Background: Existing standardized biofilm assays focus on simple mono-species or bacterial-only models. Incorporating Candida albicans into complex biofilm models can offer a more appropriate and relevant polymicrobial biofilm for the development of oral health products. Aims: This study aimed to assess the importance of interkingdom interactions in polymicrobial oral biofilm systems with or without C. albicans, and test how these models respond to oral therapeutic challenges in vitro. Materials and Methods: Polymicrobial biofilms (two models containing 5 and 10 bacterial species, respectively) were created in parallel in the presence and absence of C. albicans and challenged using clinically relevant antimicrobials. The metabolic profiles and biomasses of these complex biofilms were estimated using resazurin dye and crystal violet stain, respectively. Quantitative PCR was utilized to assess compositional changes in microbial load. Additional assays, for measurements of pH and lactate, were included to monitor fluctuations in virulence “biomarkers.” Results: An increased level of metabolic activity and biomass in the presence of C. albicans was observed. Bacterial load was increased by more than a factor of 10 in the presence of C. albicans. Assays showed inclusion of C. albicans impacted the biofilm virulence profiles. C. albicans did not affect the biofilms’ responses to the short-term incubations with different treatments. Conclusions: The interkingdom biofilms described herein are structurally robust and exhibit all the hallmarks of a reproducible model. To our knowledge, these data are the first to test the hypothesis that yeasts may act as potential “keystone” components of oral biofilms.

Aim: Endodontic infections are caused by the invasion of various microorganisms into the root canal system. Candida albicans is a biofilm forming yeast and the most prevalent eukaryotic microorganism in endodontic infections. In this study we investigated the ability of C. albicans to tolerate treatment with standard endodontic irrigants NaOCl (sodium hypochlorite), ethylenediaminetetraacetic acid (EDTA) and a combination thereof. We hypothesized that biofilm formed from a panel of clinical isolates differentially tolerate disinfectant regimens, and this may have implications for secondary endodontic infections. Methodology: Mature C. albicans biofilms were formed from 30 laboratory and oral clinical isolates and treated with either 3% NaOCl, 17% EDTA or a sequential treatment of 3% NaOCl followed by 17% EDTA for 5 min. Biofilms were then washed, media replenished and cells reincubated for an additional 24, 48 and 72 h at 37 °C. Regrowth was quantified using metabolic reduction, electrical impedance, biofilm biomass and microscopy at 0, 24, 48 and 72 h. Results: Microscopic analysis and viability readings revealed a significant initial killing effect by NaOCl, followed by a time dependent significant regrowth of C. albicans, but with inter-strain variability. In contrast to NaOCl, there was a continuous reduction in viability after EDTA treatment. Moreover, EDTA significantly inhibited regrowth after NaOCl treatment, though viable cells were still observed. Conclusions: Our results indicate that different C. albicans biofilm phenotypes grown in a non-complex surface topography have the potential to differentially tolerate standard endodontic irrigation protocols. This is the first study to report a strain dependent impact on efficacy of endodontic irrigants. Its suggested that within the complex topography of the root canal, a more difficult antimicrobial challenge, that existing endodontic irrigant regimens permit cells to regrow and drive secondary infections.

Endodontic infections are often interkingdom biofilms, though current clinical management rarely considers this phenomenon. This study aimed to evaluate new and standard endodontic antimicrobial regimens against simple and complex Candida albicans and Enterococcus faecalis mono- and dual-species biofilms. C. albicans and E. faecalis mono- and dual-species biofilms were grown upon Thermanox™ coverslips and treated for 5 min with 3% NaOCl, 3% NaOCl followed by 17% EDTA, or 9% HEDP dissolved in 3% NaOCl. The number of cells remaining immediately after treatment at 0 h and after 72 h of regrowth were assessed using real-time quantitative PCR. All three treatment arms showed a similar positive antimicrobial effect on C. albicans and E. faecalis in both mono- and dual-species biofilms following initial treatment, resulting in ≥98% reduction in colony forming equivalent (CFE). Regardless of species or biofilm type (mono- or dual- species), the antimicrobial effect of NaOCl:HEDP mixture was comparable to that of NaOCl alone, with both showing significant regrowth after 72 h, whereas sequential treatment with NaOCl and EDTA consistently prevented significant regrowth. Our data suggest that sequential irrigation with NaOCl and EDTA remains the antimicrobial strategy of choice as it significantly reduces biofilm persistence and regrowth in our experimental dual-species biofilm conditions.

Objectives: To investigate how soluble byproducts derived from a four‐species endodontic biofilm model impact the viability, transcriptomic profile, and inflammatory response of human dental pulp stem cells (DPSCs). Methods: A sterile‐filtered supernatant was extracted from an established interkingdom endodontic biofilm model comprising Streptococcus gordonii , Fusobacterium nucleatum , Porphyromonas gingivalis, and Candida albicans . DPSCs were exposed to the microbial biofilm supernatant (BSN) for 4 and 24 h. Cellular responses were evaluated via MTT, CCK‐8, LDH assays, and Annexin V/PI staining. Transcriptomic sequencing was performed to assess gene expression dynamics, with GO and KEGG pathway enrichment analyses. IL6 and IL8 expression was validated by qPCR and ELISA. Data were analyzed using t‐tests/ANOVA and RNA‐seq differential expression using DESeq. 2 with FDR adjustment. Results: BSN significantly suppressed DPSC metabolic activity without inducing apoptosis or necrosis. RNA‐seq revealed 723 significantly differentially expressed genes at 4 h and 1667 at 24 h. Early responses were dominated by upregulation of inflammatory mediators, with enrichment of TNF, NF‐κB, and JAK‐STAT signaling pathways. At 24 h, the expression profile shifted toward redox regulation and metabolic suppression, including downregulation of glycolytic and purine metabolism pathways. IL6 and IL8 expression was markedly increased at both transcript and protein levels. Conclusions: Soluble factors produced by a biofilm model representative of deep caries and carious pulp exposures induce a time‐dependent transcriptional response in DPSCs. This response is characterized by a biphasic pattern of early immune activation followed by later transcriptional metabolic adaptation. These findings highlight the capacity of soluble biofilm‐derived products associated with deep caries to modulate DPSC immune–metabolic signaling. They further emphasize the importance of vital pulp therapy strategies that not only target microorganisms but also account for their secreted byproducts.