Impact of frequency of denture cleaning on microbial and clinical parameters - a bench to chairside approach.

Objective: Robust scientific and clinical evidence of how to appropriately manage denture plaque is lacking. This two-part study (i) developed an in vitro model of denture plaque removal, and (ii) assessed effectiveness of these approaches in a randomised clinical trial. Method: (i) a complex denture plaque model was developed using the dominant microbial genera from a recent microbiome analyses. Biofilms formed on polymethylmethacrylate were brushed daily with a wet toothbrush, then either treated daily for 5 days or only on Days 1 and 5 with Polident® denture cleanser tablets (3 min soaking). Quantitative and qualitative microbiological assessments were performed. (ii), an examiner-blind, randomised, crossover study of complete maxillary denture wearers was performed (n = 19). Either once-daily for 7 days or on Day 7 only, participants soaked dentures for 15 min using Corega® denture cleansing tables, then brushed. Denture plaque microbiological assessment used sterilized filter paper discs. Results: The in vitro model showed daily cleaning with denture cleanser plus brushing significantly reduced microbial numbers compared to intermittent denture cleaning with daily brushing (p < 0.001). The clinical component of the study showed a statistically significant reduction in denture plaque microbial numbers in favour of daily versus weekly treatment (aerobic bacteria p = 0.0144). Both in vitro and in vivo studies showed that denture plaque biofilm composition were affected by different treatment arms. Conclusions: This study demonstrated that daily denture cleansing regimens are superior to intermittent denture cleansing, and that cleansing regimens can induce denture plaque compositional changes. Clinicaltrials.gov registration: NCT02780661.

Interkingdom interactions on the denture surface: Implications for oral hygiene.

BACKGROUND: Evidence to support the role of Candida species in oral disease is limited. Often considered a commensal, this opportunistic yeast has been shown to play a role in denture related disease, though whether it is an active participant or innocent bystander remains to be determined. This study sought to understand the role of Candida species alongside the bacterial microbiome in a denture patient cohort, exploring how the microbiology of the denture was affected by oral hygiene practices. MATERIALS AND METHODS: In vitro denture cleansing studies were performed on a complex 9-species interkingdom denture biofilm model, with quantitative assessment of retained bacterial and fungal viable bioburdens. Patient hygiene measures were also collected from 131 patients, including OHIP, frequency of denture cleansing, oral hygiene measure and patient demographics. The bacterial microbiome was analysed from each patient, alongside quantitative PCR assessment of ITS (fungal) and 16S (bacterial) bioburden from denture, mucosa and intact dentition. RESULTS: It was shown that following in vitro denture cleansing C. albicans were unresponsive to treatment, whereas bacterial biofilms could repopulate 100-fold, but were susceptible to subsequent treatment. Within the patient cohort, oral hygiene did not impact candidal or bacterial composition, nor diversity. The levels of Candida did not significantly influence the bacterial microbiome, though an observed gradient was suggestive of a microbial composition change in response to Candida load, indicating interkingdom interaction rather than an oral hygiene effect. Indeed, correlation analysis was able to show significant correlations between Candida species and key genera (Lactobacillus, Scardovia, Fusobacterium). CONCLUSIONS: Overall, this study has shown that the denture microbiome/mycobiome is relatively resilient to oral hygiene challenges, but that Candida species have potential interactions with key oral genera. These interactions may have a bearing on shaping community structure and a shift from health to disease when the opportunity arises.

Denture Stomatitis: Causes, Cures and Prevention.

Removable dentures are worn by 20% of the UK population and two thirds of these individuals have denture stomatitis. Poor oral hygiene is commonplace among this group, as is smoking and xerostomia, which also contribute to the development of denture stomatitis. A complex polymicrobial biofilm is able to proliferate on the surface of denture materials and matures to form visible denture plaque. This denture plaque biofilm stimulates a local inflammatory process that is detectable clinically as erythema, and hyperplasia. Systemically, denture plaque represents a potential risk factor for systemic disease, in particular aspiration pneumonia. Respiratory pathogens have been detected in the denture plaque and overnight denture wear has been linked to an increased risk of aspiration pneumonia. There is a general lack of evidence on the adequate management of denture stomatitis and we present a protocol for use in the primary care setting.

