CAM/TMA-DPH as a promising alternative to SYTO9/PI for cell viability assessment in bacterial biofilms
IntroductionAccurately assessing biofilm viability is essential for evaluating both biofilm formation and the efficacy of antibacterial treatments. Traditional SYTO9 and propidium iodide (PI) live/dead staining in biofilm viability assays often ace challenges due to non-specific staining, limiting...
Saved in:
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2025-01-01
|
| Series: | Frontiers in Cellular and Infection Microbiology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fcimb.2024.1508016/full |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1832592397258719232 |
|---|---|
| author | Tinatini Tchatchiashvili Tinatini Tchatchiashvili Mateusz Jundzill Mateusz Jundzill Mike Marquet Kamran A. Mirza Kamran A. Mirza Mathias W. Pletz Mathias W. Pletz Oliwia Makarewicz Oliwia Makarewicz Lara Thieme Lara Thieme |
| author_facet | Tinatini Tchatchiashvili Tinatini Tchatchiashvili Mateusz Jundzill Mateusz Jundzill Mike Marquet Kamran A. Mirza Kamran A. Mirza Mathias W. Pletz Mathias W. Pletz Oliwia Makarewicz Oliwia Makarewicz Lara Thieme Lara Thieme |
| author_sort | Tinatini Tchatchiashvili |
| collection | DOAJ |
| description | IntroductionAccurately assessing biofilm viability is essential for evaluating both biofilm formation and the efficacy of antibacterial treatments. Traditional SYTO9 and propidium iodide (PI) live/dead staining in biofilm viability assays often ace challenges due to non-specific staining, limiting 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.MethodsBiofilms of Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecium were matured and exposed to varying concentrations of antibiotics or sterile medium. Biofilm viability was assessed using CAM/TMA-DPH or SYTO9/PIstaining, followed by analysis with confocal laser scanning microscopy (CLSM) and ImageJ-based biofilm surface coverage quantification. Viability findings were compared with colony-forming units (CFU/mL), a standard microbial viability measure.ResultsCAM/TMA-DPH staining demonstrated strong positive correlations with CFU counts across all bacterial species (r = 0.59 - 0.91), accurately reflecting biofilm vitality. In contrast, SYTO9/PI staining consistently underestimated the viability of untreated biofilms, particularly in Klebsiella pneumoniae, where a negative correlation with CFU/mL was observed (r = –0.04). Positive correlations for SYTO9/PI staining were noted in other species (r = 0.65 - 0.79). These findings underscore the limitations of membrane integrity-based staining methods and highlight the advantages of metabolic-based probes like CAM/TMA-DPH.DiscussionOur 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, which is essential for clinical management of biofilm-associated infections. |
| format | Article |
| id | doaj-art-0ba6c50bc02d4892b9151924334eb920 |
| institution | Kabale University |
| issn | 2235-2988 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cellular and Infection Microbiology |
| spelling | doaj-art-0ba6c50bc02d4892b9151924334eb9202025-01-21T08:36:47ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882025-01-011410.3389/fcimb.2024.15080161508016CAM/TMA-DPH as a promising alternative to SYTO9/PI for cell viability assessment in bacterial biofilmsTinatini Tchatchiashvili0Tinatini Tchatchiashvili1Mateusz Jundzill2Mateusz Jundzill3Mike Marquet4Kamran A. Mirza5Kamran A. Mirza6Mathias W. Pletz7Mathias W. Pletz8Oliwia Makarewicz9Oliwia Makarewicz10Lara Thieme11Lara Thieme12Institute of Infectious Disease and Infection Control, Jena University Hospital, Friedrich-Schiller-University Jena, Jena, GermanyInstitute of Infectious Disease and Infection Control, Member of the Leibniz Center for Photonics in Infection Research (LPI), Jena, GermanyInstitute of Infectious Disease and Infection Control, Jena University Hospital, Friedrich-Schiller-University Jena, Jena, GermanyInstitute of Infectious Disease and Infection Control, Member of the Leibniz Center for Photonics in Infection Research (LPI), Jena, GermanyInstitute of Infectious Disease and Infection Control, Jena University Hospital, Friedrich-Schiller-University Jena, Jena, GermanyInstitute of Infectious Disease and Infection Control, Jena University Hospital, Friedrich-Schiller-University Jena, Jena, GermanyInstitute of Infectious Disease and Infection Control, Member of the Leibniz Center for Photonics in Infection Research (LPI), Jena, GermanyInstitute of Infectious Disease and Infection Control, Jena University Hospital, Friedrich-Schiller-University Jena, Jena, GermanyInstitute of Infectious Disease and Infection Control, Member of the Leibniz Center for Photonics in Infection Research (LPI), Jena, GermanyInstitute of Infectious Disease and Infection Control, Jena University Hospital, Friedrich-Schiller-University