The consistent application of hydrogen peroxide controls biofilm growth and removes Vermamoeba vermiformis from multi-kingdom in-vitro dental unit water biofilms.

The water systems inside a dental unit are known to be contaminated with a multi-kingdom biofilm encompassing bacteria, fungi, viruses and protozoa. Aerosolization of these micro-organisms can potentially create a health hazard for both dental staff and the patient. Very little is known on the efficacy of dental unit disinfection products against amoeba. In this study we have examined the effect of four different treatment regimens, with the hydrogen peroxide (H2O2) containing product Oxygenal, on an in-vitro multi-kingdom dental unit water system (DUWS) biofilm. The treatment efficacy was assessed in time using heterotrophic plate counts, the bacterial 16S rDNA, fungal 18S rDNA gene load and the number of genomic units for Legionella spp. the amoeba Vermamoeba vermiformis. The results indicated that a daily treatment of the DUWS with a low dose H2O2 (0.02% for 5 h), combined with a weekly shock dose (0.25% H2O2, 30 min) is necessary to reduce the heterotrophic plate count of a severely contaminated DUWS (>106 CFU.mL-1) to below 100 CFU.mL-1. A daily treatment with a low dose hydrogen peroxide alone, is sufficient for the statistically significant reduction of the total amount of bacterial 16S rDNA gene, Legionella spp. and Vermamoeba vermiformis load (p < 0.005). Also shown is that even though hydrogen peroxide does not kill the trophozoite nor the cysts of V. vermiformis, it does however result in the detachment of the trophozoite form of this amoeba from the DUWS biofilm and hereby ultimately removing the amoeba from the system.

Long-Term Analysis of Resilience of the Oral Microbiome in Allogeneic Stem Cell Transplant Recipients.

Stem cell transplantation (SCT) is associated with oral microbial dysbiosis. However, long-term longitudinal data are lacking. Therefore, this study aimed to longitudinally assess the oral microbiome in SCT patients and to determine if changes are associated with oral mucositis and oral chronic graft-versus-host disease. Fifty allogeneic SCT recipients treated in two Dutch university hospitals were prospectively followed, starting at pre-SCT, weekly during hospitalization, and at 3, 6, 12, and 18 months after SCT. Oral rinsing samples were taken, and oral mucositis (WHO score) and oral chronic graft-versus-host disease (NIH score) were assessed. The oral microbiome diversity (Shannon index) and composition significantly changed after SCT and returned to pre-treatment levels from 3 months after SCT. Oral mucositis was associated with a more pronounced decrease in microbial diversity and with several disease-associated genera, such as Mycobacterium, Staphylococcus, and Enterococcus. On the other hand, microbiome diversity and composition were not associated with oral chronic graft-versus-host disease. To conclude, dysbiosis of the oral microbiome occurred directly after SCT but recovered after 3 months. Diversity and composition were related to oral mucositis but not to oral chronic graft-versus-host disease.

Exploring the role of oral microorganisms in the pathogenesis of mucositis by assessing their impact on metabolic activity and reproductive capacity of epithelial cells in vitro.

PURPOSE: Clinical and in vitro studies showed selected oral microorganisms to be related to delayed wound healing and ulcerative oral mucositis. However, it is not known whether this effect is due to reduced metabolism and/or the reduced reproductive capacity of epithelial cells. Therefore, we studied the influence of the oral microorganisms Porphyromonas gingivalis, Candida glabrata, and Candida kefyr on cell metabolism and reproductive capacity of oral epithelial cells, aimed to further unravel the pathogenesis of oral mucositis. METHODS: Oral epithelial cells were exposed to different concentrations of P. gingivalis, C. glabrata, and C. kefyr as mono-infections or mixed together. An MTT assay was performed to determine the effect on cell metabolism. A clonogenic assay was used to study the effect on the reproductive capacity of oral epithelial cells. RESULTS: The metabolism of oral epithelial cells was reduced when the microorganisms were present in high concentrations: P. gingivalis at a multiplicity of infection (MOI) of 1000 and the Candida spp. at MOI 100. No statistical difference was observed in the ability of a single epithelial cell to grow into a colony of cells between control and P. gingivalis, C. glabrata, and C. kefyr, independent of the concentrations and combinations used. CONCLUSION: P. gingivalis, C. glabrata, and C. kefyr lowered the metabolic activity of oral epithelial cells in high concentrations, yet they did not influence the reproductive capacity of epithelial cells. Their impact on ulcerative oral mucositis is likely due to an effect on the migration, proliferation, and metabolism of epithelial cells.

Microbial changes in relation to oral mucositis in autologous hematopoietic stem cell transplantation recipients.

