(A) Quantitative analyses of biofilm formation of clinical GAS necrotizing soft tissue infection isolates on uncoated or fibronectin-coated polystyrene surfaces at indicated time points. The box and whisker plots show median values with min to max distribution (n = 4). (B) Immunofluorescence images of 5626 strain forming biofilm (wheat germ agglutinin [WGA], DAPI, Nile red positive) on uncoated (left panel) and fibronectin-coated (right panel) glass surfaces (original magnification, ×60). Individual stainings are shown in Supplemental Figure 1A. Representative images from 1 of 4 experiments are shown. (C) Representative immunofluorescence images of GAS 800 infected tissue models (original magnification, ×40). GAS is shown in green, and bacterial aggregations are indicated by arrows. Mean fluorescent intensity (MFI) of stained tissue (D), and CFU counts of bacteria recovered from skin models (E). The data represent the mean values ± SD (n ≥ 3). (F) Blinded scoring of tissue pathology of the skin model after infection. Histological severity scoring was performed in a blinded fashion using the following criteria: 0, unaffected; 0.5–1, mild injury with minor epithelial loosening; 1.5–2, moderate injury with some epithelial disruption; 2.5–3, severe injury with continuous epithelial disruption and some detachment; and > 4, extensive injury, massive epithelial disruption, and detachment. Each symbol represents one independent experiment. Horizontal lines denote median values (n = 4). (G) 3D reconstruction of immunostained biofilm in 8157 infected model (original magnification, ×60). GAS-specific antibody, WGA, and Nile red were used. Arrows indicate multilayered bacterial aggregations. (H) SEM images of bacterial biofilm in the skin tissue model 48 hours after infection with indicated clinical isolates (original magnification, ×10,000). The level of significance was determined using 1-way ANOVA with Dunnett’s multiple comparison test.