Metatranscriptome and resistome of the endodontic microbiome.

INTRODUCTION: In this study, we used metatranscriptomics for the first time to investigate microbial composition, functional signatures, and antimicrobial resistance (AR) gene expression in endodontic infections. METHODS: Root canal samples were collected from ten teeth, including five primary and five persistent/ secondary endodontic infections. RNA from endodontic samples was extracted, and RNA sequencing was performed on a NovaSeq6000 system (Illumina). Taxonomic analysis was performed using the Kraken2 bacterial database. Then, sequences with a taxonomic classification were annotated against the Universal Protein Knowledgebase for functional annotation and the Comprehensive Antibiotic Resistance Database for AR-like gene identification. RESULTS: Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria represented the dominant phyla, whereas Fusobacteria, Spirochaetes, and Synergistetes were among the non-dominant phyla. The top ten species were mainly represented by obligate (or quasi-obligate) anaerobes, including Gram-negative (e.g., Capnocytophaga sp. oral taxon 323, Fusobacterium nucleatum, Prevotella intermedia, Prevotella oris, Tannerella forsythia and Tannerella sp. oral taxon HOT-286) and Gram-positive species (e.g., Olsenella uli and Parvimonas micra). Transcripts encoding moonlighting proteins (e.g., glycolytic proteins, translational elongation factors, chaperonin, and heat shock proteins) were highly expressed, potentially affecting bacterial adhesion, biofilm formation, host defense evasion, and inflammation induction. Endodontic bacteria expressed genes conferring resistance to antibiotic classes commonly used in dentistry, with a high prevalence and expression of tetracycline and lincosamide resistance genes. Antibiotic efflux and antibiotic target alteration/ protection were the main resistance mechanisms. CONCLUSIONS: Metatranscriptomics revealed the activity of potential endodontic pathogens, which expressed putative virulence factors and a wide diversity of genes potentially involved in antimicrobial resistance.

Enhancing Antibiotic Efficacy in Regenerative Endodontics by Improving Biofilm Susceptibility.

INTRODUCTION: Various strategies have been researched to enhance the susceptibility of biofilms, given their tolerance to antibiotics. This study evaluated the effect of the anti-microbial peptide nisin in association with antibiotics used in regenerative endodontics, exploring different treatment times and biofilm growth conditions. METHODS: A mixture of 10 bacterial species was cultivated on dentin specimens anaerobically for 21 days. Biofilms were treated with 1 mL of high-purity nisin Z (nisin ZP, 200 µg/mL) and a triple antibiotic mixture (TAP: ciprofloxacin + metronidazole + minocycline, 5 mg/mL), alone or in combination. The effectiveness of antimicrobial agents was assessed after 1 and 7 days. During the 7-day period, biofilms were treated under 2 conditions: a single dose in a nutrient-depleted setting (ie, no replenishment of growth medium) and multiple doses in a nutrient-rich environment (ie, renewal of medium and antimicrobial agents every 48 h). After treatments, biofilm cells were dispersed, and total colony-forming units were counted. RESULTS: After 1 d-treatment, nisin ZP + TAP resulted in 2-log cell reduction compared to TAP alone (P < .05). After 7 d-treatment with a single dose, nisin ZP + TAP and TAP reduced bacteria to nonculturable levels (P < .05), whereas repeated antimicrobial doses did not eliminate bacteria in a nutrient-rich environment. No bacterial reduction was observed with nisin ZP alone in any treatment time. CONCLUSIONS: The additional use of nisin improved the TAP activity only after a short exposure time. Longer exposure to TAP or nisin + TAP in a nutrient-deprived environment effectively eliminated biofilms.

Initial bacterial adherence and biofilm formation on novel restorative materials used in paediatric dentistry.

