In vivo Detection of Bacteria Within a Biofilm Using a Point-of-care Fluorescence Imaging Device

Abstract Body: Introduction: Biofilm is bacteria that exists within a protective matrix, making it particularly challenging to eradicate through antimicrobial and antibiotic strategies. Biofilms must be identified and mechanically disrupted to release the bacteria, yet they are challenging to detect with standard assessment. This study tested the ability of a bacterial fluorescence imaging device* to detect bacteria from within a biofilm using a well-established in vivo biofilm model1.  Methods: A mouse wound model (n=16) was inoculated (day 0) with polymicrobial biofilms (3 biofilms1 per mouse, each with 1:1:1 parts S. aureus/E. coli/E. cloacae, 107 CFU/biofilm) that had been grown in vitro. Wounds were imaged daily for fluorescence (bacteria >104 CFU/g fluoresce red2) up to day 4, when wounds were excised, washed and re-imaged, and sent for microbiology (n=8), and histopathology (gram and matrix staining, n=4) and scanning electron microscopy (SEM, n=4) to confirm bacteria/matrix interaction.  Results: Red fluorescence was readily detected from 100% of inoculated wounds by day 1 and intensified up to day 4. Red fluorescence persisted after the excised wound tissue was washed to remove any planktonic bacteria. Microbiology confirmed high bacterial loads and SEM confirmed bacteria-matrix interaction (biofilm). Histopathologic analysis is ongoing.  Conclusions: This study is the first to demonstrate fluorescence detection of bacterial biofilm in vivo. This was achieved using a handheld device which visualized regions of high bacterial load at the point-of-care. These findings have implications for clinician targeting of their mechanical disruption (e.g. debridement) of biofilm, and may facilitate improved biofilm removal in the 50-90% of chronic wounds estimated to harbour bacterial biofilms.