Jena, Jena, GermanyInstitute of Infectious Disease and Infection Control, Member of the Leibniz Center for Photonics in Infection Research (LPI), Jena, GermanyInstitute of Infectious Disease and Infection Control, Jena University Hospital, Friedrich-Schiller-University Jena, Jena, GermanyInstitute of Infectious Disease and Infection Control, Member of the Leibniz Center for Photonics in Infection Research (LPI), Jena, GermanyIntroductionAccurately assessing biofilm viability is essential for evaluating both biofilm formation and the efficacy of antibacterial treatments. Traditional SYTO9 and propidium iodide (PI) live/dead staining in biofilm viability assays often ace challenges due to non-specific staining, limiting 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.MethodsBiofilms of Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecium were matured and exposed to varying concentrations of antibiotics or sterile medium. Biofilm viability was assessed using CAM/TMA-DPH or SYTO9/PIstaining, followed by analysis with confocal laser scanning microscopy (CLSM) and ImageJ-based biofilm surface coverage quantification. Viability findings were compared with colony-forming units (CFU/mL), a standard microbial viability measure.ResultsCAM/TMA-DPH staining demonstrated strong positive correlations with CFU counts across all bacterial species (r = 0.59 - 0.91), accurately reflecting biofilm vitality. In contrast, SYTO9/PI staining consistently underestimated the viability of untreated biofilms, particularly in Klebsiella pneumoniae, where a negative correlation with CFU/mL was observed (r = –0.04). Positive correlations for SYTO9/PI staining were noted in other species (r = 0.65 - 0.79). These findings underscore the limitations of membrane integrity-based staining methods and highlight the advantages of metabolic-based probes like CAM/TMA-DPH.DiscussionOur 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, which is essential for clinical management of biofilm-associated infections.https://www.frontiersin.org/articles/10.3389/fcimb.2024.1508016/fullbiofilmviabilitystainingmetabolismcalcein acetoxymethyllive/dead assay |
| spellingShingle | Tinatini Tchatchiashvili Tinatini Tchatchiashvili Mateusz Jundzill Mateusz Jundzill Mike Marquet Kamran A. Mirza Kamran A. Mirza Mathias W. Pletz Mathias W. Pletz Oliwia Makarewicz Oliwia Makarewicz Lara Thieme Lara Thieme CAM/TMA-DPH as a promising alternative to SYTO9/PI for cell viability assessment in bacterial biofilms Frontiers in Cellular and Infection Microbiology biofilm viability staining metabolism calcein acetoxymethyl live/dead assay |
| title | CAM/TMA-DPH as a promising alternative to SYTO9/PI for cell viability assessment in bacterial biofilms |
| title_full | CAM/TMA-DPH as a promising alternative to SYTO9/PI for cell viability assessment in bacterial biofilms |
| title_fullStr | CAM/TMA-DPH as a promising alternative to SYTO9/PI for cell viability assessment in bacterial biofilms |
| title_full_unstemmed | CAM/TMA-DPH as a promising alternative to SYTO9/PI for cell viability assessment in bacterial biofilms |
| title_short | CAM/TMA-DPH as a promising alternative to SYTO9/PI for cell viability assessment in bacterial biofilms |
| title_sort | cam tma dph as a promising alternative to syto9 pi for cell viability assessment in bacterial biofilms |
| topic | biofilm viability staining metabolism calcein acetoxymethyl live/dead assay |
| url | https://www.frontiersin.org/articles/10.3389/fcimb.2024.1508016/full |
| work_keys_str_mv | AT tinatinitchatchiashvili camtmadphasapromisingalternativetosyto9piforcellviabilityassessmentinbacterialbiofilms AT tinatinitchatchiashvili camtmadphasapromisingalternativetosyto9piforcellviabilityassessmentinbacterialbiofilms AT mateuszjundzill camtmadphasapromisingalternativetosyto9piforcellviabilityassessmentinbacterialbiofilms AT mateuszjundzill camtmadphasapromisingalternativetosyto9piforcellviabilityassessmentinbacterialbiofilms AT mikemarquet camtmadphasapromisingalternativetosyto9piforcellviabilityassessmentinbacterialbiofilms AT kamranamirza camtmadphasapromisingalternativetosyto9piforcellviabilityassessmentinbacterialbiofilms AT kamranamirza camtmadphasapromisingalternativetosyto9piforcellviabilityassessmentinbacterialbiofilms AT mathiaswpletz camtmadphasapromisingalternativetosyto9piforcellviabilityassessmentinbacterialbiofilms AT mathiaswpletz camtmadphasapromisingalternativetosyto9piforcellviabilityassessmentinbacterialbiofilms AT oliwiamakarewicz camtmadphasapromisingalternativetosyto9piforcellviabilityassessmentinbacterialbiofilms AT oliwiamakarewicz camtmadphasapromisingalternativetosyto9piforcellviabilityassessmentinbacterialbiofilms AT larathieme camtmadphasapromisingalternativetosyto9piforcellviabilityassessmentinbacterialbiofilms AT larathieme camtmadphasapromisingalternativetosyto9piforcellviabilityassessmentinbacterialbiofilms |