Micron Scale Spatial Measurement of the O2 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. IMPORTANCE O-2 is a fundamental environmental metabolite that affects all life on earth. While toxic to many microbes and obligately required by others, those that have appropriate physiological responses survive and can even benefit from various levels of O-2, particularly in biofilm communities. Although most studies have focused on measuring (O)2 within biofilms, little is known about O-2 gradients surrounding biofilms. Here, we developed electrochemical methodology based on scanning electrochemical microscopy to measure the O-2 gradients surrounding biofilms in real time on the micron scale. Our results reveal that P. aeruginosa biofilms produce a hypoxic zone that can extend hundreds of microns from the biofilm surface and that this gradient remains even after the addition of antibiotic concentrations that eradicated 99% of viable cells. Our results provide a high resolution of the O-2 gradients produced by P. aeruginosa biofilms and reveal sustained O-2 consumption in the presence of antibiotics.