The denture microbiome in health and disease: an exploration of a unique community.

The United Nations suggests the global population of denture wearers (an artificial device that acts as a replacement for teeth) is likely to rise significantly by the year 2050. Dentures become colonized by microbial biofilms, the composition of which is influenced by complex factors such as patient's age and health, and the nature of the denture material. Since colonization (and subsequent biofilm formation) by some micro-organisms can significantly impact the health of the denture wearer, the study of denture microbiology has long been of interest to researchers. The specific local and systemic health risks of denture plaque are different from those of dental plaque, particularly with respect to the presence of the opportunist pathogen Candida albicans and various other nonoral opportunists. Here, we reflect on advancements in our understanding of the relationship between micro-organisms, dentures, and the host, and highlight how our growing knowledge of the microbiome, biofilms, and novel antimicrobial technologies may better inform diagnosis, treatment, and prevention of denture-associated infections, thereby enhancing the quality and longevity of denture wearers.

Inhibition of adhesion-specific genes by Solidago virgaurea extract causes loss of Candida albicans biofilm integrity.

AIMS: Candida albicans biofilms are commonly associated with severe oral infections. We previously discovered that a crude extract from the Solidago virgaurea plant (SV extract) was a potent inhibitor of C. albicans biofilm formation. Here, we further investigate the mechanisms underlying C. albicans biofilm inhibition by the SV extract. METHODS AND RESULTS: The SV extract was shown to inhibit laboratory and clinical C. albicans isolates adherence and hyphal transition on inert support and epithelial human cells, without affecting viability and growth of planktonic yeasts. Interestingly, RT-PCR-based experiments demonstrated that some key genes involved in adhesion and hyphal morphological switch (e.g. Hwp1p, Ece1p, Als3p) were strongly down-regulated by the SV extract. Moreover, antimicrobial synergy testing (checkerboard assay) demonstrated that antifungal effects of miconazole, nystatin or a common antiseptic mouthwash were synergistically improved when used in combination with the SV extract. CONCLUSIONS: The SV extract prevents C. albicans biofilm formation through direct inhibition of key adherence and hyphae-associated genes. SIGNIFICANCE AND IMPACT OF THE STUDY: Biofilm is considered as a key virulence factor of C. albicans infection. Our discovery of an inhibitor specifically acting on genes involved in biofilm formation paves the way for the future development of a new class of antifungal product.

The epithelial cell response to health and disease associated oral biofilm models.

BACKGROUND AND OBJECTIVE: Different bacteria differentially stimulate epithelial cells. Biofilm composition and viability are likely to influence the epithelial response. In vitro model systems are commonly used to investigate periodontitis-associated bacteria and their interactions with the host; therefore, understanding factors that influence biofilm-cell interactions is essential. The present study aimed to develop in vitro monospecies and multispecies biofilms and investigate the epithelial response to these biofilms. MATERIAL AND METHODS: Bacterial biofilms were cultured in vitro and then either live or methanol-fixed biofilms were co-cultured with epithelial cells. Changes in epithelial cell viability, gene expression and cytokine content of culture supernatants were evaluated. RESULTS: Bacterial viability was better preserved within mixed-species biofilm culture than within single-species biofilm culture. Both mixed- and single-species biofilms stimulated increased expression of mRNA for interleukin 8 (IL8), C-X-C motif chemokine ligand 3 (CXCL3), C-X-C motif chemokine ligand 1 (CXCL1), interleukin 1 (IL1), interleukin 6 (IL6), colony-stimulating factor 2 (CSF2) and tumour necrosis factor (TNF), and the response was greatest in response to mixed-species biofilms. Following co-culture, cytokines detected in the supernatants included IL-8, IL-6, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, with the greatest release of cytokines found following co-culture with methanol-fixed, mixed-species biofilms. CONCLUSIONS: These data show that epithelial cells generate a distinct cytokine gene- and protein-expression signature in response to live or fixed, single- or multispecies biofilms.

Transfer of micro-organisms from dry surface biofilms and the influence of long survival under conditions of poor nutrition and moisture on the virulence of Staphylococcusaureus.