Biofilms Formed by Isolates from Recurrent Vulvovaginal Candidiasis Patients Are Heterogeneous and Insensitive to Fluconazole.

Vulvovaginal candidiasis (VVC) is a global health problem affecting ∼75% of women at least once in their lifetime. Here we examined the epidemiology of VVC in a patient cohort to identify the causative organisms associated with VVC. Biofilm-forming capacity and antifungal sensitivity profiles were also assessed. We report a shifting prevalence of Candida species with heterogeneous biofilm-forming capacity, which is associated with altered antifungal drug sensitivity.

The application of phenotypic microarray analysis to anti-fungal drug development.

Candida albicans metabolic activity in the presence and absence of acetylcholine was measured using phenotypic microarray analysis. Acetylcholine inhibited C. albicans biofilm formation by slowing metabolism independent of biofilm forming capabilities. Phenotypic microarray analysis can therefore be used for screening compound libraries for novel anti-fungal drugs and measuring antifungal resistance.

Candida albicans biofilm heterogeneity does not influence denture stomatitis but strongly influences denture cleansing capacity.

Approximately 20 % of the UK population wear some form of denture prosthesis, resulting in denture stomatitis in half of these individuals. Candida albicans is primarily attributed as the causative agent, due to its biofilm -forming ability. Recently, there has been increasing evidence of C. albicans biofilm heterogeneity and the negative impact it can have clinically; however, this phenomenon has yet to be studied in relation to denture isolates. The aims of this study were to evaluate C. albicans biofilm formation of clinical denture isolates in a denture environment and to assess antimicrobial activity of common denture cleansers against these tenacious communities. C. albicans isolated from dentures of healthy and diseased individuals was quantified using real-time PCR and biofilm biomass assessed using crystal violet. Biofilm development on the denture substratum poly(methyl methacrylate), Molloplast B and Ufi-gel was determined. Biofilm formation was assessed using metabolic and biomass stains, following treatment with denture hygiene products. Although C. albicans was detected in greater quantities in diseased individuals, it was not associated with increased biofilm biomass. Denture substrata were shown to influence biofilm biomass, with poly(methyl methacrylate) providing the most suitable environment for C. albicans to reside. Of all denture hygiene products tested, Milton had the most effective antimicrobial activity, reducing biofilm biomass and viability the greatest. Overall, our results highlight the complex nature of denture- related disease, and disease development cannot always be attributed to a sole cause. It is the distinct combination of various factors that ultimately determines the pathogenic outcome.

Viable Compositional Analysis of an Eleven Species Oral Polymicrobial Biofilm.

PURPOSE: Polymicrobial biofilms are abundant in clinical disease, particularly within the oral cavity. Creating complex biofilm models that recapitulate the polymicrobiality of oral disease are important in the development of new chemotherapeutic agents. In order to do this accurately we require the ability to undertake compositional analysis, in addition to determine individual cell viability, which is difficult using conventional microbiology. The aim of this study was to develop a defined multispecies denture biofilm model in vitro, and to assess viable compositional analysis following defined oral hygiene regimens. METHODS: An in vitro multispecies denture biofilm containing various oral commensal and pathogenic bacteria and yeast was created on poly (methyl methacrylate) (PMMA). Denture hygiene regimens tested against the biofilm model included brushing only, denture cleansing only and combinational brushing and denture cleansing. Biofilm composition and viability were assessed by culture (CFU) and molecular (qPCR) methodologies. Scanning electron microscopy and confocal laser scanning microscopy were also employed to visualize changes in denture biofilms following treatment. RESULTS: Combinational treatment of brushing and denture cleansing had the greatest impact on multispecies denture biofilms, reducing the number of live cells by more than 2 logs, and altering the overall composition in favor of streptococci. This was even more evident during the sequential testing, whereby daily sequential treatment reduced the total and live number of bacteria and yeast more than those treated intermittently. Bacteria and yeast remaining following treatment tended to aggregate in the pores of the PMMA, proving more difficult to fully eradicate the biofilm. CONCLUSIONS: Overall, we are the first to develop a method to enable viable compositional analysis of an 11 species denture biofilm following chemotherapeutic challenge. We were able to demonstrate viable cell reduction and changes in population dynamics following evaluation of various denture cleansing regimens. Specifically, it was demonstrated that daily combinational treatment of brushing and cleansing proved to be the most advantageous denture hygiene regimen, however, residual organisms still remained within the pores of PMMA surface, which could act as a reservoir for further biofilm regrowth. We have identified an industry need for denture cleansing agents with the capacity to penetrate these pores and disaggregate these complex biofilm consortia.

