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1.
J Pers Med ; 11(3)2021 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-33806927

RESUMO

This study investigated the potential of salivary bacterial and protein markers for evaluating the disease status in healthy individuals or patients with gingivitis or caries. Saliva samples from caries- and gingivitis-free individuals (n = 18), patients with gingivitis (n = 17), or patients with deep caries lesions (n = 38) were collected and analyzed for 44 candidate biomarkers (cytokines, chemokines, growth factors, matrix metalloproteinases, a metallopeptidase inhibitor, proteolytic enzymes, and selected oral bacteria). The resulting data were subjected to principal component analysis and used as a training set for random forest (RF) modeling. This computational analysis revealed four biomarkers (IL-4, IL-13, IL-2-RA, and eotaxin/CCL11) to be of high importance for the correct depiction of caries in 37 of 38 patients. The RF model was then used to classify 10 subjects (five caries-/gingivitis-free and five with caries), who were followed over a period of six months. The results were compared to the clinical assessments of dental specialists, revealing a high correlation between the RF prediction and the clinical classification. Due to the superior sensitivity of the RF model, there was a divergence in the prediction of two caries and four caries-/gingivitis-free subjects. These findings suggest IL-4, IL-13, IL-2-RA, and eotaxin/CCL11 as potential salivary biomarkers for identifying noninvasive caries. Furthermore, we suggest a potential association between JAK/STAT signaling and dental caries onset and progression.

2.
Monogr Oral Sci ; 29: 30-37, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33427216

RESUMO

More than 700 microbial species inhabit the complex environment of the oral cavity. For years microorganisms have been studied in pure cultures, a highly artificial situation because microorganisms in natural habitats grow as complex ecologies, termed biofilms. These resemble multicellular organisms and are characterized by their overall metabolic activity upon multiple cellular interactions. Microorganisms in biofilms express different genes than their planktonic counterparts, resulting in higher resistance to antimicrobials, different nutritional requirements, or creation of a low redox potential allowing the growth of strictly anaerobic bacteria in the presence of oxygen. Multiple in vitro biofilm models have been described in the literature so far. The main emphasis here will be on multispecies biofilm batch culture models developed in Zurich. The standard 6-species supragingival biofilm model has been used to study basic aspects of oral biofilms such as structure, social behavior, and spatial distribution of microorganisms, or diffusion properties. Numerous parameters related to the inhibition of dental plaque were tested illustrating the high reliability of the model to predict the in vivo efficiency of antimicrobials. Modifications and advancements led to a 10-species subgingival model often combined with human gingival epithelial cells, as an integral part of the oral innate immune system, eliciting various cell responses ranging from cytokine production to apoptosis. In conclusion, biofilm models enable a multitude of questions to be addressed that cannot be studied with planktonic monocultures. The Zurich in vitro biofilm models are reproducible and reliable and may be used for basic studies, but also for application-oriented questions that could not be addressed using culture techniques. Oral biofilm research will certainly lead to a more realistic assessment of the role of microorganisms in the oral cavity in health and disease. In this respect, substantial progress has been made, but there is still more to explore.


Assuntos
Biofilmes , Boca , Gengiva , Humanos , Plâncton , Reprodutibilidade dos Testes
3.
J Clin Med ; 9(9)2020 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-32933084

RESUMO

Oral health is maintained by a healthy microbiome, which can be monitored by state-of-the art diagnostics. Therefore, this study evaluated the presence and quantity of ten oral disease-associated taxa (P. gingivalis, T. forsythia, T. denticola, F. nucleatum, C. rectus, P. intermedia, A. actinomycetemcomitans, S. mutans, S. sobrinus, oral associated Lactobacilli) in saliva and their clinical status association in 214 individuals. Upon clinical examination, study subjects were grouped into healthy, caries and periodontitis and their saliva was collected. A highly specific point-of-care compatible dual color qPCR assay was developed and used to study the above-mentioned bacteria of interest in the collected saliva. Assay performance was compared to a commercially available microbial reference test. Eight out of ten taxa that were investigated during this study were strong discriminators between the periodontitis and healthy groups: C. rectus, T. forsythia, P. gingivalis, S. mutans, F. nucleatum, T. denticola, P. intermedia and oral Lactobacilli (p < 0.05). Significant differentiation between the periodontitis and caries group microbiome was only shown for S. mutans (p < 0.05). A clear distinction between oral health and disease was enabled by the analysis of quantitative qPCR data of target taxa levels in saliva.

