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. 2019 Nov 8;9:16284. doi: 10.1038/s41598-019-52726-0

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Oxygen stratification within the cocultured biofilm determines the spatial distribution of P. aeruginosa and S. aureus and their coexistence. (a) XZ-orthogonal view of a P. aeruginosa and S. aureus coculture biofilm grown in DMEM + BSA (see Fig. 6d). The image shows the separate position of Newman and PA14 within the ∼16 μm-biofilm and the possible oxygen gradient present in the system. (b) Graph shows the average pixel intensities of PA14 (blue) and Newman (Green) biomasses (plotted on the upper Y-axis) compared to the oxygen-related red intensity (plotted on the lower Y-axis) along the different thicknesses (μm) of a 3-day-old cocultured biofilm in flow. Pixel intensity averages were calculated from ten different images using ImageJ software. Included in the figure, is a sequential set of micrographs showing the red fluorescence emission given by the hypoxia probe through the different biofilm layers, indicating the oxygen stratification present along the thickness of the continuous-flow cocultured biofilm. Intense red emission relates to the hypoxic environment through the different biofilm depths (μm), from the bottom to the surface, additionally indicated with a schematic of the oxygen gradient present in the system.