The aim of this prospective, two center study was to investigate the dynamics of the microbial changes in relation to the development of ulcerative oral mucositis in autologous SCT (autoSCT) recipients. Fifty-one patients were diagnosed with multiple myeloma and treated with high-dose melphalan followed by autoSCT. They were evaluated before, three times weekly during hospitalization, and three months after autoSCT. At each time point an oral rinse was collected and the presence or absence of ulcerative oral mucositis (UOM) was scored (WHO scale). Oral microbiome was determined by using 16S rRNA amplicon sequencing and fungal load by qPCR. Twenty patients (39%) developed UOM. The oral microbiome changed significantly after autoSCT and returned to pre-autoSCT composition after three months. However, changes in microbial diversity and similarity were more pronounced and rapid in patients who developed UOM compared to patients who did not. Already before autoSCT, different taxa discriminated between the 2 groups, suggesting microbially-driven risk factors. Samples with high fungal load (>0.1%) had a significantly different microbial profile from samples without fungi. In conclusion, autoSCT induced significant and reversible changes in the oral microbiome, while patients who did not develop ulcerative oral mucositis had a more resilient microbial ecosystem.

Candida and Porphyromonas gingivalis: the effect on wound closure in vitro.

Microorganisms play a role in oral mucositis after cancer therapy. The current study explored the hypothesis that Candida spp. alone and together with Porphyromonas gingivalis cause delayed healing of oral ulcerations due to the inhibition of wound closure. An in vitro scratch assay model was used to study the influence of viable and heat-killed Candida glabrata, Candida kefyr, and Candida albicans on cell migration of oral epithelial cells. Separately, the effect of conditioned medium of Candida spp. and the effect of a mixed infection of Candida spp. with P. gingivalis on wound closure was studied. In the presence of 10 viable C. glabrata or C. kefyr versus one epithelial cell, with a multiplicity of infection (MOI) of 10, the relative closure of the scratch was 26% and 17%, respectively. At a MOI of 1, this was 60% for C. glabrata and 78% for C. kefyr. The inhibition of oral epithelial cell migration challenged with either C. glabrata or C. kefyr together with P. gingivalis was stronger than the inhibition caused by one of both organisms separately. Candida spp. inhibit cell migration in vitro. A combination of Candida spp. and P. gingivalis inhibited cell migration more than either microorganism separately.

Proteins and peptides in parotid saliva of irradiated patients compared to that of healthy controls using SELDI-TOF-MS.

BACKGROUND: Radiotherapy to the head and neck area damages the salivary glands. As a consequence hyposalivation may occur, but also the protein composition of saliva may be affected possibly compromising oral health. The aim of our study was to compare the relative abundance of proteins and peptides in parotid saliva of irradiated patients to that of healthy controls. METHODS: Using Lashley cups and citric acid, saliva from the parotid glands was collected from nine irradiated patients and ten healthy controls. The samples were analyzed with SELDI-TOF-MS using a NP20 and IMAC-30 chip in the molecular weight range of 1-30 kDa. RESULTS: On the NP20 chip 61 (out of 217) and on the IMAC-30 chip 32 (out of 218) peaks differed significantly in intensity between the saliva of the irradiated patients and healthy controls. 55 % of the significant peaks showed higher intensity and 45 % showed lower intensity in the saliva of irradiated patients. The peaks may represent, amongst others, the salivary proteins lysozyme, histatins, cystatin, protein S100 and PRP's. CONCLUSIONS: Large differences were found in the relative abundance of a wide range of proteins and peptides in the parotid saliva of irradiated patients compared to healthy controls.

Can the oral microflora affect oral ulcerative mucositis?

PURPOSE OF REVIEW: Oral mucositis is one of the most prevalent toxicities after hematopoietic stem cell transplantation. Mucositis is initiated by the chemotherapy or radiotherapy preceding the transplantation. It is commonly accepted that microorganisms play a role in the process of oral mucositis. Despite the upcoming techniques to determine the whole oral bacterial ecosystem, the exact role of the microflora in mucositis is not yet understood. This article provides an overview of the state-of-the-art research on the oral microflora and mucositis. RECENT FINDINGS: A shift in microflora, in both the intestine and the oral cavity, can be found after chemotherapy or radiation therapy. The presence of oral ulcerative mucositis coincides with the presence of periodontitis-associated bacteria, in particular Porphyromonas gingivalis. Moreover, this bacterium can inhibit wound healing processes in an in-vitro model. SUMMARY: We come to realize that some diseases are associated with a shift in the microflora. The role of the microflora in oral and intestinal mucositis is gaining more attention in recent literature. In the oral cavity, periodontitis-associated bacteria may influence the healing of ulcerations and the role they play in mucositis may be more subtle and complicated than was previously thought.

