Micron Scale Spatial Measurement of the O(2) Gradient Surrounding a Bacterial Biofilm in Real Time

Bacteria alter their local chemical environment through both consumption and the production of a variety of molecules, ultimately shaping the local ecology. Molecular oxygen (O(2)) is a key metabolite that affects the physiology and behavior of virtually all bacteria, and its consumption often results in O(2) gradients within sessile bacterial communities (biofilms). O(2) plays a critical role in several bacterial phenotypes, including antibiotic tolerance; however, our understanding of O(2) levels within and surrounding biofilms has been hampered by the difficulties in measuring O(2) levels in real-time for extended durations and at the micron scale. Here, we developed electrochemical methodology based on scanning electrochemical microscopy to quantify the O(2) gradients present above a Pseudomonas aeruginosa biofilm. These results reveal that a biofilm produces a hypoxic zone that extends hundreds of microns from the biofilm surface within minutes and that the biofilm consumes O(2) at a maximum rate. Treating the biofilm with levels of the antibiotic ciprofloxacin that kill 99% of the bacteria did not affect the O(2) gradient, indicating that the biofilm is highly resilient to antimicrobial treatment in regard to O(2) consumption.