In nature, bacteria usually exist as mixed-species biofilms, where they engage in a range of synergistic and antagonistic interactions that increase their resistance to environmental challenges. Biofilms are a major cause of persistent infections, and dispersal from initial foci can cause new infections at distal sites thus warranting further investigation. Studies of development and spatial interactions in mixed-species biofilms can be challenging due to difficulties in identifying the different bacterial species in situ. Here, we apply CellTrace dyes to studies of biofilm bacteria and present a novel application for multiplex labeling, allowing identification of different bacteria in mixed-species, in vitro biofilm models. Oral bacteria labeled with CellTrace dyes (far red, yellow, violet, and CFSE [green]) were used to create single- and mixed-species biofilms, which were analyzed with confocal spinning disk microscopy (CSDM). Biofilm supernatants were studied with flow cytometry (FC). Both Gram-positive and Gram-negative bacteria were well labeled and CSDM revealed biofilms with clear morphology and stable staining for up to 4 days. Analysis of CellTrace labeled cells in supernatants using FC showed differences in the biofilm dispersal between bacterial species. Multiplexing with different colored dyes allowed visualization of spatial relationships between bacteria in mixed-species biofilms and relative coverage by the different species was revealed through segmentation of the CSDM images. This novel application, thus, offers a powerful tool for studying structure and composition of mixed-species biofilms in vitro. IMPORTANCE Although most chronic infections are caused by mixed-species biofilms, much of our knowledge still comes from planktonic cultures of single bacterial species. Studies of formation and development of mixed-species biofilms are, therefore, required. This work describes a method applicable to labeling of bacteria for in vitro studies of biofilm structure and dispersal. Critically, labeled bacteria can be multiplexed for identification of different species in mixed-species biofilms using confocal spinning disk microscopy, facilitating investigation of biofilm development and spatial interactions under different environmental conditions. The study is an important step in increasing the tools available for such complex and challenging studies. IMPORTANCE Although most chronic infections are caused by mixed-species biofilms, much of our knowledge still comes from planktonic cultures of single bacterial species. Studies of formation and development of mixed-species biofilms are, therefore, required. This work describes a method applicable to labeling of bacteria for in vitro studies of biofilm structure and dispersal. Critically, labeled bacteria can be multiplexed for identification of different species in mixed-species biofilms using confocal spinning disk microscopy, facilitating investigation of biofilm development and spatial interactions under different environmental conditions. The study is an important step in increasing the tools available for such complex and challenging studies.

This commentary introduces the new Graduating European Dentist Curriculum Domain V-'Research', and outlines the international multi-stakeholder development and consultation process that was undertaken in order to develop the new Domain.

Periodontal disease affects supporting dental structures and ranks among one of the top most expensive conditions to treat in the world. Moreover, in recent years, the disease has also been linked to cardiovascular and Alzheimer's diseases. At present, there is a serious lack of accurate diagnostic tools to identify people at severe risk of periodontal disease progression. Porphyromonas gingivalis is often considered one of the most contributing factors towards disease progression. It produces the Arg- and Lys-specific proteases Rgp and Kgp, respectively. Within this work, a short epitope sequence of these proteases is immobilised onto a magnetic nanoparticle platform. These are then used as a template to produce high-affinity, selective molecularly imprinted nanogels, using the common monomers N-tert-butylacrylamide (TBAM), N-isopropyl acrylamide (NIPAM), and N-(3-aminopropyl) methacrylamide hydrochloride (APMA). N,N-Methylene bis(acrylamide) (BIS) was used as a crosslinking monomer to form the interconnected polymeric network. The produced nanogels were immobilised onto a planar gold surface and characterised using the optical technique of surface plasmon resonance. They showed high selectivity and affinity towards their template, with affinity constants of 79.4 and 89.7 nM for the Rgp and Kgp epitope nanogels, respectively. From their calibration curves, the theoretical limit of detection was determined to be 1.27 nM for the Rgp nanogels and 2.00 nM for the Kgp nanogels. Furthermore, they also showed excellent selectivity against bacterial culture supernatants E8 (Rgp knockout), K1A (Kgp knockout), and W50-d (wild-type) strains in complex medium of brain heart infusion (BHI).

INTRODUCTION: Oral health professional (OHP) education is likely to vary across Europe in accordance with an EU directive that is open to broad interpretation. It is not clear how OHP curricula are structured or delivered across Europe. The objectives of Part 2 of this paper series are: (i) to provide an overview of common practices in curriculum structure, the availability of facilities, staffing (faculty) and quality assurance processes and (ii) to consider how the existing programme structures align to stakeholder guidance documents.

