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The processing of staphylococcal biofilm images from Fluorescence and Confocal Microscopy to assess in vitro efficacy of antiseptic molecules

Grzegorz Krasowski, View ORCID ProfilePaweł Migdał, View ORCID ProfileMarta Woroszyło, View ORCID ProfileKarol Fijałkowski, View ORCID ProfileGrzegorz Chodaczek, View ORCID ProfileJoanna Czajkowska, View ORCID ProfileBartłomiej Dudek, Joanna Nowicka, View ORCID ProfileMonika Oleksy-Wawrzyniak, Bartłomiej Kwiek, View ORCID ProfileJustyna Paleczny, View ORCID ProfileMalwina Brożyna, View ORCID ProfileAdam Junka
doi: https://doi.org/10.1101/2021.11.30.470646
Grzegorz Krasowski
1Nutrikon, KCZ Surgical Ward, 47-300 Krapkowice, Poland; (G.K.)
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  • For correspondence: g.krasowski@wp.pl
Paweł Migdał
2Department of Environment Hygiene and Animal Welfare, Wroclaw University of Environmental and Life Sciences, 25 C.K. Norwida st., 51-630 Wroclaw, Poland; (P.M.)
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  • ORCID record for Paweł Migdał
  • For correspondence: pawel.migdal@upwr.pl
Marta Woroszyło
3Department of Microbiology and Biotechnology, Faculty of Biotechnology and Animal Husbandry, West Pomeranian University of Technology in Szczecin, Piastów 45, 70-311 Szczecin, Poland; (M.W.); (K.F.)
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  • For correspondence: marta.woroszylo@zut.edu.pl kfijalkowski@zut.edu.pl
Karol Fijałkowski
3Department of Microbiology and Biotechnology, Faculty of Biotechnology and Animal Husbandry, West Pomeranian University of Technology in Szczecin, Piastów 45, 70-311 Szczecin, Poland; (M.W.); (K.F.)
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  • For correspondence: marta.woroszylo@zut.edu.pl kfijalkowski@zut.edu.pl
Grzegorz Chodaczek
4Bioimaging Laboratory, Łukasiewicz Research Network—PORT Polish Center for Technology Development, 54-066 Wrocław, Poland; (G.CH.)
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  • ORCID record for Grzegorz Chodaczek
  • For correspondence: grzegorz.chodaczek@port.org.pl
Joanna Czajkowska
5Laboratory of Microbiology, Łukasiewicz Research Network–PORT Polish Center for TechnologyDevelopment, 54-066 Wrocław, Poland; (J.Cz.)
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  • For correspondence: joanna.czajkowska@port.org.pl
Bartłomiej Dudek
6Department of Microbiology, Institute of Genetics and Microbiology, University of Wrocław, Stanisława Przybyszewskiego 63, 51-148 Wrocław, Poland; (B.D.)
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  • For correspondence: bartlomiej.dudek@uwr.edu.pl
Joanna Nowicka
7Department of Microbiology, Faculty of Medicine, Wroclaw Medical University, 50-368 Wroclaw, Poland; (J.N.)
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  • For correspondence: joanna.nowicka@umed.wroc.pl
Monika Oleksy-Wawrzyniak
8Department of Pharmaceutical Microbiology and Parasitology, Faculty of Pharmacy Wrocław Medical University, 50-556 Wrocław, Poland; (A.J.); (J.P); (M.O.-W.); (M.B.)
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  • For correspondence: feliks.junka@gmail.com justyna.paleczny@student.umw.edu.pl monika.oleksy@umw.edu.pl malwina.brozyna@student.umw.edu.pl
Bartłomiej Kwiek
9Faculty of Medicine, Lazarski University, 02□662 Warszawa, Poland; (B.K)
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  • For correspondence: bartlomiej.kwiek@lazarski.pl
Justyna Paleczny
8Department of Pharmaceutical Microbiology and Parasitology, Faculty of Pharmacy Wrocław Medical University, 50-556 Wrocław, Poland; (A.J.); (J.P); (M.O.-W.); (M.B.)
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  • For correspondence: feliks.junka@gmail.com justyna.paleczny@student.umw.edu.pl monika.oleksy@umw.edu.pl malwina.brozyna@student.umw.edu.pl
Malwina Brożyna
8Department of Pharmaceutical Microbiology and Parasitology, Faculty of Pharmacy Wrocław Medical University, 50-556 Wrocław, Poland; (A.J.); (J.P); (M.O.-W.); (M.B.)
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  • For correspondence: feliks.junka@gmail.com justyna.paleczny@student.umw.edu.pl monika.oleksy@umw.edu.pl malwina.brozyna@student.umw.edu.pl
Adam Junka
5Laboratory of Microbiology, Łukasiewicz Research Network–PORT Polish Center for TechnologyDevelopment, 54-066 Wrocław, Poland; (J.Cz.)
8Department of Pharmaceutical Microbiology and Parasitology, Faculty of Pharmacy Wrocław Medical University, 50-556 Wrocław, Poland; (A.J.); (J.P); (M.O.-W.); (M.B.)
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  • For correspondence: feliks.junka@gmail.com joanna.czajkowska@port.org.pl feliks.junka@gmail.com justyna.paleczny@student.umw.edu.pl monika.oleksy@umw.edu.pl malwina.brozyna@student.umw.edu.pl
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Abstract