BACKGROUND: Biofilms on dry hospital surfaces can enhance the persistence of micro-organisms on dry harsh clinical surfaces and can potentially act as reservoirs of infectious agents on contaminated surfaces. AIM: This study was conducted to quantify the transfer of viable Staphylococcus aureus cells from dry biofilms through touching and to investigate the impact of nutrient and moisture deprivation on virulence levels in S. aureus. METHODS: Dry biofilms of S. aureus ATCC 25923 and a defective biofilm-forming ability mutant, S. aureus 1132, were formed in 24-well plates under optimized conditions mimicking dry biofilm formation on clinical surfaces. Microbial cell transfer was induced through the touching of the dry biofilms, which were quantified on nutrient agar. To investigate the impact of nutrient and moisture deprivation on virulence levels, dry and standard biofilms as well as planktonic cells of S. aureus ATCC 25923 were inoculated into Galleria mellonella and their kill rates compared. FINDINGS: Results of this study showed that viable cells from dry biofilms of S. aureus ATCC 25923 were significantly more virulent and readily transferrable from dry biofilms through a touch test, therefore representing a greater risk of infection. The biofilm-forming capability of S. aureus strains had no significant impact on their transferability with more cells transferring when biofilm surfaces were wet. CONCLUSIONS: These findings indicate that dry biofilms on hospital surfaces may serve as a reservoir for the dissemination of pathogenic micro-organisms in hospitals, thus highlighting the importance of regular cleaning and adequate disinfection of hospital surfaces.

The role of streptococcal plasmin(ogen) binding in infective endocarditis.

The ability of viridans group streptococci (VGS) to bind human plasminogen and its subsequent activation into plasmin may contribute to the pathogenesis of streptococcal endocarditis. The increased proteolytic activity acquired through cell-bound plasmin may lead to a decreased stability of the streptococcal vegetation and possible embolisation. Twenty-two infective endocarditis isolates and 16 non-infective endocarditis isolates were screened for their ability to bind plasminogen through the quantification of its active form plasmin, using the colorimetric substrate D-Val-Leu-Lys p-nitroanilide. The species of the VGS assessed expressed a universal capability to bind human plasminogen, although they did so with differing affinities and independently of the site of isolation.

Identification of clinically relevant viridans group streptococci by phenotypic and genotypic analysis.

Two phenotypic and three molecular methods were assessed for their ability to identify viridans group streptococci (VGS) to the species level. A panel of 23 clinical isolates, comprising strains isolated from infective endocarditis, blood cultures, pleural and peritoneal fluid, and 19 type/reference strains were analyzed. Identification was performed using two conventional phenotypic methods: API® rapid ID 32 Strep and the VITEK® 2 system, and genotypic analysis of the nucleotide sequence of the housekeeping gene sodA, restriction patterns generated by restriction fragment length polymorphism (RFLP) of the 16S rRNA gene and multilocus sequence analysis (MLSA) of seven housekeeping genes. The API® rapid ID 32 Strep accurately speciated 79% of the strains assessed, while the VITEK® 2 generated a successful identification for 55%, presenting limitations particularly with regard to species belonging to the mitis group. RFLP of the 16S rRNA gene correctly speciated 24% of the strains, having failed to allocate a species for 36% of the isolates examined. In contrast, sequence analysis of the sodA gene provided a correct identification for 95% of the strains assessed, while identification using the MLSA technique was unsuccessful due to practical limitations. The results generated herein indicate that no single methodology can be used to provide an accurate identification to the species level of all VGS, although nucleotide sequence analysis of the sodA gene proved to be useful in providing reliable speciation.

Increased plasma levels epithelial cell-derived neutrophil-activating peptide 78/CXCL5 in periodontitis patients undergoing supportive therapy.