4.
J Endod ; 46(8): 1105-1112, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32497654

RESUMO

INTRODUCTION: Because active bacteria present a higher abundance of ribosomal RNA (rRNA) than DNA (rRNA gene), the rRNA/DNA ratio of next-generation sequencing (NGS) data was measured to search for active bacteria in endodontic infections. METHODS: Paired complementary DNA and DNA samples from 5 root canals of teeth with apical periodontitis were subjected to polymerase chain reaction with bar-coded primers amplifying the 16S rRNA gene hypervariable regions V4-V5. High-throughput sequencing was performed using MiSeq (Illumina, San Deigo, CA), and data were analyzed using Quantitative Insights Into Microbial Ecology and Human Oral Microbiome Database. Statistical analysis was performed for relative abundance of bacteria in the DNA- and rRNA-based NGS data using the Mann-Whitney test, whereas differences in the diversity and richness indexes were assessed using a nonparametric 2-sample t test (P < .05). For bacterial taxa detected in both approaches, the rRNA/DNA ratios were calculated by dividing the average abundance of individual species in the respective analysis. RESULTS: Although no significant difference was found in the indexes of bacterial richness and diversity, the relative abundance of bacterial members varied in both analyses. Comparing rRNA with DNA data, there was a significant decrease in the relative abundance of Firmicutes (P < .05). The bacterial taxa Bacteroidales [G-2] bacterium HMT 274, Porphyromonas endodontalis, Tannerella forsythia, Alloprevotella tannerae, Prevotella intermedia, Pseudoramibacter alactolyticus, Olsenella sp. HMT 809, Olsenella sp. HMT 939, Olsenella uli, and Fusobacterium nucleatum subsp. animalis were both dominant (DNA ≥ 1%) and active (rRNA/DNA ≥ 1). CONCLUSIONS: The integrated DNA- and rRNA-based NGS strategy was particularly important to disclose the activity of as-yet-uncultivated or difficult-to-culture bacteria in endodontic infections.


Assuntos
Infecções Bacterianas , Sequenciamento de Nucleotídeos em Larga Escala , Actinobacteria , Bactérias , Clostridiales , DNA Bacteriano , Humanos , RNA Ribossômico 16S
5.
Microorganisms ; 8(5)2020 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-32384777

RESUMO

Oral bacteria possess the ability to form biofilms on solid surfaces. After the penetration of oral bacteria into the pulp, the contact between biofilms and pulp tissue may result in pulpitis, pulp necrosis and/or periapical lesion. Depending on the environmental conditions and the availability of nutrients in the pulp chamber and root canals, mainly Gram-negative anaerobic microorganisms predominate and form the intracanal endodontic biofilm. The objective of the present study was to investigate the role of different substrates on biofilm formation as well as the separate and collective incorporation of six endodontic pathogens, namely Enterococcus faecalis, Staphylococcus aureus, Prevotella nigrescens, Selenomonas sputigena, Parvimonas micra and Treponema denticola into a nine-species "basic biofilm". This biofilm was formed in vitro as a standard subgingival biofilm, comprising Actinomyces oris, Veillonella dispar, Fusobacterium nucleatum, Streptococcus anginosus, Streptococcus oralis, Prevotella intermedia, Campylobacter rectus, Porphyromonas gingivalis, and Tannerella forsythia. The resulting endodontic-like biofilms were grown 64 h under the same conditions on hydroxyapatite and dentin discs. After harvesting the endodontic-like biofilms, the bacterial growth was determined using quantitative real-time PCR, were labeled using fluorescence in situ hybridization (FISH) and analyzed by confocal laser scanning microscopy (CLSM). The addition of six endodontic pathogens to the "basic biofilm" induced a decrease in the cell number of the "basic" species. Interestingly, C. rectus counts increased in biofilms containing E. faecalis, S. aureus, P. nigrescens and S. sputigena, respectively, both on hydroxyapatite and on dentin discs, whereas P. intermedia counts increased only on dentin discs by addition of E. faecalis. The growth of E. faecalis on hydroxyapatite discs and of E. faecalis and S. aureus on dentin discs were significantly higher in the biofilm containing all species than in the "basic biofilm". Contrarily, the counts of P. nigrescens, S. sputigena and P. micra on hydroxyapatite discs as well as counts of P. micra and T. denticola on dentin discs decreased in the all-species biofilm. Overall, all bacterial species associated with endodontic infections were successfully incorporated into the standard multispecies biofilm model both on hydroxyapatite and dentin discs. Thus, future investigations on endodontic infections can rely on this newly established endodontic-like multispecies biofilm model.