The microbiome associated with equine periodontitis and oral health.

Equine periodontal disease is a common and painful condition and its severe form, periodontitis, can lead to tooth loss. Its aetiopathogenesis remains poorly understood despite recent increased awareness of this disorder amongst the veterinary profession. Bacteria have been found to be causative agents of the disease in other species, but current understanding of their role in equine periodontitis is extremely limited. The aim of this study was to use high-throughput sequencing to identify the microbiome associated with equine periodontitis and oral health. Subgingival plaque samples from 24 horses with periodontitis and gingival swabs from 24 orally healthy horses were collected. DNA was extracted from samples, the V3-V4 region of the bacterial 16S rRNA gene amplified by PCR and amplicons sequenced using Illumina MiSeq. Data processing was conducted using USEARCH and QIIME. Diversity analyses were performed with PAST v3.02. Linear discriminant analysis effect size (LEfSe) was used to determine differences between the groups. In total, 1308 OTUs were identified and classified into 356 genera or higher taxa. Microbial profiles at health differed significantly from periodontitis, both in their composition (p < 0.0001, F = 12.24; PERMANOVA) and in microbial diversity (p < 0.001; Mann-Whitney test). Samples from healthy horses were less diverse (1.78, SD 0.74; Shannon diversity index) and were dominated by the genera Gemella and Actinobacillus, while the periodontitis group samples showed higher diversity (3.16, SD 0.98) and were dominated by the genera Prevotella and Veillonella. It is concluded that the microbiomes associated with equine oral health and periodontitis are distinct, with the latter displaying greater microbial diversity.

One step closer to understanding the role of bacteria in diabetic foot ulcers: characterising the microbiome of ulcers.

BACKGROUND: The aim of this study was to characterise the microbiome of new and recurrent diabetic foot ulcers using 16S amplicon sequencing (16S AS), allowing the identification of a wider range of bacterial species that may be important in the development of chronicity in these debilitating wounds. Twenty patients not receiving antibiotics for the past three months were selected, with swabs taken from each individual for culture and 16S AS. DNA was isolated using a combination of bead beating and kit extraction. Samples were sequenced on the Illumina Hiseq 2500 platform. RESULTS: Conventional laboratory culture showed positive growth from only 55 % of the patients, whereas 16S AS was positive for 75 % of the patients (41 unique genera, representing 82 different operational taxonomic units (OTU's). S. aureus was isolated in 72 % of culture-positive samples, whereas the most commonly detected bacteria in all ulcers were Peptoniphilus spp., Anaerococcus spp. and Corynebacterium spp., with the addition of Staphylococcus spp. in new ulcers. The majority of OTU's residing in both new and recurrent ulcers (over 67 %) were identified as facultative or strict anaerobic Gram-positive organisms. Principal component analysis (PCA) showed no difference in clustering between the two groups (new and recurrent ulcers). CONCLUSIONS: The abundance of anaerobic bacteria has important implications for treatment as it suggests that the microbiome of each ulcer "starts afresh" and that, although diverse, are not distinctly different from one another with respect to new or recurrent ulcers. Therefore, when considering antibiotic therapy the duration of current ulceration may be a more important consideration than a history of healed ulcer.

Dentures are a Reservoir for Respiratory Pathogens.