AIM: To investigate the influence of cigarette smoking on plasma epithelial cell-derived neutrophil-activating peptide-78 (CXCL5/ENA-78) and interleukin-6 (IL-6) in supportive therapy periodontitis patients. MATERIALS AND METHODS: Plasma concentrations of CXCL5/ENA-78 and IL-6 were evaluated in 167 systemically healthy subjects (54 smokers and 113 non-smokers) divided into four groups: non-smokers with periodontitis (n=90), smokers with periodontitis (n=49), healthy non smokers (n=23) and healthy smokers (n=5). RESULTS: Clinical probing depth (CPD) of smokers with periodontitis were significantly greater than those of non-smoking patients (p<0.05). Although clinical attachment loss (CAL) and the number of deep sites affected were greater in the smokers with periodontitis, these differences were not significant. Periodontitis patients had significantly higher plasma IL-6 and ENA-78 than healthy subjects (p<0.05). There was no significant difference in IL-6 between smokers and non-smokers with periodontitis but CXCL5/ENA-78 concentrations were significantly greater in smokers with periodontitis (p=0.006). Plasma CXCL5/ENA-78 correlated with CPD, CAL and tobacco consumption (all p<0.05). CONCLUSION: Plasma CXCL5/ENA-78 concentrations are a good systemic indicator of the inflammatory process and disease severity in subjects with periodontitis and in addition are potential indicator of inflammatory effects of cigarette smoking. Further studies are required to elucidate the biological mechanisms underlining this increase in CXCL5/ENA-78.

'Secondary biofilms' could cause failure of peracetic acid high-level disinfection of endoscopes.

INTRODUCTION: The reduced susceptibility of biofilms to disinfectants presents a challenge to the successful reprocessing of medical equipment. This study examined the effect of residual biomass remaining after previous disinfection with peracetic acid (PAA) on the tolerance of subsequent mature Pseudomonas aeruginosa biofilms to PAA. The effect of enzymatic degradation of specific components of the extracellular polymeric substance (EPS) of P. aeruginosa biofilm on the effectiveness of PAA disinfection was also evaluated. METHODS: The susceptibility of biofilm grown on the biomass of PAA-killed biofilm to PAA was compared with the PAA susceptibility of biofilm grown in wells of a 24-well plate by evaluating their viability using the plate count assay. The effect of PAA on biofilm biomass was measured using crystal violet quantification of total biofilm biomass, while its effect on the polysaccharide and protein components of biofilm EPS was quantified using the phenol-sulphuric acid assay or Bradford assay, respectively. A confocal microscope was used to visualize the distribution of living and dead cells in biofilms grown on residual biofilm biomass. FINDINGS: The presence of residual biomass from previously disinfected biofilms significantly enhanced the tolerance of subsequent biofilms. A 96-h-old 'secondary biofilm' formed on disinfected biomass survived PAA concentrations of 4000 ppm, which exceeds the concentrations used in practice for high-level disinfection. CONCLUSION: These observations indicate that, under certain circumstances, recolonization of residual EPS can cause failure of disinfection of medical equipment such as endoscopes, and emphasizes the importance of cleaning endoscopes prior to disinfection.

Mechanical biofilm disruption causes microbial and immunological shifts in periodontitis patients.

Periodontitis is characterized by subgingival biofilm dysbiosis, inflammation and tissue destruction. Current treatment involves mechanical biofilm disruption known as non-surgical periodontal therapy (NSPT). This study sought to characterise the impact of treatment on microbial diversity and overall community, and the parallel impact on host inflammation in the oral cavity. Fourty-two periodontitis patients were included in this study, with periodontal clinical parameters, subgingival plaque and saliva samples collected at baseline and 90 days after treatment. Salivary cytokines were quantified, and subgingival plaque was analysed using 16S rRNA sequencing. After treatment, there were marked health-associated alterations in microbial composition and diversity, including differential abundance of 42 genera and 61 species. These changes were accompanied by substantial clinical improvement (pockets ≥ 5 mm, 27.50% to 9.00%, p < 0.001) and a decrease in salivary IL-1ß (p < 0.001)-a putative marker of periodontal inflammation. Despite significant reductions in disease associated anaerobes, several genera (Fusobacterium, Prevotella, Tanenerella, Treponema) remained present and formed a distinct subnetwork associated with residual disease. Collectively, this study shows that current periodontal treatment results in partial restoration of a healthy microbial ecosystem, but features of biofilm dysbiosis and host inflammation remain in some patients, which were surprisingly independent of clinical response.

Candida auris exhibits resilient biofilm characteristics in vitro: implications for environmental persistence.