6.
J Clin Med ; 9(4)2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32244784

RESUMO

Modification of oral biofilms adhering to dental hard tissues could lead to new treatment approaches in cariology and periodontology. In this study the impact of DNase I and/or proteinase K on the formation of a simulated supragingival biofilm was investigated in vitro. Six-species biofilms were grown anaerobically in the presence of DNase I and proteinase K. After 64 h biofilms were either harvested and quantified by culture analysis or proceeded to staining followed by confocal laser scanning microscopy. Microbial cells were stained using DNA-dyes or fluorescent in situ hybridization. Exopolysaccharides, eDNA and exoproteins were stained with Calcofluor, anti-DNA-antibody, and SyproTM Ruby, respectively. Overall, results showed that neither DNase I nor proteinase K had an impact on total colony-forming units (CFUs) compared to the control without enzymes. However, DNase I significantly suppressed the growth of Actinomyces oris, Fusobacterium nucleatum, Streptococcus mutans, Streptococcus oralis and Candida albicans. Proteinase K treatment induced significant increase in S. mutans and S. oralis CFUs (p < 0.001), whereas C. albicans and V. dispar showed lower CFUs compared to the control. Interestingly, confocal images visualized the biofilm degradation caused by DNase I and proteinase K. Thus, enzymatic treatment should be combined with conventional antimicrobial agents aiming at both bactericidal effectiveness and biofilm dispersal.

7.
Antibiotics (Basel) ; 8(4)2019 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-31817588

RESUMO

The aim of this study was to determine the antibiotic susceptibility patterns of 57 Aggregatibacter actinomycetemcomitans and 56 Porphyromonas gingivalis strains isolated from subgingival biofilm samples of periodontitis patients in Switzerland from 1980 to 2017. The minimal inhibitory concentrations (MIC) of the most commonly used antibiotics in periodontal therapy (amoxicillin, metronidazole, azithromycin, and doxycycline) or in severe body infections (amoxicillin/clavulanic acid, clindamycin, ertapenem, and moxifloxacin) were determined. Furthermore, all the strains were screened for beta-lactamase activity and the presence of selected resistance genes (cfxA, ermF, and tetQ). Overall, there was no significant increase in MIC values over the 37­year period. Two of the most recent P. gingivalis isolates yielded the highest MIC values. The first isolate was ermF-positive with MIC values >8 µg/mL, 2 µg/mL, and 0.25 µg/mL for clindamycin, azithromycin, and moxifloxacin, respectively. The second isolate showed a high MIC value of 4 µg/mL for moxifloxacin, which was associated with a confirmed single-point mutation in the quinolone resistance-determining region (QRDR) of the gyrA gene. Although there was no significant increase in the antibiotic resistance among the oral bacterial isolates tested, the detection of resistant P. gingivalis isolates underlines the need to optimize the antibiotic therapeutic protocols in dentistry.

8.
Front Microbiol ; 10: 1716, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31417514

RESUMO

Fusobacteria are common obligately anaerobic Gram-negative bacteria of the oral cavity that may act as a bridge between early and late colonizing bacteria in dental plaque and have a role in oral and extra-oral infections. Fusobacterium nucleatum has a crucial role in oral biofilm structure and ecology, as revealed in experimental and clinical biofilm models. The aim of this study was to investigate the impact of various Fusobacterium species on in vitro biofilm formation and structure in three different oral biofilm models namely a supragingival, a supragingival "feeding", and a subgingival biofilm model. The standard six-species supragingival and "feeding" biofilm models employed contained Actinomyces oris, Candida albicans, Streptococcus mutans, Streptococcus oralis, Veillonella dispar, and Fusobacterium sp. The subgingival biofilm model contained 10 species (A. oris, Campylobacter rectus, F. nucleatum ssp. nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Streptococcus anginosus, S. oralis, Tannerella forsythia, Treponema denticola, and V. dispar). Six different Fusobacterium species or subspecies, respectively, were tested namely F. nucleatum ssp. fusiforme, F. nucleatum ssp. nucleatum, F. nucleatum ssp. polymorphum, F. nucleatum ssp. vincentii, F. naviforme, and F. periodonticum). Biofilms were grown anaerobically on hydroxyapatite disks in 24-well culture dishes. After 64 h, biofilms were either harvested and quantified by culture analysis or proceeded to fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). All Fusobacterium species tested established well in the biofilms, with CFUs ranging from 1.4E+04 (F. nucleatum ssp. fusiforme) to 5.6E+06 (F. nucleatum ssp. nucleatum). The presence of specific Fusobacterium sp./ssp. induced a significant decrease in C. albicans levels in the supragingival model and in V. dispar levels in the "feeding" supragingival model. In the subgingival model, the counts of A. oris, S. oralis, P. intermedia, P. gingivalis, and C. rectus significantly decreased in the presence of specific Fusobacterium sp./ssp. Collectively, this study showed variations in the growing capacities of different fusobacteria within biofilms, affecting the growth of surrounding species and potentially the biofilm architecture. Hence, clinical or experimental studies need to differentiate between Fusobacterium sp./ssp., as their biological properties may well vary.