OBJECTIVE: To evaluate the initial bacterial adherence and biofilm formation on novel restorative materials in paediatric dentistry and compare the results to stainless steel crown and primary enamel. MATERIALS AND METHODS: Twenty-five samples (Diameter = 4 mm) from five restorative materials (Tetric Power Fill light cured for 3 s or 10 s, Fuji II LC, Equia Forte HT Fil, Cention Forte, Stainless-steel crown) and primary enamel were prepared. Four samples served for recording of surface roughness (Ra) using a contact profilometer, 21 samples were incubated in stimulated human saliva for 2 h (initial bacterial adherence) and 72 h (biofilm formation) and served to determine ion releasing and bacterial growth. After 2 and 72 h, the number of colony-forming units (CFU) per ml was counted and expressed in Log10 CFU/ml. Data were analysed with two-way ANOVA and Tuckey's multiple comparisons test (p < 0.05). RESULTS: All tested materials showed similar initial bacterial adherence (p > 0.1). Stainless steel crown showed statistically significantly less biofilm formation than all other tested materials (p ≤ 0.02), except for Fuji II LC (p = 0.06). In terms of biofilm formation, the differences between all tested materials were not statistically significant (p ≥ 0.9). SIGNIFICANCE: Novel restorative materials in paediatric dentistry show similar initial bacterial adherence and biofilm formation. However, compared to other restorative materials, stainless steel crowns demonstrate the lowest level of biofilm formation. Ion-releasing materials may not necessarily show better antimicrobial properties than conventional materials.

Dynamic interactions between Candida albicans and different streptococcal species in a multispecies oral biofilm.

The oral cavity is colonized by a plethora of bacteria, fungi, and archaea, including streptococci of the mitis group (MSG) and the yeast Candida albicans. This study aims to investigate the role of streptococcal species in the development of oral biofilm and the cross-kingdom interactions between some of the members of the commensal MSG and the pathogen yeast C. albicans using a multispecies supragingival biofilm model. A total of nine different in vitro biofilms were grown, quantified with culture analyses, and visually examined with confocal laser scanning microscopy (CLSM). A four-species biofilm without any streptococcal species was used as a basic biofilm. In each subsequent inoculum, one species of MSG was added and afterward combined with Streptococcus mutans. The eight-species biofilm contained all eight strains used in this study. Culture analyses showed that the presence of S. mutans in a four-species biofilm with Streptococcus oralis or S. oralis subsp. tigurinus did not differ significantly in C. albicans colony-forming unit (CFU) counts compared to biofilms without S. mutans. However, compared to other mitis species, Streptococcus gordonii combined with S. mutans resulted in the lowest CFUs of C. albicans. Visual observation by CLSM showed that biofilms containing both S. mutans and one species of MSG seemed to induce the formation of filamentous form of C. albicans. However, when several species of MSG were combined with S. mutans, C. albicans was again found in its yeast form.

Antibiotic Resistance among Fusobacterium, Capnocytophaga, and Leptotrichia Species of the Oral Cavity.

PURPOSE: Antibiotics play an important role in treating periodontal diseases. Due to the effectiveness of antibiotic therapies, their usage in dentistry has significantly increased. The aim of this study focused on the in-vitro susceptibility of different gram-negative oral bacteria species - which are associated with periodontal diseases (Fusobacterium spp., Capnocytophaga spp. and Leptotrichia buccalis) and have different geographical origins (Asia and Europe) - against antimicrobials that are clinically relevant in dental therapy. MATERIALS AND METHODS: A total of 45 strains were tested (29 Fusobacterium spp., 13 Capnocytophaga spp. and 3 L. buccalis) that were either isolated from Chinese patients or were obtained from different strain collections. Their antimicrobial susceptibility to the antimicrobial agents benzylpenicillin, amoxicillin, amoxicillin-clavulanic acid, ciprofloxacin, moxifloxacin, clindamycin, doxycycline, tetracycline and metronidazole was tested using the E-Test. Strains with particular resistance to penicillin, clindamycin and metronidazole were further analysed for resistance genes. RESULTS: All tested bacterial isolates were sensitive to amoxicillin, amoxicillin-clavulanic acid, doxycycline and tetracycline, but showed variable sensitivity towards other antibiotics such as benzylpenicillin, ciprofloxacin, moxifloxacin, clindamycin and metronidazole. CONCLUSION: The results of the present study suggest that certain periodontal disease-related bacterial strains can be resistant towards antimicrobial agents commonly used in adjuvant periodontal therapy.

