(232f) Optical Nanosensors for Monitoring 3D Oxygen Gradients in Pseudomonas Aeruginosa Biofilms

Bacterial biofilms are the cause of chronic infections of
wounds, medical implants, and immunocompromised patients such as those with
cystic fibrosis. These biofilms often exhibit antibiotic resistance that makes
them medically difficult to treat. Being able to monitor target analytes within
the structure of the biofilm is critical for understanding biofilm disease
biology and its response to treatment. Oxygen consumption can be used as a
measure of metabolic activity in facultative aerobes, but current methods such
as microelectrodes limit monitoring to one dimension and cannot adequately
capture dynamics. However, biofilms are complex three
dimensional structures that require higher resolution approaches to gain
a more complete understanding of activity. Traditional measurements require
physically moving the microelectrode, which is inherently disruptive to biofilm
structure and obscures rapid dynamics within the system, which can potentially
alter results. Here, we demonstrate optical oxygen-sensitive nanosensors that were used to measure 3D oxygen gradients
in Pseudomonas 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 quantitative 3 dimensional oxygen gradients
could be determined, as shown in Figure 1.

Figure 1. a) Raw confocal microscopy data; b) ratiometric fluorescence signal; and c) rendered 3D oxygen
gradient.

Using this approach, we improved on traditional
electrode-based 1D methods of measuring oxygen profiles by investigating both the
spatial and temporal variation in oxygen concentration during biofilm growth
and under antibiotic attack. 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 or a transition to anaerobic
respiration. This new approach to biological interrogation provides higher
resolution data, with improved temporal resolution, while minimizing the impact
of the measurement on the biofilm itself. Increased resolution allows for
better in vitro study and screening
of antibiotic treatment relevant to treating P. aeruginosa biofilm infections.