Candida albicans biofilm heterogeneity and tolerance of clinical isolates: implications for secondary endodontic infections

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

Comparison of three endodontic irrigant regimens against dual-species interkingdom biofilms: considerations for maintaining the status quo

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

Candida albicans and Enterococcus faecalis biofilm frenemies: when the relationship sours

The opportunistic yeast Candida albicans and lactic acid bacteria Enterococcus faecalis are frequently co-isolated from various infection sites on the human body, suggesting a common interkingdom interaction. While some reports suggest an antagonism, the reason for their co-isolation therefore remains unclear. The purpose of this study was to undertake a detailed characterisation of this dual-species interaction. We used standard biofilm characterisation methodologies alongside an RNASeq analysis to assess the response of C. albicans to E. faecalis. We evaluated the relevance of pH to dual-species biofilm interactions and demonstrated that E. faecalis rapidly and significantly impacted C. albicans morphogenesis and biofilm formation, which was mirrored by levels of gene expression. These transcripts were enriched in amino acids biosynthesis and metabolism pathways in co-cultures, a finding that guided our investigation into pH related mechanism. We were able to demonstrate the direct role of E. faecalis induced low pH, which inhibited C. albicans hyphal morphogenesis and biofilm formation. The results suggest that the anti-candidal effect of E. faecalis is not based solely on a single mechanism, instead it may involve various mechanisms, which collectively reflects the complexity of interaction between C. albicans and E. faecalis and impacts treatment outcomes

Candida albicans as an essential "keystone" component within polymicrobial oral biofilm models?

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. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Candida albicans and Enterococcus faecalis interkingdom interaction: implications for management of endodontic infections

Endodontic disease is inflammation or infection of the pulp tissue lying within the root canal space of teeth and the associated periapical tissue, also known as periapical periodontitis. It is estimated that around 52% of adults worldwide have at least one tooth with periapical periodontitis. Root canal infections are usually debilitating, associated with pain, discomfort and decreased quality of life. The role of microorganisms in the pathogenesis of periapical periodontitis is well established. Root canal infections are characterised by biofilms, i.e. polymicrobial infections encased in polymers that are isolated from infection sites. Candida albicans and Enterococcus faecalis are common endodontic pathogens that have been linked with persistent endodontic infections. Deeper understanding of their response to endodontic procedures and how they interact with each other may enhance our understanding of their role and lead to better treatment outcomes. Chapter 1: reviews the literature in relation to endodontic infections and their related microbiology, basics of endodontic treatment, C. albicans and E. faecalis as endodontic pathogens and C. albicans and E. faecalis in mixed biofilms. Chapter 2: is presented in the traditional chapter format. It aimed at assessing a panel of C. albicans oral clinical isolates for their ability to form biofilms using the crystal violet assay (biomass). Following the selection of representative low and high biofilm forming isolates, the response of different C. albicans phenotypes to NaOCl was assessed using XTT (metabolism), crystal violet and microscopic imaging. C. albicans showed an ability to tolerate NaOCl treatment and was able to regrow when a nutrient source is provided. In addition, C. albicans heterogeneity influenced the response to NaOCl treatment, where low biofilm forming isolates showed increased tolerance compared with high biofilm forming isolates. Chapter 3: is presented as a published journal article. It further analysed the response of single species of C. albicans biofilms to NaOCl. It also assessed the effect of sequential treatment with sodium hypochlorite followed by EDTA on C. albicans biofilms viability using XXT and biofilm biomass using crystal violet assays. In this analysis, the immediate post treatment effect as well as the regrowth potential over 72h of re-incubation with fresh media were investigated. Treatment with NaOCl alone resulted in a significant regrowth of the treated population while regrowth was significantly inhibited with sequential treatment. Chapter 4: is presented as a published journal article. In this chapter the complexity of the used model was increased by incorporating E. faecalis into the subsequent analysis. C. albicans and E. faecalis mono- and dual-species biofilms were assessed against sequential (NaOCl followed by EDTA) or continuous chelation protocols (NaOCl combined with HEDP) using qPCR and microscopic examination. Sequential treatment with NaOCl and EDTA were more effective in inhibiting C. albicans and E. faecalis regrowth compared with continuous chelation. In terms of the difference between mono- and dual-species, C. albicans was more susceptible to endodontic irrigants when co-cultured with E. faecalis, while E. faecalis remained unaffected. The regrowth of C. albicans was significantly inhibited when cocultured with E. faecalis. Chapter 5: is presented as a published journal article. Firstly, the effect of E. faecalis on C. albicans biomarkers was assessed using crystal violet, microscopy and real time PCR. Afterwards, RNA seq was employed to investigate the molecular basis of the interaction between C. albicans and E. faecalis. The expression of EntV across twelve E. faecalis strains was also measured and correlated with the degree of C. albicans biofilm inhibition. Finally, the effect of pH modified and ultrafiltered E. faecalis supernatant on C. albicans hyphal morphogenesis and biofilm formation was assessed. The results show that E. faecalis inhibited C. albicans growth, hyphal morphogenesis and biofilm formation. The transcriptional analysis showed rapid and significant changes in the C. albicans transcriptome characterized by upregulation in amino acid biosynthesis and metabolism genes. This guided further analysis on EntV and pH dependent mechanisms where C. albicans biofilm inhibition were shown to be related to an E. faecalis induced environmental pH drop. In conclusion, this thesis highlights the importance of considering the tolerance potential of the treated microorganisms and the role it may have in the aetiology of disease persistence. It also contributes to our knowledge of the interaction between microorganisms in polymicrobial infections and how the ultimate outcome of this interaction may impact disease progression and prognosis