In vitro versus in situ biofilms for evaluating the antimicrobial effectiveness of herbal mouthrinses.

For centuries, diverse mouthrinses have been applied for medicinal purposes in the oral cavity. In view of the growing resistance of oral microorganisms against conventional antimicrobial agents e.g. chlorhexidine, the implementation of alternative treatments inspired by nature has lately gained increasing interest. The aim of the present study was to compare in vitro biofilm models with in situ biofilms in order to evaluate the antimicrobial potential of different natural mouthrinses. For the in vitro study a six-species supragingival biofilm model containing A. oris, V. dispar, C. albicans, F. nucleatum, S. mutans and S. oralis was used. Biofilms were grown anaerobically on hydroxyapatite discs and treated with natural mouthrinses Ratanhia, Trybol and Tebodont. 0.9% NaCl and 10% ethanol served as negative controls, while 0.2% CHX served as positive control. After 64h hours, biofilms were harvested and quantified by cultural analysis CFU. For the in situ study, individual test splints were manufactured for the participants. After 2h and 72h the biofilm-covered samples were removed and treated with the mouthrinses and controls mentioned above. The biofilms were quantified by CFU and stained for vitality under the confocal laser scanning microscope. In the in vitro study, 0.2% CHX yielded the highest antimicrobial effect. Among all mouthrinses, Tebodont (4.708 ± 1.294 log10 CFU, median 5.279, p<0.0001) compared with 0.9% NaCl showed the highest antimicrobial potential. After 72h there was no significant reduction in CFU after 0.2% CHX treatment. Only Trybol showed a statistically significant reduction of aerobic growth of microorganisms in situ (5.331 ± 0.7350 log10 CFU, median 5.579, p<0.0209). After treatment with the positive control 0.2% CHX, a significant percentage of non-vital bacteria (42.006 ± 12.173 log10 CFU, median 42.150) was detected. To sum up, a less pronounced effect of all mouthrinses was shown for the in situ biofilms compared to the in vitro biofilms.

Examining the Composition of the Oral Microbiota as a Tool to Identify Responders to Dietary Changes.

BACKGROUND: The role of diet and nutrition in the prevention of oral diseases has recently gained increasing attention. Understanding the influence of diet on oral microbiota is essential for developing meaningful prevention approaches to oral diseases, and the identification of typical and atypical responders may contribute to this. METHODS: We used data from an experimental clinical study in which 11 participants were exposed to different dietary regimens in five consecutive phases. To analyse the influence of additional nutritional components, we examined changes in bacterial concentrations measured by culture techniques compared to a run-in phase. A measure of correspondence between the mean and individual patterns of the bacterial composition is introduced. RESULTS: The distance measures introduced showed clear differences between the subjects. In our data, two typical and three atypical responders appear to have been identified. CONCLUSIONS: The proposed method is suitable to identify typical and atypical responders, even in small datasets. We recommend routinely performing such analyses.

Antibiofilm Activity of LL-37 Peptide and D-Amino Acids Associated with Antibiotics Used in Regenerative Endodontics on an Ex Vivo Multispecies Biofilm Model.

