed over 48 (black) and 72 h (red) at 34°C on prefixed epithelial H292 cells and evaluated for biomass (A) or gentamicin killing (B) by measuring the log₁₀ CFU per ml or the log₁₀ death (i.e., the total biomass [CFU/ml] − biofilm biomass [CFU/ml] after treatment with 500 μg/ml gentamicin for 3 h), respectively. (C and D) Biofilms of M3 and M6 strains expressing wild-type capsule (WT) or lacking capsule (ΔhasA) were formed over 48 and 72 h at 34°C on prefixed epithelial H292 cells and evaluated for biomass (C) and gentamicin killing (D), as described above. Data are from three separate experiments with three individual biofilms each. Biomass (A and C) is plotted as individual data points (with SD; n = 9), and groups were compared using the Mann-Whitney U test. Gentamicin killing data (B and D) are means (with SD; n = 3), and groups were compared using Student's t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant difference. (E) Structure of biofilms formed by the M3ΔhasA mutant on different epithelial cells (respiratory H292 cells or SCC13 keratinocytes) for 72 h, viewed using SEM (bar = 10 μm). The underlying cell substratum is indicated by a green arrow, the red arrow highlights bacterial chains, and blue and yellow arrows indicate ECM aggregates alone and cells coated with ECM, respectively.