PURPOSE: Recent studies have established a relationship between dental plaque and pulmonary infection, particularly in elderly individuals. Given that approximately one in five adults in the UK currently wears a denture, there remains a gap in our understanding of the direct implications of denture plaque on systemic health. The aim of this study was to undertake a comprehensive evaluation of putative respiratory pathogens residing upon dentures using a targeted quantitative molecular approach. MATERIALS AND METHODS: One hundred and thirty patients' dentures were sonicated to remove denture plaque biofilm from the surface. DNA was extracted from the samples and was assessed for the presence of respiratory pathogens by quantitative polymerase chain reaction (qPCR). Ct values were then used to approximate the number of corresponding colony forming equivalents (CFEs) based on standard curves. RESULTS: Of the dentures, 64.6% were colonized by known respiratory pathogens. Six species were identified: Streptococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, Haemophilus influenzae B, Streptococcus pyogenes, and Moraxella catarrhalis. P. aeruginosa was the most abundant species followed by S. pneumoniae and S. aureus in terms of average CFE and overall proportion of denture plaque. Of the participants, 37% suffered from denture stomatitis; however, there were no significant differences in the prevalence of respiratory pathogens on dentures between healthy and inflamed mouths. CONCLUSIONS: Our findings indicate that dentures can act as a reservoir for potential respiratory pathogens in the oral cavity, thus increasing the theoretical risk of developing aspiration pneumonia. Implementation of routine denture hygiene practices could help to reduce the risk of respiratory infection among the elderly population.

The Oral Microbiome of Denture Wearers Is Influenced by Levels of Natural Dentition.

OBJECTIVES: The composition of dental plaque has been well defined, whereas currently there is limited understanding of the composition of denture plaque and how it directly influences denture related stomatitis (DS). The aims of this study were to compare the microbiomes of denture wearers, and to understand the implications of these towards inter-kingdom and host-pathogen interactions within the oral cavity. METHODS: Swab samples were obtained from 123 participants wearing either a complete or partial denture; the bacterial composition of each sample was determined using bar-coded illumina MiSeq sequencing of the bacterial hypervariable V4 region of 16S rDNA. Sequencing data processing was undertaken using QIIME, clustered in Operational Taxonomic Units (OTUs) and assigned to taxonomy. The dentures were sonicated to remove the microbial flora residing on the prosthesis, sonicate was then cultured using diagnostic colorex Candida media. Samples of unstimulated saliva were obtained and antimicrobial peptides (AMP) levels were measured by ELISA. RESULTS: We have shown that dental and denture plaques are significantly distinct both in composition and diversity and that the oral microbiome composition of a denture wearer is variable and is influenced by the location within the mouth. Dentures and mucosa were predominantly made up of Bacilli and Actinobacteria. Moreover, the presence of natural teeth has a significant impact on the overall microbial composition, when compared to the fully edentulous. Furthermore, increasing levels of Candida spp. positively correlate with Lactobacillus spp. AMPs were quantified, though showed no specific correlations. CONCLUSIONS: This is the first study to provide a detailed understanding of the oral microbiome of denture wearers and has provided evidence that DS development is more complex than simply a candidal infection. Both fungal and bacterial kingdoms clearly play a role in defining the progression of DS, though we were unable to show a defined role for AMPs.

Polymicrobial Candida biofilms: friends and foe in the oral cavity.

The role of polymicrobial biofilm infections in medicine is becoming more apparent. Increasing number of microbiome studies and deep sequencing has enabled us to develop a greater understanding of how positive and negative microbial interactions influence disease outcomes. An environment where this is particularly pertinent is within the oral cavity, a rich and diverse ecosystem inhabited by both bacteria and yeasts, which collectively occupy and coexist within various niches as biofilm communities. Studies within this environment have however tended to be subject to extensive independent investigation, in the context of either polymicrobial bacterial communities or yeast biofilms, but rarely both together. It is clear however that they are not mutually exclusive. Therefore, this review aims to explore the influence of candidal populations on the composition of these complex aggregates and biofilm communities, to investigate their mechanistic interactions to understand how these impact clinical outcomes, and determine whether we can translate how this knowledge can be used to improve patient management.