9.
Sci Rep ; 9(1): 20325, 2019 12 30.
Artigo em Inglês | MEDLINE | ID: mdl-31889168

RESUMO

Due to increasing antibiotic resistance, the application of antimicrobial photodynamic therapy (aPDT) is gaining increasing popularity in dentistry. The aim of this study was to investigate the antimicrobial effects of aPDT using visible light (VIS) and water-filtered infrared-A (wIRA) in combination with a Hypericum perforatum extract on in situ oral biofilms. The chemical composition of H. perforatum extract was analyzed using ultra-high-performance liquid chromatography coupled with high resolution mass spectrometry (UPLC-HRMS). To obtain initial and mature oral biofilms in situ, intraoral devices with fixed bovine enamel slabs (BES) were carried by six healthy volunteers for two hours and three days, respectively. The ex situ exposure of biofilms to VIS + wIRA (200 mWcm-2) and H. perforatum (32 mg ml-1, non-rinsed or rinsed prior to aPDT after 2-min preincubation) lasted for five minutes. Biofilm treatment with 0.2% chlorhexidine gluconate solution (CHX) served as a positive control, while untreated biofilms served as a negative control. The colony-forming units (CFU) of the aPDT-treated biofilms were quantified, and the surviving microorganisms were identified using MALDI-TOF biochemical tests as well as 16 S rDNA-sequencing. We could show that the H. perforatum extract had significant photoactivation potential at a concentration of 32 mg ml-1. When aPDT was carried out in the presence of H. perforatum, all biofilms (100%) were completely eradicated (p = 0.0001). When H. perforatum was rinsed off prior to aPDT, more than 92% of the initial viable bacterial count and 13% of the mature oral biofilm were killed. Overall, the microbial composition in initial and mature biofilms was substantially altered after aPDT, inducing a shift in the synthesis of the microbial community. In conclusion, H. perforatum-mediated aPDT using VIS + wIRA interferes with oral biofilms, resulting in their elimination or the substantial alteration of microbial diversity and richness. The present results support the evaluation of H. perforatum-mediated aPDT for the adjunctive treatment of biofilm-associated oral diseases.


Assuntos
Anti-Infecciosos/farmacologia , Biofilmes/efeitos dos fármacos , Biofilmes/efeitos da radiação , Hypericum/química , Raios Infravermelhos , Luz , Extratos Vegetais/farmacologia , Anti-Infecciosos/química , Bactérias/efeitos dos fármacos , Bactérias/efeitos da radiação , Aderência Bacteriana , Cromatografia Líquida de Alta Pressão , Humanos , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/efeitos da radiação , Mucosa Bucal/microbiologia , Compostos Fitoquímicos/química , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/química , Espectrometria de Massas por Ionização por Electrospray
10.
Dent J (Basel) ; 6(4)2018 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-30347835

RESUMO

On account of its proven clinical efficacy, the combination of systemically administered amoxicillin and metronidazole is frequently adjuncted to non-operative periodontal therapy and well documented. Potential drawbacks of this regimen, e.g., side effects and problems with the compliance, led to an ongoing search for alternatives. Azithromycin, an antibiotic extensively used in general medicine, has recently found its niche in periodontal therapy as well. This systematic review aimed to analyze the in vitro antimicrobial efficacy of amoxicillin plus metronidazole versus azithromycin. For this purpose, a systematic literature search was performed, and studies published up to 29 March 2018 referenced in Medline, Embase, Cochrane, and Biosis were independently screened by two authors. An additional hand search was performed and studies focusing on the evaluation of in vitro antimicrobial efficacy of amoxicillin + metronidazole or azithromycin on bacteria from the subgingival biofilm were included. English and German language research reports were considered. From 71 identified articles, only three articles were eligible for inclusion. These studies showed heterogeneity in terms of analytical methods and strains explored. However, all studies used multispecies biofilm models for analysis of the antimicrobial activity. Unanimously, studies reported on more pronounced antimicrobial effects when applying the combination of amoxicillin + metronidazole, compared to azithromycin. Based on the few studies available, the combination of amoxicillin + metronidazole seemed to display higher antimicrobial efficacy in vitro than azithromycin.