The antimicrobial peptide LL-37 and D-amino acids (D-AAs) have been proposed as antibiofilm agents. Therefore, this study aimed to test the antimicrobial effect of antibiofilm agents associated with antibiotics used in regenerative endodontic procedures (the triple antibiotic paste­TAP: ciprofloxacin + metronidazole + minocycline). An endodontic-like biofilm model grown on bovine dentin discs was used in this study. After 21-day growth, the biofilms were treated with 1 mg/mL TAP, 10 µM LL-37, an association of LL-37 + TAP, 40 mM D-AAs solution, an association of D-AAs + TAP, and phosphate-buffered saline (negative control). Colony forming unit (CFU) data were analyzed by two-way ANOVA and Tukey's multiple comparison test (p < 0.05). LL-37 + TAP showed the best antibacterial activity (7-log10 CFU/mL ± 0.5), reaching a 1 log reduction of cells in relation to the negative control (8-log10 CFU/mL ± 0.7) (p < 0.05). In turn, no significant reduction in bacterial cells was observed with TAP, LL-37, D-AAs, and D-AAs + TAP compared to the negative control. In conclusion, the combination of antibiotics and LL-37 peptide showed mild antibacterial activity, while the combination of antibiotics and D-AAs showed no activity against complex biofilms.

On the biocompatibility of endodontic sealers.

Periapical tissue may be exposed to root canal filling materials in consequence of root canal therapy. There is scant scientific data about the biocompatibility of root canal filling materials of various chemistry on the periapical area. This study aimed to investigate the effects of different root canal sealers and their eluates on human alveolar osteoblasts in terms of cell proliferation, adhesion, morphology and gene expression in vitro. Five endodontic sealers (AH Plus®, Apexit®, Tubli-Seal®, Real Seal SE®, EndoRez®) and one gutta-percha obturation material (BeeFill®) were tested. Human alveolar osteoblasts derived from 3 different donors following incubation with sealer eluates after 24 h and 72 h were investigated by means of qPCR (gene expression). Morphological reactions of the alveolar osteoblasts were measured by culturing the cells for 3 d, and 7 d and 14 d, respectively, followed by scanning electron microscopy (morphology, adhesion) and fluorescence imaging of the actin cytoskeleton (morphology, proliferation). A repeated measures analysis was performed and p-values were adjusted by Tukey. While all sealers influenced the cell morphology and the expression of genes associated with apoptosis (Casp3), proliferation (histone H3), and inflammation (interleukin-6 and matrix metalloproteinases 1 and 3), mainly AH Plus® and Apexit® yielded a regular actin cytoskeleton and beneficial gene expression patterns. Regarding cell adhesion, only AH Plus® supported proper anchorage for alveolar osteoblasts. Our results provide evidence for the biocompatibility of epoxy resin-based endodontic sealers, i.e. AH Plus®, while other sealers proved cytotoxic for alveolar osteoblasts. Further studies are needed for understanding the bone cell reactions after endodontic treatment and the clinical decision-making regarding the sealer of choice for root canal fillings.

Microbial approaches for the assessment of toothpaste efficacy against oral species: A method comparison.

Antibacterial properties of toothpastes enable chemical plaque control in limited-access tooth regions that are mechanically not sufficiently reached by toothbrushes. Therefore, this study aimed to compare different microbial methods to assess antimicrobial toothpaste properties and evaluate different toothpastes in terms of their antibacterial efficacy against different oral microorganisms in an in vitro setting. Six toothpaste suspensions with varying antibacterial supplements were applied to a multispecies biofilm model (Actinomyces oris, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, and Streptococcus mutans) as well as to each microorganism. A culture method was used to assess the anti-biofilm effects and two different agar diffusion assays were performed for testing the antimicrobial effect on each microorganism. The measurements of the culture and diffusion analyses were statistically normalized and compared and toothpastes were ranked according to their antimicrobial efficacy. The results of both agar diffusion assays showed a high correlation across all tested species (Spearman correlation coefficients ρs > 0.95). The results of the multispecies biofilm model, however, substantially differed in its assessment of antibacterial properties (ρs ranging from 0.22 to 0.87) compared to the results of both diffusion assays. Toothpastes with amine fluoride (with and without stannous fluoride), and toothpastes with triclosan resulted in the highest antimicrobial efficacy. Activated carbon supplements in toothpastes were comparable in their antimicrobial action to the negative control NaCl. The appropriate selection of a broad range of oral microorganisms seems crucial when testing the chemical impact of toothpaste and toothpaste supplements.

