Figure 1. Establishment of mixed-species biofilm infection in a full thickness porcine burn wound model.

Six 2 × 2 sq inch size burn wounds were created with a burning device (Fig S1). Co- infection of the burn wounds was performed day 3 post-burn with P. aeruginosa PAO1 and A. baumannii 19606. A, representative digital photograph of wounds on the day of burn, days 7 and 14 post-infection. Note signs of active infection with localized erythema, yellowish exudates, and friable wound edges on days 7 and 14 post-infection. B, scanning electron microscope (SEM) images of biopsies collected from the wounds on the day of burn and days 7 & 14 post-infection. The images clearly demonstrate a mix of extracellular tissue matrix, fibers, and red blood cells over the surface of burn wounds before bacterial inoculation. Large aggregates of rods attached to the wound surface encased in a layer of extracellular amorphous material was noted on days 7 & 14 post-infection. Upper panel, scale bar = 20 μm, 2500× magnification. Lower panel, magnification of the red dashed boxes in the upper panels. Scale bar = 5 μm, 10000× magnification. C, Gram stained images of inoculated wounds shows presence of bacterial aggregates. Upper panel, representative mosaic image of a day 7 post infection wound. Images collected under 400× magnification using microscope supported with a motorized stage. Scale bar = 200 μm. Es= eschar, Grn= granulation tissue. Lower panels, Zoom of three boxed area in upper panel image showing Gram negative bacilli & coccobacilli over the surface of the burn wound while large Gram negative clumps colonizing the wound tissues (yellow arrows). Scale bar = 50 μm. D, both P. aeruginosa and A. baumannii on the burn wounds were visualized using anti-pseudomonas (green) and anti-acinetobactor (red) antibody and confocal laser scanning (CLSM) microscopy. Merged (red & green) immunofluorescence images of day 7 (post-infection) wound biopsies show heavy colonization of wound tissues with both strains. Mosaic images were collected under 400× magnification using fluorescent microscope supported with a motorized stage. Scale bar = 100 μm. Z-stack images of boxed areas of the lower panel surface of burn wound tissues. Inset, zoom of the boxed area in merged image. The image was created by merging serial scans of thick tissue section (20 μm), viewed under 600× magnification in a confocal laser scanning microscope (CLSM). Dense micro-colonies of P. aeruginosa and A. baumannii with some co-localization in the wound tissues were noted. x/z and y/z planes display the thickness of the microbial clumps within the tissue section. E, representative SEM image of wound biopsies from human chronic pressure ulcers showing that the bacterial colonization and biofilm established in experimental porcine biofilm model is comparable to that of human chronic wounds. left panel, scale bar = 20 μm, 2500× magnification. Right panel, scale bar = 5 μm, 10000× magnification.