Acetylcholine Protects against Candida albicans Infection by Inhibiting Biofilm Formation and Promoting Hemocyte Function in a Galleria mellonella Infection Model.

Both neuronal acetylcholine and nonneuronal acetylcholine have been demonstrated to modulate inflammatory responses. Studies investigating the role of acetylcholine in the pathogenesis of bacterial infections have revealed contradictory findings with regard to disease outcome. At present, the role of acetylcholine in the pathogenesis of fungal infections is unknown. Therefore, the aim of this study was to determine whether acetylcholine plays a role in fungal biofilm formation and the pathogenesis of Candida albicans infection. The effect of acetylcholine on C. albicans biofilm formation and metabolism in vitro was assessed using a crystal violet assay and phenotypic microarray analysis. Its effect on the outcome of a C. albicans infection, fungal burden, and biofilm formation were investigated in vivo using a Galleria mellonella infection model. In addition, its effect on modulation of host immunity to C. albicans infection was also determined in vivo using hemocyte counts, cytospin analysis, larval histology, lysozyme assays, hemolytic assays, and real-time PCR. Acetylcholine was shown to have the ability to inhibit C. albicans biofilm formation in vitro and in vivo. In addition, acetylcholine protected G. mellonella larvae from C. albicans infection mortality. The in vivo protection occurred through acetylcholine enhancing the function of hemocytes while at the same time inhibiting C. albicans biofilm formation. Furthermore, acetylcholine also inhibited inflammation-induced damage to internal organs. This is the first demonstration of a role for acetylcholine in protection against fungal infections, in addition to being the first report that this molecule can inhibit C. albicans biofilm formation. Therefore, acetylcholine has the capacity to modulate complex host-fungal interactions and plays a role in dictating the pathogenesis of fungal infections.

Rate of pathologic complete responses to docetaxel, cisplatin, and fluorouracil induction chemotherapy in patients with squamous cell carcinoma of the head and neck.

OBJECTIVE: To report the rate of pathological complete response after induction chemotherapy with the docetaxel, cisplatin, and fluorouracil (TPF) combination. DESIGN: Retrospective cohort analysis. SETTING: Tertiary care academic cancer center, between June 1999 and May 2004. PATIENTS: Seventy-two patients with newly diagnosed squamous cell carcinoma of the head and neck; 68 (95%) of the patients had stage IV, locally advanced disease. INTERVENTIONS: Three cycles of induction chemotherapy followed by a biopsy of the primary site. All patients subsequently underwent chemotherapy with 3 cycles of TPF. MAIN OUTCOME MEASURE: Rate of pathological complete response at the primary site after induction chemotherapy with 3 cycles of TPF. RESULTS: Biopsy results were negative for cancer in 64 patients (89%) and positive in 8 patients (11%). The median follow-up was 2 years. In the positive biopsy result group, 2 (25%) of 8 patients died of disease vs 3 (4%) of 64 patients in the negative biopsy result group. Twenty-nine neck dissections were performed; results were positive in 7 patients (all alive with no evidence of disease) and negative in 22 patients (21 alive with no evidence of disease). The overall 2- and 5-year progression-free survival is currently projected at 85% and 85%, respectively; the overall 2- and 5-year survival, at 95% and 90%, respectively. Importantly, T4 presentation did not predict a positive biopsy result at the primary site or a positive neck dissection result (P = .60 and P = .56, respectively). N3 presentation (12 patients) did not predict a positive biopsy result at the primary site (P = .87) but did correlate with positive neck dissection results in 6 of 12 patients (P<.001). CONCLUSIONS: Induction chemotherapy with the TPF regimen results in a high pathological complete response rate (89%). This rate is higher than with the cisplatin plus fluorouracil combination therapy, which was reported to be between 25% and 50% in previous studies. Chemoradiotherapy is currently an accepted standard of care, but induction chemotherapy continues to be investigated. Based on recent phase 3 trial results and the data presented herein, we propose that the 3-drug combination be used as the new platform when administering induction chemotherapy.