METHODS: A total of 27 questions from a 91-item questionnaire were used for this manuscript. The questionnaire was developed following the Delphi method to establish consensus from a group of experts. Members of the research team and colleagues from other countries in Europe completed a multi-step piloting process. An online data hub was created to allow the respondents to be data controllers and respond to the questionnaire. ADEE member schools (n = 144) were invited to provide data.

RESULTS: Totally, 71 institutions from 25 European countries provided data between June 2021 and April 2023, which represents a response rate of 49.3% of ADEE members. Data on curriculum approaches, teaching methods, integration of topics of interest, clinical education, staff-student ratios, access to facilities and new technologies, teaching staff (faculty) and quality assurance processes are presented for Primary Dental Degree Programmes.

CONCLUSION: To the best of our knowledge, this series of papers are the first attempts to provide a comprehensive overview of OHP education in Europe. Results showed that the majority of European dental programmes are engaged in providing innovative and scientifically grounded education in order to develop quality future OHPs. Nevertheless, significant variability in the delivery of clinical education across the European OHP schools was notable in this dataset. A comprehensive view of the state of OHP education in Europe is not yet available but the O-Health-Edu data hub provides a means for all education providers in Europe to contribute data to reach this goal. It is anticipated that the data hub will be updated and built upon over time to continually establish a clearer picture of the state of OHP education in Europe.

Introduction: Current legislation leaves Oral Health Professional (OHP) education open to wide interpretation and may result in significant variation in educational practice and resultant professional attributes across Europe. Data regarding the current state of OHP education across Europe is limited. The aim of Part 1 of this series is to provide programme-level data for Primary Dental Degree Programmes, Dental Hygiene and Postgraduate Education.Methods: A 91-item questionnaire was developed following the Delphi method. The questionnaire and the Articulate glossary of OHP education terms were developed concurrently to facilitate a common understanding of language. Piloting was performed in multiple stages and included institutions internal and external to the research group. The questionnaire was uploaded online and converted to a data hub, allowing dental schools to control their own data and update the data provided whenever they wish. All ADEE member schools (n = 144) were invited to provide data. Forty questions relating to school details, Primary Dental Degree Programmes, Dental Hygiene and Postgraduate Education were included in this part of the series.Results: Seventy-one institutions from 25 European countries provided data between June 2021 and April 2023, which represents a response rate of 49.3% of ADEE members. Programme-level data for Primary Dental Degree Programmes, Dental Hygiene and Postgraduate Education is presented including programme length, funding, languages and fees, student numbers and demographics, student admission and selection processes and permission to practice after graduation.Conclusion: This series of papers, as far as the authors are aware, are the first attempts to build a comprehensive picture of the current state of OHP education in Europe. A comprehensive view of the state of OHP education in Europe is not yet available but the O-Health-Edu data hub provides a means for all education providers in Europe to contribute data to reach this goal. It is anticipated that the data hub will be updated and built upon over time to continually establish a clearer picture of the state of OHP education in Europe.

Introduction: During development of dental caries, oral biofilms undergo changes in microbial composition and phenotypical traits. The aim of this study was to compare the acid tolerance (AT) of plaque from two groups of children: one with severe caries (CA) and one with no caries experience (CF) and to correlate this to the microbial composition and metabolic profile of the biofilms.

Methods: Dental plaque samples from 20 children (2–5 years) in each group were studied. The AT was analyzed by viability assessment after exposure to an acid challenge (pH 3.5), using LIVE/DEAD® BacLight™ stain and confocal microscopy. Levels of acid tolerance (AT) were evaluated using a scoring system ranging from 1 (no/low AT), to 5 (high/all AT). Metabolic profiles were investigated following a 20 mM glucose pulse for one hour through Nuclear Magnetic Resonance (NMR). Microbial composition was characterized by 16S rRNA Illumina sequencing.

Results: The mean AT score of the CA group (4.1) was significantly higher than that of the CF group (2.6, p < 0.05). When comparing the end-products of glucose metabolism detected after a glucose-pulse, the CA samples showed a significantly higher lactate to acetate, lactate to formate, lactate to succinate and lactate to ethanol ratio than the CF samples (p < 0.05). The bacterial characterization of the samples revealed 25 species significantly more abundant in the CA samples, including species of Streptococcus, Prevotella, Leptotrichia and Veillonella (p < 0.05).

Discussion: Our results show that AT in pooled plaque from the oral cavity of children with severe caries is increased compared to that in healthy subjects and that this can be related to differences in the metabolic activity and microbial composition of the biofilms. Thus, the overall phenotype of dental plaque appears to be a promising indicator of the caries status of individuals. However, longitudinal studies investigating how the AT changes over time in relation to caries development are needed before plaque AT could be considered as a prediction method for the development of dental caries.

