Compositional shifts within the denture-associated bacteriome in pneumonia - an analytical cross-sectional study.

Introduction. Bacterial pneumonia is a common cause of morbidity and mortality in elderly individuals. While the incidence of edentulism is falling, approximately 19 % of the UK population wear a full or partial removable denture. Despite advances in denture biomaterials, the majority of dentures are fabricated using polymethyl-methacrylate. Growing evidence suggests that colonization of the oral cavity by putative respiratory pathogens predisposes individuals to respiratory infection, by translocation of these microorganisms along the respiratory tract.Hypothesis/Gap Statement. We hypothesized that denture surfaces provide a susceptible colonization site for putative respiratory pathogens, and thus could increase pneumonia risk in susceptible individuals.Aim. This study aimed to characterize the bacterial community composition of denture-wearers in respiratory health compared with individuals with a confirmed diagnosis of pneumonia.Methodology. This was an analytical cross-sectional study, comparing frail elderly individuals without respiratory infection (n=35) to hospitalized patients with pneumonia (n=26). The primary outcome was the relative abundance of putative respiratory pathogens identified by 16S rRNA metataxonomic sequencing, with quantitative PCR used to identified Streptococcus pneumoniae.Results. There was a statistically significant increase in the overall relative abundance of putative respiratory pathogens (P<0.0001), with a greater than 20-fold increase in the bioburden of these microorganisms. In keeping with these findings, there were significant shifts in bacterial community diversity (Chao index, P=0.0003) and richness (Inverse Simpson index P<0.0001) in the denture-associated microbiota of pneumonia patients compared with control subjects.Conclusion. Within the limitations of this study, our evidence supports the role of denture acrylic biomaterials as a potential colonization site for putative respiratory pathogens, which may lead to an increased risk of pneumonia in susceptible individuals. These findings support prior observational studies which have found denture-wearers to be at increased risk of respiratory infection. Further research is needed to confirm the sequence of colonization and translocation to examine potential causal relationships.

Molecular community profiling of the bacterial microbiota associated with denture-related stomatitis.

Denture-associated stomatitis (DS) affects over two-thirds of denture-wearers. DS presents as erythema of the palatal mucosa in areas where denture-surface associated polymicrobial biofilms containing the fungus Candida albicans exist. The contribution of the oral bacterial microbiota toward the infection is unknown. Therefore, this study characterised the bacterial microbiota of sites within the oral cavity to identify potential associations with occurrence of DS. Denture-wearing patients were recruited (denture stomatitis (DS) n = 8; non-denture stomatitis (NoDS) n = 11) and the oral bacterial microbiota of the tongue, palate and denture-fitting surface was characterised using next-generation sequencing. Operational taxonomic units (OTUs) were identified to bacterial genera and species, and presence/absence and relative abundances were examined. A significant (P = 0.007) decrease in the number of OTUs and thus, diversity of the microbiota was observed in tongue samples of DS patients (vs non-DS). The microbiota of denture-fitting surfaces and palatal mucosae were similar. Large differences in the abundance of bacterial genera and species were observed at each sample site, and unique presence/absence of bacteria was noted. Presence/absence and relative abundance of specific bacteria associated with DS warrants further in vitro and in vivo evaluation, particularly as our previous work has shown C. albicans virulence factor modulation by oral bacteria.

Denture-associated biofilm infection in three-dimensional oral mucosal tissue models.

PURPOSE: In vitro analyses of virulence, pathogenicity and associated host cell responses are important components in the study of biofilm infections. The Candida-related infection, denture-associated oral candidosis, affects up to 60 % of denture wearers and manifests as inflammation of palatal tissues contacting the denture-fitting surface. Commercially available three-dimensional tissue models can be used to study infection, but their use is limited for many academic research institutions, primarily because of the substantial purchase costs. The aim of this study was to develop and evaluate the use of in vitro tissue models to assess infections by biofilms on acrylic surfaces through tissue damage and Candida albicans virulence gene expression. METHODOLOGY: In vitro models were compared against commercially available tissue equivalents (keratinocyte-only, SkinEthic; full-thickness, MatTek Corporation). An in vitro keratinocyte-only tissue was produced using a cancer-derived cell line, TR146, and a full-thickness model incorporating primary fibroblasts and immortalised normal oral keratinocytes was also generated. The in vitro full-thickness tissues incorporated keratinocytes and fibroblasts, and have potential for future further development and analysis. RESULTS: Following polymicrobial infection with biofilms on acrylic surfaces, both in-house developed models were shown to provide equivalent results to the SkinEthic and MatTek models in terms of tissue damage: a significant (P<0.05) increase in LDH activity for mixed species biofilms compared to uninfected control, and no significant difference (P>0.05) in the expression of most C. albicans virulence genes when comparing tissue models of the same type. CONCLUSION: Our results confirm the feasibility and suitability of using these alternative in vitro tissue models for such analyses.

Respiratory pathogen colonization of dental plaque, the lower airways, and endotracheal tube biofilms during mechanical ventilation.