The antimicrobial effect of Rosmarinus officinalis extracts on oral initial adhesion ex vivo.

OBJECTIVE: In the last few decades, there has been a growing worldwide interest in the use of plant extracts for the prevention of oral diseases. The main focus of this interest lies in the identification and isolation of substances that limit the formation of microbial biofilm which plays a major role in the development of caries, periodontitis, and peri-implantitis. In this clinical ex vivo study, we investigated the antimicrobial effects of Rosmarinus officinalis extract against oral microorganisms within in situ initial oral biofilms. MATERIALS AND METHODS: Initial in situ biofilm samples (2 h) from six healthy volunteers were treated ex vivo with R. officinalis extract at concentrations of 20 mg/ml and 30 mg/ml. The number of viable bacterial cells was determined by counting the colony-forming units. All surviving bacteria were isolated in pure cultures and identified using MALDI-TOF and biochemical testing procedures. Additionally, live/dead staining in combination with epifluorescence microscopy was used for visualizing the antimicrobial effects in the initial biofilms. RESULTS: The number of colony-forming units in the R. officinalis-treated biofilms was significantly lower than in the untreated controls (p < 0.001). The reduction range of log10 was 1.64-2.78 and 2.41-3.23 for aerobic and anaerobic bacteria, respectively. Regarding the bacterial composition, large intra- and interindividual variability were observed. Except for Campylobacter spp., the average amount of all bacterial taxa was lower after treatment with R. officinalis than in the untreated biofilms. A total of 49 different species were detected in the untreated biofilms, while only 11 bacterial species were detected in the R. officinalis-treated biofilms. Live/dead staining confirmed that the R. officinalis-treated biofilms had significantly lower numbers of surviving bacteria than the untreated biofilms. CONCLUSIONS: The treatment with R. officinalis extract has a significant potential to eliminate microbial oral initial biofilms. CLINICAL RELEVANCE: The results of this study encourage the use of R. officinalis extracts in biofilm control and thus in the treatment of caries and periodontitis as a herbal adjuvant to synthetic substances.

Biodentine Inhibits the Initial Microbial Adhesion of Oral Microbiota In Vivo.

This study aimed to evaluate the in vivo initial microbial adhesion of oral microorganisms on the biomaterial Biodentine compared to MTA and AH Plus. Cylindrical samples of the materials were prepared, and dentin slabs served as a control. An individual intraoral lower jaw splint served as a carrier for the samples and was worn by six volunteers. The specimens were worn for 120 min. Adherent bacteria were quantified by determining the colony-forming units (CFUs), while the visualization and quantification of total adherent microorganisms were facilitated by using DAPI and live/dead staining combined with fluorescence microscopy. Bovine dentin had a significantly higher number of aerobic CFUs compared to Biodentine (p = 0.017) and MTA (p = 0.013). The lowest amounts of DAPI-stained adherent microorganisms were quantified for Biodentine (15% ± 9%) and the control (18% ± 9%), while MTA showed the highest counts of initially adherent microorganisms (38% ± 10%). Significant differences were found for MTA and Biodentine (p = 0.004) as well as for MTA and the control (p = 0.021) and for AH Plus and the control (p = 0.025). Biodentine inhibited microbial adherence, thereby yielding an antimicrobial effectivity similar to that of MTA.

Antimicrobial Effects of Inula viscosa Extract on the In Situ Initial Oral Biofilm.

