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. 2020 Feb 26;11:291. doi: 10.3389/fmicb.2020.00291

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Copyright © 2020 Jordana-Lluch, Garcia, Kingdon, Singh, Alexander, Williams and Hardie.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Localization of bacteria within the polymicrobial keratinocyte colonization model is species dependent. Using the protocol outlined in Figure 1, either S. aureus, S. epidermidis or M. luteus was combined with P. aeruginosa to form a polymicrobial biofilm on top of a HaCat monolayer. 3D reconstructions of the polymicrobial biofilms are shown using 40 × magnification. HaCat cells were not stained. (A) Dual species intercalated biofilm of S. aureus (GFP tagged, green) combined with P. aeruginosa (mCherry tagged, red). (B) Dual species layered biofilm of S. epidermidis (GFP tagged, green) with P. aeruginosa (mCherry tagged, red). (C) Triple species biofilm containing a layer of M. luteus intercalated with S. epidermidis between P. aeruginosa (above) and the keratinocyte monolayer (below). Bacteria were detected by FISH using pan-bacterial (green) and Pseudomonas-specific (red) probes.