Rapid fluorescence amplified biofilm detection

• Main Author: Herzog, Dylan Benjamin
• Other Authors: Festy, Frederic ; Watson, Timothy F.
• Published: King's College London (University of London) 2017
• Subjects: 579
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.724291

Bacterial biofilms are the cause of many diseases linked to persistent bacterial infection. When remaining in the root canal space following endodontic treatment, bacteria can lead to a secondary infection, resulting in treatment failure and the subsequent need for re-intervention. Currently, there are no methods in use to detect bacterial presence within the root canal space in a fast and reliable manner. Such methods would be especially beneficial in determining the end point of root canal treatments. Here, we make use of molecular fluorescent dyes and fluorescent microscopy/spectroscopy to optically detect remnant live bacteria within the root canal space. Calcein AM was evaluated as a fast acting vital cell stain, suitable for rapid staining of stressed and mature biofilms. Calcein AM staining produced fluorescence only when vital bacteria were present and showed significantly higher ratios for vital/non-vital bacteria as well as vital bacteria/ substrates when compared to other stains tested (p < 0.001). Detection was achieved with an ex-situ approach, where root canals were sampled with endodontic paper points for subsequent staining and analysis using a spectrometer coupled to a fluorescence microscope. Detection of vital cells in a stressed in-vitro endodontic biofilm was shown to be more sensitive than culturing: fluorescence detection was achieved for up to 40 seconds of 1% NaOCl exposure, compared to 15 seconds for culturing. The methodology was then applied to clinical trial involving 53 teeth and 114 roots. Detection immediately preobturation was achieved in 18.4% of roots and in 35.8% of teeth. The feasibility of remote in-situ spectral analysis was shown with preliminary studies using fluorescent beads as well as in-vitro studies using a 200 μm diameter probe on biofilms. Ratiometric analysis of live/dead stained nutritionally starved endodontic biofilms provided a simple alternative to conventional live/dead staining. Using software for spectral unmixing provided a method for fast and objective detection. The creation of a prototype, which couples a fluorescence spectrometer to a wide-field microscope, allowed for a successful patient trial, demonstrating the potential of this system to be miniaturised and commercialised for a chair-side use.