Quantitative analysis of in vivo mucosal bacterial biofilms

Abstract Background: Quantitative assays of mucosal biofilms on ex vivo samples are challenging using the currently applied specialized microscopic techniques to identify them. The COMSTAT2 computer program has been applied to in vitro biofilm models for quantifying biofilm structures seen on confocal scanning laser microscopy (CSLM). The aim of this study was to quantify Staphylococcus aureus ( S. aureus ) biofilms seen via CSLM on ex situ samples of sinonasal mucosa, using the COMSTAT2 program. Methods: S. aureus biofilms were grown in frontal sinuses of 4 merino sheep as per a previously standardized sheep sinusitis model for biofilms. Two sinonasal mucosal samples, 10 mm × 10 mm in size, from each of the 2 sinuses of the 4 sheep were analyzed for biofilm presence with Baclight stain and CSLM. Two random image stacks of mucosa with S. aureus biofilm were recorded from each sample, and analyzed using COMSTAT2 software that translates image stacks into a simplified 3‐dimensional matrix of biofilm mass by eliminating surrounding host tissue. Three independent observers analyzed images using COMSTAT2 and 3 repeated rounds of analyses were done to calculate biofilm biomass. Results: The COMSTAT2 application uses an observer‐dependent threshold setting to translate CSLM biofilm images into a simplified 3‐dimensional output for quantitative analysis. Intraclass correlation coefficient (ICC) between thresholds set by the 3 observers for each image stacks was 0.59 ( p = 0.0003). Threshold values set at different points of time by a single observer also showed significant correlation as seen by ICC of 0.80 ( p < 0.001). Conclusion: COMSTAT2 can be applied to quantify and study the complex 3‐dimensional biofilm structures that are recorded via CSLM on mucosal tissue like the sinonasal mucosa. © 2011 ARS‐AAOA, LLC.