The in vitro efficacy of locally applied antiseptic molecules against staphylococcal biofilm is frequently assessed by a set of standard quantitative and semi-quantitative methods. The development of software for parametric image processing allowed to obtain parametric data also from microscopic images of biofilm dyed with a variety of dyes, especially with propidium iodine and SYTO-9, differentiating dead from live cells. In this work, using confocal/epifluorescent microscopy, we analyzed such major properties of staphylococcal biofilm in vitro as its thickness, cellular density and share of Live/Dead cells within its individual parts. We also scrutinized the impact of sample preparation and antiseptic introduction on the outcome obtained. As a result of our analyses we developed a revelatory method of assessment of the impact of antiseptic agents on staphylococcal biofilm in vitro, in which the microscopic images are processed with the use of ABE formula (Antiseptic’s Biofilm Eradication) which implements all the data and phenomena detected and revealed within the course of this study. We tested ABE with regard to polyhexanide, pov idone-iodine and hypochlorous antiseptics and found a high correlation between this parameter and the results obtained by means of traditional techniques. Taking into account the fact that in vitro results of the efficacy of antiseptic agents against staphylococcal biofilm are frequently applied to back up their use in hospitals and ambulatory units, our work should be considered an important tool providing reliable, parametric data with this regard.

Competing Interest Statement

The authors have declared no competing interest.

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Posted November 30, 2021.
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The processing of staphylococcal biofilm images from Fluorescence and Confocal Microscopy to assess in vitro efficacy of antiseptic molecules
Grzegorz Krasowski, Paweł Migdał, Marta Woroszyło, Karol Fijałkowski, Grzegorz Chodaczek, Joanna Czajkowska, Bartłomiej Dudek, Joanna Nowicka, Monika Oleksy-Wawrzyniak, Bartłomiej Kwiek, Justyna Paleczny, Malwina Brożyna, Adam Junka
bioRxiv 2021.11.30.470646; doi: https://doi.org/10.1101/2021.11.30.470646
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The processing of staphylococcal biofilm images from Fluorescence and Confocal Microscopy to assess in vitro efficacy of antiseptic molecules
Grzegorz Krasowski, Paweł Migdał, Marta Woroszyło, Karol Fijałkowski, Grzegorz Chodaczek, Joanna Czajkowska, Bartłomiej Dudek, Joanna Nowicka, Monika Oleksy-Wawrzyniak, Bartłomiej Kwiek, Justyna Paleczny, Malwina Brożyna, Adam Junka
bioRxiv 2021.11.30.470646; doi: https://doi.org/10.1101/2021.11.30.470646

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