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. 2018 Apr 9;8:5681. doi: 10.1038/s41598-018-23830-4

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© The Author(s) 2018

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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B. pseudohinzii (strain 3227) attaches to and intermingles with cilia in an in vitro-infection model. Tracheal explants were cultured for 24 h, with following addition of bacteria for 4 h (a–i) or 24 h (j–r). (a–c) Semithin sections of tracheal explants cultured for 24 + 4 h, light microscopy. (a) Without adding of bacteria, cilia are normally orientated and no bacteria are visible. (b) B. pseudohinzii (0.4 × 10⁴ CFU) was added for 4 h. No bacteria are seen. (c) B. pseudohinzii (2.3 × 10⁸ CFU) was added for 4 h. Bacteria attach to cilia (dotted arrows). (d–i) Transmission electron microscopy. (d,g) Control experiment. Tracheas were cultured for 24 + 4 h without bacteria. Cilia appear normal in length and orientation. No signs of cell damage. (e,h) No obvious difference to controls 4 h after incubation with 0.4 × 10⁴ CFU B. pseudohinzii. (f,i) B. pseudohinzii (2.3 × 10⁸ CFU) was added for 4 h. Bacteria are located between the cilia (dotted arrow), (j,m,p) Control. Explants cultivated for 24 h without bacteria demonstrate unaltered ciliated cells in light (j) and transmission electron microscopy (m,p). (k,n,q). B. pseudohinzii (0.4 × 10⁴ CFU) was added for 24 h. Bacteria (dotted arrows) intermingle with kinocilia. They are not seen at non-ciliated cells. (l,o,r) B. pseudohinzii (2.3 × 10⁸ CFU) was added for 24 h. The number of bacteria (dotted arrows) is increased, cells with shortened of cilia can be observed in transmission electron microscopy (o,r).