Robust, well-characterised biofilm models exist for both P. This acidification leads to the formation of dental caries 34, 35, 36.
mutans ferments available carbohydrates, readily found in a sugar-laden diet, which results in the production of organic acids 33. This enables bacteria to continue to colonise and establish more biofilm 30, 31, 32.
mutans uses sucrose as a substrate to synthesise glucan via glucosyltransferases, in order to provide anchoring sites on the tooth’s enamel. mutans lies in their participation in dental caries, the most common infection affecting humans, through the production of organic acids 29. mutans is both an aciduric and acidogenic bacterium and a pre-dominant species in late stage oral biofilms 27, 28. Oral biofilms are characterised by a bacterial shift from early colonisers of the tooth towards increasingly acid-producing (acidogenic) and acid-tolerant (aciduric) species 26. Oral biofilms play a crucial role in the aetiology of oral diseases, such as dental caries and periodontitis, which can lead to increased economic burden and reduced quality of life 25. Secondly, we chose the Gram-positive oral pathogen Streptococcus mutans. aeruginosa is particularly important for it to establish chronic infections and has therefore been studied intensively. aeruginosa exerts its pathogenicity through the production of several factors, including those shown to play a key role in biofilm attachment such as extracellular DNA 21, 22, 23, 24. aeruginosa is prevalent in the environment creating numerous potential reservoirs of infection. aeruginosa infections being the leading cause of morbidity for CF sufferers. These infections are challenging to treat, due to the intrinsic antibiotic resistance of P. 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.įirstly, we selected the Gram-negative bacterium Pseudomonas aeruginosa, an opportunistic pathogen, causing infections in immunocompromised patients, including those suffering from burns, wounds and cystic fibrosis (CF) 17, 18, 19, 20. Although a number of analytical tools have been applied to static biofilms, for real-time monitoring, tools that are suitably sensitive at the required resolution now require testing in appropriate flow biofilm models 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16. While there is evidence for fluid channels and microcolonies within in vitro biofilms, the characterisation of the environmental microniches these create has been limited. Although 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. The architecture of biofilms and the identity of extracellular components have been characterised in vitro for a variety of biofilms. The biofilm communities that bacteria form on surfaces are dynamic and complex. The detection of sugar metabolism in real time by nanosensors provides a potential application to identify therapeutic solutions to improve oral health. 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. Nanosensors comprised two pH-sensitive fluorophores covalently encapsulated with a reference pH-insensitive fluorophore in an inert polyacrylamide nanoparticle matrix. We describe the application of ratiometric fluorescent pH-sensitive nanosensors, as a tool, to observe physiological pH changes in biofilms in real time. This is primarily because of the absence of tools with the required measurement sensitivity and resolution to detect these changes. The components and architecture of biofilms have been interrogated in depth however, little is known about the environmental microniches present. Understanding the dynamic environmental microniches of biofilms will permit us to detect, manage and exploit these communities.