Abstract
Understanding the dynamic environmental microniches of biofilms will permit us to detect, manage and exploit these communities. The components and architecture of biofilms have been interrogated in depth; however, little is known about the environmental microniches present. This is primarily because of the absence of tools with the required measurement sensitivity and resolution to detect these changes. We describe the application of ratiometric fluorescent pH-sensitive nanosensors, as a tool, to observe physiological pH changes in biofilms in real time. Nanosensors comprised two pH-sensitive fluorophores covalently encapsulated with a reference pH-insensitive fluorophore in an inert polyacrylamide nanoparticle matrix. The nanosensors were used to analyse the real-time three-dimensional pH variation for two model biofilm formers: (i) opportunistic pathogen Pseudomonas aeruginosa and (ii) oral pathogen Streptococcus mutans. The detection of sugar metabolism in real time by nanosensors provides a potential application to identify therapeutic solutions to improve oral health.
Highlights
The biofilm communities that bacteria form on surfaces are dynamic and complex
Superresolution microscopy of planktonic PAO1-N grown overnight with positively charged or neutral nanosensors was performed to confirm that the nanosensors stay extracellular and to ensure that all subsequent pH measurements report on pH changes within the biofilm and the matrix rather than inside the cells
The addition of positive nanoparticles without the inclusion of fluorophores led to the similar increases in biofilm thickness and robustness as the positively charged nanosensors, suggesting that the charge of the particles was responsible for the change in the biofilm formation
Summary
The architecture of biofilms and the identity of extracellular components have been characterised in vitro for a variety of biofilms. It is not yet clear how representative this is of in vivo bacterial communities[1,2,3,4,5], further in vitro investigation of dynamic biofilms will help deliver the tools required to interrogate the emerging more realistic models. While there is evidence for fluid channels and microcolonies within in vitro biofilms, the characterisation of the environmental microniches these create has been limited. In this article we demonstrate the application of fluorescent nanosensors capable of dynamic pH monitoring of the environmental microniches within biofilms using two important pathogens
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