Surfaces within healthcare play a key role in the transmission of drug-resistant pathogens. Candida auris is an emerging multidrug-resistant yeast which can survive for prolonged periods on environmental surfaces. Here we show that the ability to form cellular aggregates increases survival after 14 days, which coincides with the upregulation of biofilm-associated genes. Additionally, the aggregating strain demonstrated tolerance to clinical concentrations of sodium hypochlorite and remained viable 14 days post treatment. The ability of C. auris to adhere to and persist on environmental surfaces emphasizes our need to better understand the biology of this fungal pathogen.

Mixed Candida albicans and Candida glabrata populations associated with the pathogenesis of denture stomatitis.

INTRODUCTION: Oral yeasts are an important component of the resident microbial ecology of the oral cavity, but they are also associated with various forms of oral candidosis, such as denture stomatitis. Although Candida albicans is the predominant oral fungal pathogen, other species may also play an integral role in pathogenesis. The aim of this study was to examine the mycological ecology in patients with denture stomatitis, using an improved sampling technique, to determine whether species diversity and species quantity were related to oral pathology. METHODS: Thirty-seven patients attending the Glasgow Dental Hospital were enrolled in this study following informed consent. A full clinical history was obtained, including details of their oral hygiene practices and the levels of erythema based on Newton's classification scale. Oral rinse, denture sonicate, and swab samples were taken, which were processed for quantitative and qualitative analysis of oral yeasts. RESULTS: The proportion of patients with no inflammation or Newton's Types I, II, and III were 31, 33, 25, and 14%, respectively. Denture sonication was a superior sampling procedure, with statistically greater quantities of yeasts isolated using this methodology (P < 0.01). The predominant oral yeasts isolated were C. albicans (75%) and Candida glabrata (30%), which were isolated in higher proportions in patients with the highest grades of inflammation (100 and 80%), and in combination from 80% of these patients. CONCLUSIONS: This study has demonstrated that mixed C. albicans and C. glabrata biofilms may play an important role in the pathogenesis associated with severe inflammation in denture wearers.

Combined analysis of the salivary microbiome and host defence peptides predicts dental disease.

Understanding the triad of host response, microbiome and disease status is potentially informative for disease prediction, prevention, early intervention and treatment. Using longitudinal assessment of saliva and disease status, we demonstrated that partial least squares modelling of microbial, immunological and clinical measures, grouped children according to future dental disease status. Saliva was collected and dental health assessed in 33 children aged 4 years, and again 1-year later. The composition of the salivary microbiome was assessed and host defence peptides in saliva were quantified. Principal component analysis of the salivary microbiome indicated that children clustered by age and not disease status. Similarly, changes in salivary host defence peptides occurred with age and not in response to, or preceding dental caries. Partial least squares modelling of microbial, immunological and clinical baseline measures clustered children according to future dental disease status. These data demonstrate that isolated evaluation of the salivary microbiome or host response failed to predict dental disease. In contrast, combined assessment of both host response together with the microbiome revealed clusters of health and disease. This type of approach is potentially relevant to myriad diseases that are modified by host-microbiome interactions.

Surface disinfection challenges for Candida auris: an in-vitro study.

The emerging pathogenic multidrug-resistant yeast Candida auris is an important source of healthcare-associated infections and of growing global clinical concern. The ability of this organism to survive on surfaces and withstand environmental stressors creates a challenge for eradicating it from hospitals. A panel of C. auris clinical isolates was evaluated on different surface environments against the standard disinfectant sodium hypochlorite and high-level disinfectant peracetic acid. C. auris was shown to selectively tolerate clinically relevant concentrations of sodium hypochlorite and peracetic acid in a surface-dependent manner, which may explain its ability to successfully persist within the hospital environment.

Tolerance of Pseudomonas aeruginosa in in-vitro biofilms to high-level peracetic acid disinfection.

