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ORIGINAL RESEARCH article

Front. Cell. Infect. Microbiol.
Sec. Biofilms
Volume 14 - 2024 | doi: 10.3389/fcimb.2024.1508016
This article is part of the Research Topic Fighting Microbial Biofilms: Novel Therapeutics and Antibiofilm Strategies View all 10 articles

CAM/TMA-DPH as a promising alternative to SYTO9/PI for cell viability assessment in bacterial biofilms

Provisionally accepted
Tinatini Tchatchiashvili Tinatini Tchatchiashvili *Mateusz Jundzill Mateusz Jundzill Mike Marquet Mike Marquet Kamran Ahmed Mirza Kamran Ahmed Mirza Mathias Wilhelm Pletz Mathias Wilhelm Pletz Oliwia Makarewicz Oliwia Makarewicz Lara Thieme Lara Thieme
  • University Hospital Jena, Jena, Germany

The final, formatted version of the article will be published soon.

    Accurately assessing biofilm viability is essential for evaluating both biofilm formation and the efficacy of antibacterial treatments. However, the reliability of SYTO9 and propidium iodide (PI) live/dead staining in biofilm viability assays is often compromised by non-specific staining, which can hinder precise differentiation between live and dead cells. To address this limitation, we investigated an alternative staining method employing calcein acetoxymethyl (CAM) to detect viable cells based on esterase activity, and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) to assess the remaining biofilm population.Biofilms of Pseudomonas aeruginosa (P. aeruginosa), Klebsiella pneumoniae (K. pneumoniae), Staphylococcus aureus (S. aureus), and Enterococcus faecium (E. faecium) were matured and exposed to varying concentrations of antibiotics or sterile medium, followed by staining with CAM/TMA-DPH or SYTO9/PI. Using confocal laser scanning microscopy (CLSM) and ImageJ-based biofilm viability analysis, biofilm surface coverage was quantified and compared against colony-forming units (CFU/mL), a standard measure of microbial viability.Our results revealed that SYTO9/PI staining consistently underestimated the viability of untreated biofilms across most species, while CAM/TMA-DPH, more accurately reflected vitality and showed strong positive correlations with CFU counts across all bacterial species (r = 0.59 -0.91). In contrast, SYTO9/PI staining demonstrated a negative correlation with CFU/mL in K. pneumoniae biofilms (r = -0.04) but positive correlations in other species (r = 0.65 -0.79).Our findings suggest that CAM/TMA-DPH staining provides a promising alternative to SYTO9/PI for cell viability assessment in bacterial biofilms, highlighting the advantages of metabolic-based probes over traditional membrane integrity assays. The consistency of CAM/TMA-DPH staining across different bacterial species underscores its potential to advance studies on biofilm and contribute to the development of more effective anti-biofilm treatments.

    Keywords: Biofilm, viability, Staining, Metabolism, calcein acetoxymethyl, Live/dead assay

    Received: 08 Oct 2024; Accepted: 23 Dec 2024.

    Copyright: © 2024 Tchatchiashvili, Jundzill, Marquet, Mirza, Pletz, Makarewicz and Thieme. 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) or licensor 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.

    * Correspondence: Tinatini Tchatchiashvili, University Hospital Jena, Jena, Germany

    Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.