Given the undesirable side effects of commercially used mouth rinses that include chemically synthesized antimicrobial compounds such as chlorhexidine, it is essential to discover novel antimicrobial substances based on plant extracts. The aim of this study was to examine the antimicrobial effect of Inula viscosa extract on the initial microbial adhesion in the oral cavity. Individual test splints were manufactured for the participants, on which disinfected bovine enamel samples were attached. After the initial microbial adhesion, the biofilm-covered oral samples were removed and treated with different concentrations (10, 20, and 30 mg/mL) of an I. viscosa extract for 10 min. Positive and negative controls were also sampled. Regarding the microbiological parameters, the colony-forming units (CFU) and vitality testing (live/dead staining) were examined in combination with fluorescence microscopy. An I. viscosa extract with a concentration of 30 mg/mL killed the bacteria of the initial adhesion at a rate of 99.99% (log10 CFU value of 1.837 ± 1.54). Compared to the negative control, no killing effects were determined after treatment with I. viscosa extract at concentrations of 10 mg/mL (log10 CFU value 3.776 ± 0.831; median 3.776) and 20 mg/mL (log10 CFU value 3.725 ± 0.300; median 3.711). The live/dead staining revealed a significant reduction (p < 0.0001) of vital adherent bacteria after treatment with 10 mg/mL of I. viscosa extract. After treatment with an I. viscosa extract with a concentration of 30 mg/mL, no vital bacteria could be detected. For the first time, significant antimicrobial effects on the initial microbial adhesion in in situ oral biofilms were reported for an I. viscosa extract.

Necrotizing Gingivitis: Microbial Diversity and Quantification of Protein Secretion in Necrotizing Gingivitis.

Necrotizing gingivitis (NG) is a necrotizing periodontal disease that differs from chronic gingivitis (CG). To date, both the microbiological causes and the involved host cytokine response of NG still remain unclear. Here, we investigated corresponding interdental plaque and serum samples from two groups of Chinese patients with CG (n = 21) or NG (n = 21). The microbiota were studied by 16S rRNA Illumina MiSeq sequencing of the microbial metagenome and by assessing quantitatively the abundance of the phylum Bacteroidetes, the genus Prevotella and the species T. forsythia, P. endodontalis, and P. gingivalis using fluorescence in situ hybridization (FISH). With respect to the associated host response, the levels of 30 inflammatory mediators were quantified by multiplex immunoassay analysis. Differential microbial abundance analysis of the two disease groups revealed at the phylum level that Proteobacteria accounted for 67% of the differentially abundant organisms, followed by organisms of Firmicutes (21%) and Actinobacteria (9%). At the species level, significant differences in abundance were seen for 75 species of which 58 species were significantly more abundant in CG patients. Notably, the FISH analysis revealed that Bacteroidetes was the most prevalent phylum in NG. The multiplex cytokine assay showed significant quantitative differences between the disease groups for eight analytes (GM-CSF, G-CSF, IFN-α, IL-4, IL-13, TNF-α, MIG, and HGF). The G-CSF was found to be the most significantly increased inflammatory protein marker in NG. The next-generation sequencing (NGS) data supported the understanding of NG as a multi-microbial infection with distinct differences to CG in regard to the microbial composition.

Low Concentrations of Chlorhexidine Inhibit the Formation and Structural Integrity of Enzyme-Treated Multispecies Oral Biofilms.

The self-produced matrix of biofilms, consisting of extracellular polymeric substances, plays an important role in biofilm adhesion to surfaces and the structural integrity of biofilms. In dentistry, biofilms cause multiple diseases such as caries, periodontitis, and pulpitis. Disruption of these biofilms adhering to dental hard tissues may pose a major challenge since biofilms show higher tolerance to antimicrobials and antibiotics than planktonic cells. In this study, the effect of low concentrations of chlorhexidine (CHX) on enzyme-treated multispecies oral biofilm was investigated in an in vitro model. Six-species biofilms were enzymatically treated by anaerobic growth in a medium containing DNase I and proteinase K. Biofilms were exposed to a low concentration of CHX at defined time points. After 64h, biofilms were either harvested and quantified by cultural analyses or stained for confocal laser scanning microscopy (CLSM) analyses using either Live/Dead kit or different fluorescent dyes. A mixture of YoPro1 and SYTOX™ Green, Fluorescent Brightener 28 (Calcofluor), and SYPRO™ Ruby Protein Gel Stain was used to stain total DNA, exopolysaccharides, and extracellular proteins, respectively. Extracellular DNA (eDNA) was visualized via an indirect immunofluorescence assay (Mouse anti-DNA IgG, Goat anti-Mouse IgG, Streptavidin-Cy3). Overall, the total colony-forming units significantly decreased after combined treatment with a low concentration of CHX and enzymes compared to the group treated with CHX alone (p<0.001). These findings also apply to five species individually (Streptococcus mutans, Streptococcus oralis, Actinomyces oris, Veillonella dispar, and Candida albicans) occurring in the biofilms, with Fusobacterium nucleatum being the only exception. Furthermore, CLSM images showed less dense biofilms and a reduction in cell numbers after combined treatment compared to the group without enzymes. The combination of enzymes capable of disturbing the matrix integrity with antimicrobial agents thus appears to be a promising approach for biofilm disruption and killing.