BACKGROUND: Biofilm has been suggested as a cause of disinfection failures in flexible endoscopes where no lapses in the decontamination procedure can be identified. To test this theory, the activity of peracetic acid, one of the widely used disinfectants in the reprocessing of flexible endoscopes, was evaluated against both planktonic and sessile communities of Pseudomonas aeruginosa. AIM: To investigate the ability of P. aeruginosa biofilm to survive high-level peracetic acid disinfection. METHOD: The susceptibility of planktonic cells of P. aeruginosa and biofilms aged 24, 48, 96, and 192 h to peracetic acid was evaluated by estimating their viability using resazurin viability and plate count methods. The biomass of the P. aeruginosa biofilms was also quantified using Crystal Violet assay. Planktonic cells of P. aeruginosa were treated with 5-30 ppm concentration of peracetic acid in the presence of 3.0 g/L of bovine serum albumin (BSA) for 5 min. Biofilms of P. aeruginosa were also treated with various peracetic acid concentrations (100-3000 ppm) for 5 min. FINDINGS: Planktonic cells of P. aeruginosa were eradicated by 20 ppm of peracetic acid, whereas biofilms showed an age-dependent tolerance to peracetic acid, and 96 h biofilm was only eradicated at peracetic acid concentration of 2500 ppm. CONCLUSION: Ninety-six-hour P. aeruginosa biofilm survives 5 min treatment with 2000 ppm of peracetic acid, which is the working concentration used in some endoscope washer-disinfectors. This implies that disinfection failure of flexible endoscopes might occur when biofilms build up in the lumens of endoscopes.

The Porphyromonas gingivalis hemagglutinins HagB and HagC are major mediators of adhesion and biofilm formation.

Porphyromonas gingivalis is a bacterium associated with chronic periodontitis that possesses a family of genes encoding hemagglutinins required for heme acquisition. In this study we generated ΔhagB and ΔhagC mutants in strain W83 and demonstrate that both hagB and hagC are required for adherence to oral epithelial cells. Unexpectedly, a double ΔhagB/ΔhagC mutant had less severe adherence defects than either of the single mutants, but was found to exhibit increased expression of the gingipain-encoding genes rgpA and kgp, suggesting that a ΔhagB/ΔhagC mutant is only viable in populations of cells that exhibit increased expression of genes involved in heme acquisition. Disruption of hagB in the fimbriated strain ATCC33277 demonstrated that HagB is also required for stable attachment of fimbriated bacteria to oral epithelial cells. Mutants of hagC were also found to form defective single and multi-species biofilms that had reduced biomass relative to biofilms formed by the wild-type strain. This study highlights the hitherto unappreciated importance of these genes in oral colonization and biofilm formation.

Mast Cells Contribute to Porphyromonas gingivalis-induced Bone Loss.

Periodontitis is a chronic inflammatory and bone-destructive disease. Development of periodontitis is associated with dysbiosis of the microbial community, which may be caused by periodontal bacteria, such as Porphyromonas gingivalis Mast cells are sentinels at mucosal surfaces and are a potent source of inflammatory mediators, including tumor necrosis factors (TNF), although their role in the pathogenesis of periodontitis remains to be elucidated. This study sought to determine the contribution of mast cells to local bone destruction following oral infection with P. gingivalis Mast cell-deficient mice (Kit(W-sh/W-sh)) were protected from P. gingivalis-induced alveolar bone loss, with a reduction in anti-P. gingivalis serum antibody titers compared with wild-type infected controls. Furthermore, mast cell-deficient mice had reduced expression of Tnf, Il6, and Il1b mRNA in gingival tissues compared with wild-type mice. Mast cell-engrafted Kit(W-sh/W-sh) mice infected with P. gingivalis demonstrated alveolar bone loss and serum anti-P. gingivalis antibody titers equivalent to wild-type infected mice. The expression of Tnf mRNA in gingival tissues of Kit(W-sh/W-sh) mice was elevated following the engraftment of mast cells, indicating that mast cells contributed to the Tnf transcript in gingival tissues. In vitro, mast cells degranulated and released significant TNF in response to oral bacteria, and neutralizing TNF in vivo abrogated alveolar bone loss following P. gingivalis infection. These data indicate that mast cells and TNF contribute to the immunopathogenesis of periodontitis and may offer therapeutic targets.

Biofilm formation is a risk factor for mortality in patients with Candida albicans bloodstream infection-Scotland, 2012-2013.