Clinical management of fungal biofilm infections

Fungal biofilms are highly resilient to antifungal therapies, of which there are relatively few licensed options available in clinical medicine. Nonetheless, there is a vibrant research culture aimed at enhancing and expanding the arsenal of antifungals capable of inhibiting, killing, and disrupting fungal biofilms. This chapter aims to explore the wide variety of fungal biofilms affecting human health and to discuss the clinical options for existing and novel chemotherapeutics

Detection, treatment and prevention of endodontic biofilm infections: what's new in 2020?

Endodontic disease, a biofilm infection of the root canal space, is a significant cause of dental morbidity worldwide. Endodontic treatment, or root canal treatment, as it is commonly known is founded on the ability to eradicate microbial biofilm infection and prevent re-infection of the highly complex root canal space. Despite many “advances” in clinical endodontics we have seen little improvement in outcomes. The aim of this critical review paper is to provide a contemporary view of endodontic microbiology and biofilm polymicrobiality, provide an understanding of the host response, and how together these impact upon clinical treatment. Ultimately, it is intended to provide insight into novel opportunities and strategies for the future diagnostics, treatment, and prevention of endodontic disease

Small molecule based anti-virulence approaches against Candida albicans infections

Fungi are considered “silent killers” due to the difficulty of, and delays in diagnosis of infections and lack of effective antifungals. This challenge is compounded by the fact that being eukaryotes, fungi share several similarities with human cellular targets, creating obstacles to drug discovery. Candida albicans, a ubiquitous microbe in the human body is well-known for its role as an opportunistic pathogen in immunosuppressed people. Significantly, C. albicans is resistant to all the three classes of antifungals that are currently clinically available. Over the past few years, a paradigm shift has been recommended in the management of C. albicans infections, wherein anti-virulence strategies are considered an alternative to the discovery of new antimycotics. Small molecules, with a molecular weight <900 Daltons, can easily permeate the cell membrane and modulate the signal transduction pathways to elicit desired virulence inhibitory actions against pathogens. This review dissects in-depth, the discoveries that have been made with small-molecule anti-virulence approaches to tackle C. albicans infections

Screening the Tocriscreen™ bioactive compound library in search for inhibitors of Candida biofilm formation

Background and purpose: Biofilms formed by Candida species present a significant clinical problem due to the ineffectiveness of many conventional antifungal agents, in particular the azole class. We urgently require new and clinically approved antifungal agents quickly for treatment of critically ill patients. Methods: To improve efficiency in antifungal drug development, we utilized a library of 1280 biologically active molecules within the Tocriscreen 2.0 Micro library. C. auris NCPF 8973 and C. albicans SC5314 were initially screened for biofilm inhibitory activity using metabolic and biomass quantitative assessment methods, followed up by targeted evaluation of five selected hits. Results: The initial screening (80% metabolic inhibition rate) revealed that there was 90 and 87 hits (approx. 7%) for C. albicans and C. auris, respectively. Additionally, all five compounds selected from the initial hits exhibited a biofilm inhibition effect against several key Candida species tested, including C. glabrata and C. krusei. Toyocamycin displayed the most potent activity at concentrations as low as 0.5 µg/mL, though was limited to inhibition. Darapladib demonstrated an efficacy for biofilm inhibition and treatment at a concentration range from 8 to 32 µg/mL and from 16 to 256 µg/mL, respectively. Combinational testing with conventional antifungals against C. albicans strains demonstrated a range of synergies for planktonic cells, and notably an anti-biofilm synergy for darapladib and caspofungin. Interpretation: Together, these data provide new insights into antifungal management possibilities for Candida biofilms