Antibacterial Effect of Sodium Hypochlorite and EDTA in Combination with High-Purity Nisin on an Endodontic-like Biofilm Model.

Antimicrobial peptides have been proposed as antibiofilm agents. Therefore, we evaluated the effect of endodontic irrigants combined or not with the antimicrobial peptide nisin against an endodontic biofilm model composed of eleven bacterial species. Biofilms were grown on hydroxyapatite discs for 3, 15 and 21 days and treated with 1.5% sodium hypochlorite (NaOCl) or 17% EDTA followed by high-purity nisin (nisin ZP) or saline for 5 min each. Differences between groups were tested by two-way ANOVA and Tukey's multiple comparisons test (p < 0.05). Treatment with 1.5% NaOCl completely eliminated 3-d and 15-d biofilms but did not eradicate 21-d biofilms. Treatment with 1.5% NaOCl and 17% EDTA was equally effective against 21-d biofilms, showing 5-log and 4-log cell reduction, respectively, compared to the untreated control (9 log10, p < 0.05). No significant difference was found between 1.5% NaOCl + nisin ZP and 1.5% NaOCl in 21-d biofilms (p > 0.05). Likewise, no significant difference was found between 17% EDTA + nisin ZP and 17% EDTA treatments (p > 0.05). In conclusion, 1.5% NaOCl or 17% EDTA were effective strategies to combat mature biofilms. The additional use of nisin did not improve the activity of conventional irrigants against multispecies biofilms.

Single DNase or Proteinase Treatment Induces Change in Composition and Structural Integrity of Multispecies Oral Biofilms.

Biofilm virulence is mainly based on its bacterial cell surrounding biofilm matrix, which contains a scaffold of exopolysaccharides, carbohydrates, proteins, lipids, and nucleic acids. Targeting these nucleid acids or proteins could enable an efficient biofilm control. Therefore, the study aimed to test the effect of deoxyribonuclease I (DNase I) and proteinase K on oral biofilms. Six-species biofilms (Streptococcus mutans, Streptococcus oralis, Actinomyces oris, Fusobacterium nucleatum, Veillonella dispar, and Candida albicans) were exposed to DNase I (0.001 mg/mL, 0.002 mg/mL) or proteinase K (0.05 mg/mL, 0.1 mg/mL) for 1 h during biofilm formation. After 64 h, biofilms were harvested, quantified by culture analysis and visualized by image analysis using CLSM (confocal laser scanning microscopy). Statistical analysis was performed by ANOVA, followed by the Tukey test at a 5% significance level. The biofilm treatment with proteinase K induced a significant increase of Logs10 counts in S. mutans and a decrease in C. albicans, while biofilm thickness was reduced from 28.5 µm (control) to 9.07 µm (0.05 mg/mL) and 7.4 µm (0.1 mg/mL). Treatment with DNase I had no effect on the total bacterial growth within the biofilm. Targeting proteins of biofilms by proteinase K are promising adjunctive tool for biofilm control.

A Log Ratio-Based Analysis of Individual Changes in the Composition of the Oral Microbiota in Different Dietary Phases.