Bloodstream infections caused by Candida species remain a significant cause of morbidity and mortality in hospitalized patients. Biofilm formation by Candida species is an important virulence factor for disease pathogenesis. A prospective analysis of patients with Candida bloodstream infection (n = 217) in Scotland (2012-2013) was performed to assess the risk factors associated with patient mortality, in particular the impact of biofilm formation. Candida bloodstream isolates (n = 280) and clinical records for 157 patients were collected through 11 different health boards across Scotland. Biofilm formation by clinical isolates was assessed in vitro with standard biomass assays. The role of biofilm phenotype on treatment efficacy was also evaluated in vitro by treating preformed biofilms with fixed concentrations of different classes of antifungal. Available mortality data for 134 patients showed that the 30-day candidaemia case mortality rate was 41%, with predisposing factors including patient age and catheter removal. Multivariate Cox regression survival analysis for 42 patients showed a significantly higher mortality rate for Candida albicans infection than for Candida glabrata infection. Biofilm-forming ability was significantly associated with C. albicans mortality (34 patients). Finally, in vitro antifungal sensitivity testing showed that low biofilm formers and high biofilm formers were differentially affected by azoles and echinocandins, but not by polyenes. This study provides further evidence that the biofilm phenotype represents a significant clinical entity, and that isolates with this phenotype differentially respond to antifungal therapy in vitro. Collectively, these findings show that greater clinical understanding is required with respect to Candida biofilm infections, and the implications of isolate heterogeneity.

Combined fluorescent in situ hybridisation and immunolabelling of Bacteroides fragilis.

We have demonstrated that pure cultures of Bacteroides fragilis can be riboprobed with the oligoprobes BAC303 and EUB338, whilst simultaneously immunolabelled with either the mAb QUBF7, or polyclonal antiserum specific for a common antigen of B. fragilis. We were also able to distinguish between pure cultures of B. fragilis and Escherichia coli, by means of combined immunolabelling and riboprobing. The success of the combined technique is critically dependent on the size of the bacterial capsules, bacterial growth phase, antibody diluent and the length of the washing steps. The combined FISH and immunolabelling of bacteria has potential applications in studies of bacteria of medical and veterinary importance, as well as bacteria from other environments, as it yields information about both the identity and antigen expression of individual bacterial cells.

Improved detection of infection in hip replacements. A currently underestimated problem.

Our aim was to determine if the detection rate of infection of total hip replacements could be improved by examining the removed prostheses. Immediate transfer of prostheses to an anaerobic atmosphere, followed by mild ultrasonication to dislodge adherent bacteria, resulted in the culture of quantifiable numbers of bacteria, from 26 of the 120 implants examined. The same bacterial species were cultured by routine microbiological techniques from only five corresponding tissue samples. Tissue removed from 18 of the culture-positive implants was suitable for quantitative tissue pathology and inflammatory cells were present in all samples. Furthermore, inflammatory cells were present in 87% of tissue samples taken from patients whose implants were culture-negative. This suggests that these implants may have been infected by bacteria which were not isolated by the techniques of culture used. The increased detection of bacteria from prostheses by culture has improved postoperative antibiotic therapy and should reduce the need for further revision.

Characteristics of biofilm formation by Candida albicans.

A variety of manifestations of Candida albicans infections are associated with the formation of biofilms on the surface of biomaterials. Cells in biofilms display phenotypic traits that are dramatically different from their free-floating planktonic counterparts, such as increased resistance to anti-microbial agents and protection form host defenses. Here, we describe the characteristics of C. albicans biofilm development using a 96 well microtitre plate model, microscopic observations and a colorimetric method based on the use of a modified tetrazolium salt (2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide, XTT) to monitor metabolic activities of cells within the biofilm. C. albicans biofilm formation was characterized by initial adherence of yeast cells (0-2 h), followed by germination and micro-colony formation (2-4 h), filamentation (4-6 h), monolayer development (6-8 h), proliferation (8-24 h) and maturation (24-48 h). The XTT-reduction assay showed a linear relationship between cellular density of the biofilm and metabolic activity. Serum and saliva pre-conditioning films increased the initial attachment of C. albicans, but had minimal effect on subsequent biofilm formation. Scanning electron microscopy and confocal scanning laser microscopy were used to visualize C. albicans biofilms. Mature C. albicans biofilms consisted of a dense network of yeasts cells and hyphal elements embedded within exopolymeric material. C. albicans biofilms displayed a complex three dimensional structure which demonstrated spatial heterogeneity and a typical architecture showing microcolonies with ramifying water channels. Antifungal susceptibility testing demonstrated the increased resistance of sessile C. albicans cells against clinically used fluconazole and amphotericin B as compared to their planktonic counterparts.