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. 2021 Jun 25;12:672975. doi: 10.3389/fmicb.2021.672975

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Copyright © 2021 Reigada, San-Martin-Galindo, Gilbert-Girard, Chiaro, Cerullo, Savijoki, Nyman, Fallarero and Miettinen.

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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(A) Monitoring of the bacterial density of Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa PAO1 in mono- and co-cultured biofilms at different time points. (B–D) Cytographs corresponding to cells recovered from the biofilm formed by P. aeruginosa (B) and S. aureus (C) in monoculture and the dual-species biofilm (D). SYTO 9 was used to stain viable cells, while PI was utilized to stain cells with disrupted cell membrane integrity. The results are expressed as the mean of at least two biological repetitions with their ± SD. Statistical differences were assessed by comparing the mono- and co-cultured biofilms of each bacterial species (Student’s t-test, ^∗∗p < 0.01).