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Mapping of a Subgingival Dual-Species Biofilm Model Using Confocal Raman Microscopy

Techniques for continuously monitoring the formation of subgingival biofilm, in relation to the determination of species and their accumulation over time in gingivitis and periodontitis, are limited. In recent years, advancements in the field of optical spectroscopic techniques have provided an alte...

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Autores principales: Kriem, Lukas Simon, Wright, Kevin, Ccahuana-Vasquez, Renzo Alberto, Rupp, Steffen
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8525910/
https://www.ncbi.nlm.nih.gov/pubmed/34675902
http://dx.doi.org/10.3389/fmicb.2021.729720
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author Kriem, Lukas Simon
Wright, Kevin
Ccahuana-Vasquez, Renzo Alberto
Rupp, Steffen
author_facet Kriem, Lukas Simon
Wright, Kevin
Ccahuana-Vasquez, Renzo Alberto
Rupp, Steffen
author_sort Kriem, Lukas Simon
collection PubMed
description Techniques for continuously monitoring the formation of subgingival biofilm, in relation to the determination of species and their accumulation over time in gingivitis and periodontitis, are limited. In recent years, advancements in the field of optical spectroscopic techniques have provided an alternative for analyzing three-dimensional microbiological structures, replacing the traditional destructive or biofilm staining techniques. In this work, we have demonstrated that the use of confocal Raman spectroscopy coupled with multivariate analysis provides an approach to spatially differentiate bacteria in an in vitro model simulating a subgingival dual-species biofilm. The present study establishes a workflow to evaluate and differentiate bacterial species in a dual-species in vitro biofilm model, using confocal Raman microscopy (CRM). Biofilm models of Actinomyces denticolens and Streptococcus oralis were cultured using the “Zürich in vitro model” and were analyzed using CRM. Cluster analysis was used to spatially differentiate and map the biofilm model over a specified area. To confirm the clustering of species in the cultured biofilm, confocal laser scanning microscopy (CLSM) was coupled with fluorescent in vitro hybridization (FISH). Additionally, dense bacteria interface area (DBIA) samples, as an imitation of the clusters in a biofilm, were used to test the developed multivariate differentiation model. This confirmed model was successfully used to differentiate species in a dual-species biofilm and is comparable to morphology. The results show that the developed workflow was able to identify main clusters of bacteria based on spectral “fingerprint region” information from CRM. Using this workflow, we have demonstrated that CRM can spatially analyze two-species in vitro biofilms, therefore providing an alternative technique to map oral multi-species biofilm models.
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spelling pubmed-85259102021-10-20 Mapping of a Subgingival Dual-Species Biofilm Model Using Confocal Raman Microscopy Kriem, Lukas Simon Wright, Kevin Ccahuana-Vasquez, Renzo Alberto Rupp, Steffen Front Microbiol Microbiology Techniques for continuously monitoring the formation of subgingival biofilm, in relation to the determination of species and their accumulation over time in gingivitis and periodontitis, are limited. In recent years, advancements in the field of optical spectroscopic techniques have provided an alternative for analyzing three-dimensional microbiological structures, replacing the traditional destructive or biofilm staining techniques. In this work, we have demonstrated that the use of confocal Raman spectroscopy coupled with multivariate analysis provides an approach to spatially differentiate bacteria in an in vitro model simulating a subgingival dual-species biofilm. The present study establishes a workflow to evaluate and differentiate bacterial species in a dual-species in vitro biofilm model, using confocal Raman microscopy (CRM). Biofilm models of Actinomyces denticolens and Streptococcus oralis were cultured using the “Zürich in vitro model” and were analyzed using CRM. Cluster analysis was used to spatially differentiate and map the biofilm model over a specified area. To confirm the clustering of species in the cultured biofilm, confocal laser scanning microscopy (CLSM) was coupled with fluorescent in vitro hybridization (FISH). Additionally, dense bacteria interface area (DBIA) samples, as an imitation of the clusters in a biofilm, were used to test the developed multivariate differentiation model. This confirmed model was successfully used to differentiate species in a dual-species biofilm and is comparable to morphology. The results show that the developed workflow was able to identify main clusters of bacteria based on spectral “fingerprint region” information from CRM. Using this workflow, we have demonstrated that CRM can spatially analyze two-species in vitro biofilms, therefore providing an alternative technique to map oral multi-species biofilm models. Frontiers Media S.A. 2021-10-05 /pmc/articles/PMC8525910/ /pubmed/34675902 http://dx.doi.org/10.3389/fmicb.2021.729720 Text en Copyright © 2021 Kriem, Wright, Ccahuana-Vasquez and Rupp. https://creativecommons.org/licenses/by/4.0/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.
spellingShingle Microbiology
Kriem, Lukas Simon
Wright, Kevin
Ccahuana-Vasquez, Renzo Alberto
Rupp, Steffen
Mapping of a Subgingival Dual-Species Biofilm Model Using Confocal Raman Microscopy
title Mapping of a Subgingival Dual-Species Biofilm Model Using Confocal Raman Microscopy
title_full Mapping of a Subgingival Dual-Species Biofilm Model Using Confocal Raman Microscopy
title_fullStr Mapping of a Subgingival Dual-Species Biofilm Model Using Confocal Raman Microscopy
title_full_unstemmed Mapping of a Subgingival Dual-Species Biofilm Model Using Confocal Raman Microscopy
title_short Mapping of a Subgingival Dual-Species Biofilm Model Using Confocal Raman Microscopy
title_sort mapping of a subgingival dual-species biofilm model using confocal raman microscopy
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8525910/
https://www.ncbi.nlm.nih.gov/pubmed/34675902
http://dx.doi.org/10.3389/fmicb.2021.729720
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