BACKGROUND: Investigating the influence of nutrition on oral health has a long scientific history. Due to recent technical advances like sequencing techniques for the oral microbiota, this topic has gained scientific interest again. A basic challenge is to understand the influence of nutrition on the oral microbiota and on the interaction between the oral bacteria, which is also statistically challenging. METHODS: Log-transformed ratios of two bacteria concentrations are introduced as the basic analytic tool. The framework is illustrated by application in an experimental study exposing eleven participants to different nutrition schemes in five consecutive phases. RESULTS: The method could be sufficiently used to analyse the interrelation between the bacteria and to identify some bacterial groups with the same as well as different reactions to additional dietary components. It was found that the strongest changes in bacterial concentrations were achieved by the additional consumption of dairy products. CONCLUSION: A log ratio-based analysis offers insights into the relation of different bacteria while taking specific features of compositional data into account. The presented methods allow becoming independent of the behaviour of other bacteria, which is a disadvantage of common analysis methods of compositions. The results indicate that modulations of the oral biofilm microbiota due to nutrition change can be attained.

Antibacterial Effect of High-Purity Nisin Alone and in Combination with D-Amino Acids or Chlorhexidine in an Endodontic-Like Biofilm Model.

New strategies to eradicate endodontic biofilms are needed. Therefore, we evaluated the effect of high-purity nisin alone and in combination with D-amino acids (D-AAs) or chlorhexidine (CHX) against an "endodontic-like" biofilm model. Biofilms were grown on hydroxyapatite discs for 64 h and treated with nisin, eight D-AAs mixture, nisin + eight D-AAs, 2% CHX, and nisin + 2% CHX. After the 5 min and 24 h treatments, biofilm cells were harvested and total colony-forming units were counted. Differences between groups were tested by two-way ANOVA followed by Tukey's multiple comparisons test (p < 0.05). Nisin and D-AAs, alone or in combination, were not effective in reducing bacteria after short or long exposure times. After 5 min, treatment with 2% CHX and nisin + 2% CHX resulted in 2 and 2.4-log cell reduction, respectively, compared with the no treatment control (p < 0.001). After 24 h, 2% CHX and nisin + 2% CHX drastically reduced bacterial counts. In conclusion, high-purity nisin alone or in combination with D-AAs did not show antibacterial activity against multispecies biofilms. Moreover, combined treatment using nisin and CHX showed similar antibiofilm activity compared with the use of CHX alone.

Antimicrobial Photoinactivation of In Situ Oral Biofilms by Visible Light Plus Water-Filtered Infrared A and Tetrahydroporphyrin-tetratosylate (THPTS).

The aim of this study was to examine the effect of aPDT with visual light (VIS) + water-filtered infrared A (wIRA) as a light source, and tetrahydroporphyrin-tetratosylate (THPTS) as a photosensitizer on in situ initial and mature oral biofilms. The samples were incubated, ex situ, with THPTS for two minutes, followed by irradiation with 200 mW cm - 2 VIS + wIRA for five minutes at 37 °C. The adherent microorganisms were quantified, and the biofilm samples were visualized using live/dead staining and confocal laser scanning microscopy (CLSM). The THPTS-mediated aPDT resulted in significant decreases in both the initially adherent microorganisms and the microorganisms in the mature oral biofilms, in comparison to the untreated control samples (>99.99% each; p = 0.018 and p = 0.0066, respectively). The remaining vital bacteria significantly decreased in the aPDT-treated biofilms during initial adhesion (vitality rate 9.4% vs. 71.2% untreated control, 17.28% CHX). Of the mature biofilms, 25.67% remained vital after aPDT treatment (81.97% untreated control, 16.44% CHX). High permeability of THPTS into deep layers could be shown. The present results indicate that the microbial reduction in oral initial and mature oral biofilms resulting from aPDT with VIS + wIRA in combination with THPTS has significant potential for the treatment of oral biofilm-associated diseases.