PURPOSE: In mechanically ventilated patients, the endotracheal tube is an essential interface between the patient and ventilator, but inadvertently, it also facilitates the development of ventilator-associated pneumonia (VAP) by subverting pulmonary host defenses. A number of investigations suggest that bacteria colonizing the oral cavity may be important in the etiology of VAP. The present study evaluated microbial changes that occurred in dental plaque and lower airways of 107 critically ill mechanically ventilated patients. MATERIALS AND METHODS: Dental plaque and lower airways fluid was collected during the course of mechanical ventilation, with additional samples of dental plaque obtained during the entirety of patients' hospital stay. RESULTS: A "microbial shift" occurred in dental plaque, with colonization by potential VAP pathogens, namely, Staphylococcus aureus and Pseudomonas aeruginosa in 35 patients. Post-extubation analyses revealed that 70% and 55% of patients whose dental plaque included S aureus and P aeruginosa, respectively, reverted back to having a predominantly normal oral microbiota. Respiratory pathogens were also isolated from the lower airways and within the endotracheal tube biofilms. CONCLUSIONS: To the best of our knowledge, this is the largest study to date exploring oral microbial changes during both mechanical ventilation and after recovery from critical illness. Based on these findings, it was apparent that during mechanical ventilation, dental plaque represents a source of potential VAP pathogens.

Microbial profiling of dental plaque from mechanically ventilated patients.

Micro-organisms isolated from the oral cavity may translocate to the lower airways during mechanical ventilation (MV) leading to ventilator-associated pneumonia (VAP). Changes within the dental plaque microbiome during MV have been documented previously, primarily using culture-based techniques. The aim of this study was to use community profiling by high throughput sequencing to comprehensively analyse suggested microbial changes within dental plaque during MV. Bacterial 16S rDNA gene sequences were obtained from 38 samples of dental plaque sampled from 13 mechanically ventilated patients and sequenced using the Illumina platform. Sequences were processed using Mothur, applying a 97% gene similarity cut-off for bacterial species level identifications. A significant 'microbial shift' occurred in the microbial community of dental plaque during MV for nine out of 13 patients. Following extubation, or removal of the endotracheal tube that facilitates ventilation, sampling revealed a decrease in the relative abundance of potential respiratory pathogens and a compositional change towards a more predominantly (in terms of abundance) oral microbiota including Prevotella spp., and streptococci. The results highlight the need to better understand microbial shifts in the oral microbiome in the development of strategies to reduce VAP, and may have implications for the development of other forms of pneumonia such as community-acquired infection.

Antimicrobial resistance and virulence traits of Enterococcus faecalis from primary endodontic infections.

OBJECTIVES: To determine the phenotypic and molecular characteristics of Enterococcus faecalis recovered from primary endodontic infections in Brazilian patients. METHODS: Twenty isolates of E. faecalis recovered from 43 Brazilian patients with primary endodontic infections were identified by biochemical profiling (API20Strep) and 16S rDNA sequencing. Antimicrobial susceptibility was ascertained by agar dilution, using the recommended protocol of the Clinical and Laboratory Standards Institute (CLSI). PCR with validated primers was used to detect genes associated with antibiotic resistance and specific virulence factors. RESULTS: All isolates were deemed susceptible to penicillin G, erythromycin and vancomycin. However, nine isolates had a minimum inhibitory concentration of 4µg/mL to vancomycin (the resistance breakpoint). Fourteen isolates (70% of isolates) were also resistant to tetracycline with MICs of >64µg/mL. PCR products for tetracycline resistance genes were detected in test isolates, while erythromycin and vancomycin resistance genes were not evident. Gelatinase, aggregation substance and enteroccocal surface protein genes were detected in 20, 18 and 12 isolates, respectively. CONCLUSIONS: Endodontic E. faecalis isolates exhibit high level of resistance to tetracycline, an antibiotic that has use in local treatment of dental infections. This opens up a much-needed debate on the role and efficacy of this antibiotic for oral infections. Furthermore, these isolates were shown to possess genes that could contribute to pathogenicity in the pulp cavity.

Real-time monitoring of the adherence of Streptococcus anginosus group bacteria to extracellular matrix decorin and biglycan proteoglycans in biofilm formation.

Members of the Streptococcus anginosus group (SAGs) are significant pathogens. However, their pathogenic mechanisms are incompletely understood. This study investigates the adherence of SAGs to the matrix proteoglycans decorin and biglycan of soft gingival and alveolar bone. Recombinant chondroitin 4-sulphate(C4S)-conjugated decorin and biglycan were synthesised using mammalian expression systems. C4S-conjugated decorin/biglycan and dermatan sulphate (DS) decorin/biglycan were isolated from ovine alveolar bone and gingival connective tissue, respectively. Using surface plasmon resonance, adherence of the SAGs S. anginosus, Streptococcus constellatus and Streptococcus intermedius to immobilised proteoglycan was assessed as a function of real-time biofilm formation. All isolates adhered to gingival proteoglycan, 59% percent of isolates adhered to alveolar proteoglycans, 70% to recombinant decorin and 76% to recombinant biglycan. Higher adherence was generally noted for S. constellatus and S. intermedius isolates. No differences in adherence were noted between commensal and pathogenic strains to decorin or biglycan. DS demonstrated greater adherence compared to C4S. Removal of the glycosaminoglycan chains with chondroitinase ABC resulted in no or minimal adherence for all isolates. These results suggest that SAGs bind to the extracellular matrix proteoglycans decorin and biglycan, with interaction mediated by the conjugated glycosaminoglycan chain.