INTRODUCTION: The Erasmus+O-Health-EDU project aims to gain a comprehensive view of oral health professional (OHP) education in Europe, through the development of web-based surveys and online toolkits. A glossary to facilitate a common language through which academic teams could cooperate and communicate more accurately was identified as a key need within the project. The aim of ARTICULATE was thus to create a shared language, with a European focus, for terms and concepts used in the field of OHP education.

METHODS: The methodology was developed from those published for construction of other glossaries with a circular and iterative process: the creation of content and definitions by a group of experts in OHP education, the testing of "fitness for purpose" of the content, and stakeholder consultation. All creation steps were followed by refinements based on testing results and stakeholder comments. The final glossary was then launched as an online resource including a built-in mechanism for user feedback.

RESULTS: The scope and structure of the glossary were mapped out at a workshop with 12 dental education experts from 7 European countries. A total of 328 terms were identified, of which 171 were finally included in ARTICULATE. After piloting with a close group of other colleagues, the glossary was opened for external input. Thirty European Deans or Heads of Education assessed the definition of each term as "clear" or "not clear." A total of 86 definitions were described as "clear" by all individuals. Terms deemed unclear by at least one individual were revisited and changes made to 37 of the definitions. In conjunction with the launch of the glossary, a range of stakeholder organisations were informed and asked to participate in an open global consultation by providing feedback online. Since its launch in June 2021, the ARTICULATE website (https://o-health-edu.org/articulate) has had an average of 500 visits/month. To promote community ownership, forms embedded on the ARTICULATE webpage allow users to give feedback and suggest new terms. A standing taskforce will meet regularly to consider amendments and make changes to ensure that the glossary remains a relevant and up-to-date resource over time.

CONCLUSION: ARTICULATE is a unique, evolving, online glossary of terms relating to OHP education, created as a resource for all interested OHP educators. The glossary is a key output of the O-Health-Edu project, which relies on a comprehensive vision of OHP education to address the future oral health needs of the European population.

The oral microbiota, which is known to include at least 600 different bacterial species, is found on the teethand mucosal surfaces as multi-species communities or biofilms. The oral surfaces are covered with a pellicleof proteins absorbed from saliva, and biofilm formation is initiated when primary colonizers, which expresssurface adhesins that bind to specific salivary components, attach to the oral tissues. Further developmentthen proceeds through co-aggregation of additional species. Over time, the composition of oral biofilms,which varies between different sites throughout the oral cavity, is determined by a combination ofenvironmental factors such as the properties of the underlying surface, nutrient availability and oxygenlevels, and bacterial interactions within the community. A complex equilibrium between biofilm communities and the host is responsible for the maintenance of a healthy biofilm phenotype (eubiosis). In the faceof sustained environmental perturbation, however, biofilm homeostasis can break down giving rise todysbiosis, which is associated with the development of oral diseases such as caries and periodontitis.In vitro models have an important part to play in increasing our understanding of the complex processesinvolved in biofilm development in oral health and disease, and the requirements for experimental system,microbial complexity, and analysis techniques will necessarily vary depending on the question posed. In thischapter we describe some current and well-established methods used in our laboratory for studying oralbacteria in biofilm models which can be adapted to suit the needs of individual users. 

Probiotic bacteria show promising results in prevention of the biofilm-mediated disease caries, but the mechanisms are not fully understood. The acid tolerance response (ATR) allows biofilm bacteria to survive and metabolize at low pH resulting from microbial carbohydrate fermentation. We have studied the effect of probiotic strains: Limosilactobacillus reuteri and Lacticaseibacillus rhamnosus on ATR induction in common oral bacteria. Communities of L. reuteri ATCC PTA5289 and Streptoccus gordonii, Streptococcus oralis, Streptococcus mutans or Actinomyces naeslundii in the initial stages of biofilm formation were exposed to pH 5.5 to allow ATR induction, followed by a low pH challenge. Acid tolerance was evaluated as viable cells after staining with LIVE/ DEAD & REG;BacLightTM. The presence of L. reuteri ATCC PTA5289 caused a significant reduction in acid tolerance in all strains except S. oralis. When S. mutans was used as a model organism to study the effects of additional probiotic strains (L. reuteri SD2112, L. reuteri DSM17938 or L. rhamnosus GG) as well as L. reuteri ATCC PTA5289 supernatant on ATR development, neither the other probiotic strains nor supernatants showed any effect. The presence of L. reuteri ATCC PTA5289 during ATR induction led to down-regulation of three key genes involved in tolerance of acid stress (luxS, brpA and ldh) in Streptococci. These data suggest that live cells of probiotic L. reuteri ATCC PTA5289 can interfere with ATR development in common oral bacteria and specific strains of L. reuteri may thus have a role in caries prevention by inhibiting development of an acid-tolerant biofilm microbiota.