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.

<p><strong>Objectives:</strong> Confocal Raman microscopy can give inside knowledge on the composition and structure of biofilms and give some understanding on the strain&#8217;s role in the development and then maturation of biofilm structures. This information can then be helpful in understanding the impact of structure on both disease formation and the impact of therapies on biofilms. While different techniques like CLSM real-time PCR, SEM and others have been used for biofilm analysis successfully, confocal Raman microscopy has the advantage to be non-destructive and can thus consider the construction of biofilms over time.</p> <p><strong>Methods:</strong> S.oralis (DSM20066), V.dispar (DSM20735) and A.denticolens (DSM20671) were cultivated in brain-heart infusion broth under anaerobic conditions (37&#176;C, 80% N₂, 15% CO₂, 5% H₂) for 96h. Biofilms were formed on hydroxyapatite discs under the same conditions by using the static &#8216;Z&#252;rich&#8217; model. A Renishaw inVia Qontor instrument was used for confocal Raman analysis of planktonic cells and biofilms (532nm Laser, 50mW laser power, 1s exposure time, 15 scans, spectral detection range from 268-2017cm^-1). After spectral processing a bacterial database was created using planktonic bacteria. Predictions of species in a biofilm and mapping was performed using multivariate statistical analysis.</p> <p><strong>Results:</strong> The Raman spectra show a specific fingerprint region (600-1800 cm^-1) where differentiation between different strains are apparent. Using multivariate statistical methods it is possible to differentiate the strains of interest. In combination with structural spectral analysis, it is possible to predict species in artificially grown biofilms. Two-dimensional mapping allowed the visualization of the distribution based on their location of the acquired spectra in a specified random window.</p> <p><strong>Conclusion:</strong> Confocal Raman microscopy was able to differentiate clusters of oral bacteria in a subgingival biofilm model. This preliminary analysis is showing the potential of this technology in biofilm assessment and bacterial differentiation and may be used as an alternative to confocal laser scanning microscopy in the future. In addition, the developed methodology has the potential to be applied to different multi-species biofilms beyond the scope of oral biofilms.</p>

In the present study, the early stage of bacteria biofilm formation has been studied as a function of different nutrients. Infrared spectra of Pseudomonas fluorescens (PF) and Staphylococcus epidermidis (SE), on germanium ATR crystal, were collected under deionized water H2O, phosphate buffered solution (PBS) and PBS with glucose (PBS-G). In H2O, protein bands of PF increased while, no difference in PBS and PBS-G were observed until 135 min. SE strain showed a low sensitivity to PBS composition starting to expose proteins on surfaces after 120 min. SE shows a low polysaccharides increase in H2O while, in bare and enriched PBS their intensity increases after 120 and 75 min. in PBS and PBS-G respectively. PF exhibits a peculiar behavior in H2O where the saccharide bands increased strongly after 100 min, while under all the other conditions, the intensity of polysaccharide bands increased up to the plateau probably because the layer of the biofilm exceeded the penetration capability of FTIR technique. All data suggest that, under lack of nutrients, both the bacteria tend to firmly anchor themselves to the support using proteins.

The study of oral disease progression, in relation to the accumulation of subgingival biofilm in gingivitis and periodontitis is limited, due to either the ability to monitor plaque in vitro. When compared, optical spectroscopic techniques offer advantages over traditional destructive or biofilm staining approaches, making it a suitable alternative for the analysis and continued development of three-dimensional structures. In this work, we have developed a confocal Raman spectroscopy analysis approach towards in vitro subgingival plaque models. The main objective of this study was to develop a method for differentiating multiple oral subgingival bacterial species in planktonic and biofilm conditions, using confocal Raman microscopy. Five common subgingival bacteria (Fusobacterium nucleatum, Streptococcus mutans, Veillonella dispar, Actinomyces naeslundii and Prevotella nigrescens) were used and differentiated using a 2-way orthogonal Partial Least Square with Discriminant Analysis (O2PLS-DA) for the collected spectral data. In addition to planktonic growth, mono-species biofilms cultured using the 'Zürich Model' were also analyzed. The developed method was successfully used to predict planktonic and mono-species biofilm species in a cross validation setup. The results show differences in the presence and absence of chemical bands within the Raman spectra. The O2PLS-DA model was able to successfully predict 100% of all tested planktonic samples and 90% of all mono-species biofilm samples. Using this approach we have shown that Confocal Raman microscopy can analyse and predict the identity of planktonic and mono-species biofilm species, thus enabling its potential as a technique to map oral multi-species biofilm models.