Discussion

For millennia humans have used analytical approaches to study what is happening in biological systems to better understand them. The typical approach is to pull out a sample and measure it using offline analysis. This works amazingly well for many applications - including clinical diagnostics, field sampling, and lab research. However, this approach can be more limited when it comes to understanding dynamics in smaller systems such as microbial biofilms. There are a few key challenges. 1) Taking a sample out from the system can perturb the sample itself. 2) For some analytes and samples you are interested in, you can't get a big enough sample to analyze. For example, if you are trying to sample between microbes there isn't much volume to sample. 3) It can be hard to get continuous data with sampling - so if you are interested in kinetic results or changes over time, sampling isn't optimal. 4) It is hard to get spatial imaging with sampling approaches - so spatial variations can get lost.

Our group is studying metabolic dynamics in microbial systems such as biofilms, and we accomplish this by flipping this sampling paradigm. Rather than bring the sample out of the system to the diagnostic, we bring the diagnostic to the sample. We developed luminescent nanoparticle-based sensors that are embedded throughout biofilms that we study. While this approach is applicable to other systems, I will talk about our efforts in monitoring Pseudomonas aeruginosa, where we use both typical lab strains and patient isolates from our clinical cystic fibrosis collaborators.

I will discuss our optical oxygen-sensitive nanosensors that were used to measure 3D oxygen gradients in P. aeruginosa biofilms with minimal disruption to the biofilm structure. We created and characterized optical nanosensors for detection of molecular oxygen which can be incorporated into the biofilm structure during growth. Using these, we obtained confocal microscopy data from which we can determine quantitative 3-dimensional oxygen gradients. We observed spatial gradation in oxygen concentration during biofilm growth, attributed to nutrient consumption at the edges of the biofilm. We also studied 3D oxygen gradients during antibiotic attack and found that oxygen was present at greater depths compared to untreated controls, consistent with cell death (as confirmed with follow-up plating). This approach can be used to determine strain specific metabolic responses to antibiotics – demonstrated with individual metabolic profiling of clinical isolates from three different patients. We can also use this nanosensor platform for higher throughput assays in multiwell plates to test antibiotic susceptibility in biofilms, measuring the strain dependent response to antibiotics in a simple assay. Overall, the use of optical nanosensors unlocks new areas of measurement in microbial systems to better study their function.