The influence of oral bacteria on epithelial cell migration in vitro.

Oral ulcerations often arise as a side effect from chemo- and radiation therapy. In a previous clinical study, Porphyromonas gingivalis was identified as a positive predictor for oral ulcerations after hematopoetic stem cell transplantation, possibly incriminating P. gingivalis in delayed healing of the ulcerations. Therefore, it was tested whether P. gingivalis and its secreted products could inhibit the migration of oral epithelial cells in an in vitro scratch assay. To compare, the oral bacteria Prevotella nigrescens, Prevotella intermedia, Tannerella forsythia, and Streptococcus mitis were included. A standardized scratch was made in a confluent layer of human oral epithelial cells. The epithelial cells were challenged with bacterial cells and with medium containing secretions of these bacteria. Closure of the scratch was measured after 17 h using a phase contrast microscope. P. gingivalis, P. nigrescens, and secretions of P. gingivalis strongly inhibited cell migration. A challenge with 1000 heat-killed bacteria versus 1 epithelial cell resulted in a relative closure of the scratch of 25% for P. gingivalis and 20% for P. nigrescens. Weaker inhibitory effects were found for the other bacteria. The results confirmed our hypothesis that the oral bacteria may be involved in delayed wound healing.

Oral bacteria and yeasts in relationship to oral ulcerations in hematopoietic stem cell transplant recipients.

BACKGROUND: Oral mucositis is a serious and debilitating side effect of conditioning regimens for hematopoietic stem cell transplant (HSCT). Through HSCT, the homeostasis in the oral cavity is disrupted. The contribution of the oral microflora to mucositis remains to be clarified. The aim of our study was to investigate the relationship between yeasts, bacteria associated with periodontitis, and oral ulcerations in HSCT recipients. METHODS: This prospective observational study included 49 adult HSCT recipients. Twice weekly, oral ulcerations were scored, and oral rinsing samples were obtained. Samples were evaluated for the total bacterial load; the Gram-negative bacteria: Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Parvimonas micra, Fusobacterium nucleatum, Tannerella forsythia, and Treponema denticola; and the yeasts: Candida albicans, Candida glabrata, Candida kefyr, Candida krusei, Candida parapsilosis, and Candida tropicalis using real-time polymerase chain reaction with specific primers and probes. Explanatory variables for oral ulcerations were calculated using the multilevel generalized estimated equations (GEE) technique. RESULTS: None of the samples was positive for A. actinomycetemcomitans, while F. nucleatum was found most often (66 % of samples). C. albicans was the most isolated yeast (88 % of samples), whereas C. parapsilosis was found in only 8 % of the samples. Multivariate GEE analyses identified P. gingivalis, P. micra, T. denticola, F. nucleatum, C. glabrata, and C. kefyr as significant explanatory variables of oral ulcerations. CONCLUSIONS: Our data indicate that P. gingivalis in particular, but also P. micra, T. denticola, F. nucleatum, C. glabrata, and C. kefyr may play a role in ulcerative oral mucositis in patients undergoing HSCT.

Highly specific protease-based approach for detection of porphyromonas gingivalis in diagnosis of periodontitis.

Porphyromonas gingivalis is associated with the development of periodontitis. Here we describe the development of a highly specific protease-based diagnostic method for the detection of P. gingivalis in gingival crevicular fluid. Screening of a proteolytic peptide substrate library, including fluorogenic dipeptides that contain d-amino acids, led to the discovery of five P. gingivalis-specific substrates. Due to the presence of lysine and arginine residues in these substrates, it was hypothesized that the cleavage was mediated by the gingipains, a group of P. gingivalis-specific proteases. This hypothesis was confirmed by the observation that P. gingivalis gingipain knockout strains demonstrated clearly impaired substrate cleavage efficacy. Further, proteolytic activity on the substrates was increased by the addition of the gingipain stimulators dithiothreitol and l-cysteine and decreased by the inhibitors leupeptin and N-ethylmaleimide. Screening of saliva and gingival crevicular fluid of periodontitis patients and healthy controls showed the potential of the substrates to diagnose the presence of P. gingivalis proteases. By using paper points, a sensitivity of approximately 10(5) CFU/ml was achieved. P. gingivalis-reactive substrates fully composed of l-amino acids and Bz-l-Arg-NHPhNO(2) showed a relatively low specificity (44 to 85%). However, the five P. gingivalis-specific substrates that each contained a single d-amino acid showed high specificity (96 to 100%). This observation underlines the importance of the presence of d-amino acids in substrates used for the detection of bacterial proteases. We envisage that these substrates may improve the specificity of the current enzyme-based diagnosis of periodontitis associated with P. gingivalis.