11.
Artigo em Inglês | MEDLINE | ID: mdl-29844920

RESUMO

Aggregatibacter actinomycetemcomitans is a Gram-negative organism, strongly associated with aggressive forms of periodontitis. An important virulence property of A. actinomycetemcomitans is its ability to form tenacious biofilms that can attach to abiotic as well as biotic surfaces. The histone-like (H-NS) family of nucleoid-structuring proteins act as transcriptional silencers in many Gram-negative bacteria. To evaluate the role of H-NS in A. actinomycetemcomitans, hns mutant derivatives of serotype a strain D7S were generated. Characteristics of the hns mutant phenotype included shorter and fewer pili, and substantially lower monospecies biofilm formation relative to the wild type. Furthermore, the D7S hns mutant exhibited significantly reduced growth within a seven-species oral biofilm model. However, no apparent difference was observed regarding the numbers and proportions of the remaining six species regardless of being co-cultivated with D7S hns or its parental strain. Proteomics analysis of the strains grown in monocultures confirmed the role of H-NS as a repressor of gene expression in A. actinomycetemcomitans. Interestingly, proteomics analysis of the multispecies biofilms indicated that the A. actinomycetemcomitans wild type and hns mutant imposed different regulatory effects on the pattern of protein expression in the other species, i.e., mainly Streptococcus spp., Fusobacterium nucleatum, and Veillonella dispar. Gene ontology analysis revealed that a large portion of the differentially regulated proteins was related to translational activity. Taken together, our data suggest that, apart from being a negative regulator of protein expression in A. actinomycetemcomitans, H-NS promotes biofilm formation and may be an important factor for survival of this species within a multispecies biofilm.

12.
Caries Res ; 52(6): 447-453, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29617682

RESUMO

Antimicrobial photodynamic therapy (aPDT) may be useful as a supportive antimicrobial measure for caries-active subjects. In this study, the antimicrobial efficacy of aPDT with a phenalen-1-one photosensitizer was evaluated in a novel in vitro biofilm model comprising Actinomyces naeslundii, Actinomyces odontolyticus, and Streptococcus mutans and was compared to chlorhexidine. The proposed biofilm model allows high-throughput screening for antimicrobial efficacy while exhibiting a differentiated response to different antimicrobial approaches. While chlorhexidine 0.2% showed a reduction of ≈4 log10 for all species, aPDT led to a more pronounced reduction of S. mutans (2.8 log10) than of Actinomyces spp. (1.2 or 1.3 log10). A similar effect was also observed in monospecies biofilms. Therefore, aPDT may be more effective against S. mutans than against Actinomyces spp. when in biofilms, and this antimicrobial approach merits further investigations.


Assuntos
Anti-Infecciosos/uso terapêutico , Biofilmes/efeitos dos fármacos , Clorexidina/uso terapêutico , Cárie Dentária/tratamento farmacológico , Fenalenos/uso terapêutico , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/uso terapêutico , Actinomyces/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Humanos , Streptococcus mutans/efeitos dos fármacos
13.
Front Microbiol ; 9: 688, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29681899

RESUMO

In light of increasing resistance toward conventional antibiotics and antiseptics, antimicrobial photodynamic therapy (aPDT) may be a valuable alternative, especially for use in dentistry. In this regard, photosensitizers (PS) based on a phenalen-1-one structure seem to be especially favorable due to their high singlet oxygen quantum yield. However, the actual target structures of phenalen-1-one-mediated aPDT are still unclear. The aim of the present study was to investigate the antimicrobial efficacy of aPDT mediated by phenalen-1-one derivatives SAPYR and SAGUA for inactivation of a polymicrobial biofilm consisting of three putative periodontal pathogens in vitro and to get first insights in the mechanism of action of phenalen-1-one-mediated aPDT by assessing damage of cytoplasmic membranes. aPDT with SAPYR exhibited identical antimicrobial efficacy as compared to chlorhexidine (CHX) [4.4-6.1 log10 reduction of colony forming units (CFUs) depending on bacterial species] while aPDT with SAGUA was less effective (2.0-2.8 log10). Flow cytometric analysis combined with propidium iodide (PI) staining revealed no damage of cytoplasmic membranes after aPDT with both phenalen-1-one derivatives, which was confirmed by spectroscopic measurements for release of nucleic acids after treatment. Spectrophotometric PS-uptake measurements showed no uptake of SAPYR by bacterial cells. Despite the inability to pinpoint the actual target of phenalen-1-one-mediated aPDT, this study shows the high antimicrobial potential of phenalen-1-on mediated aPDT (especially when using SAPYR) and represents a first step for getting insights in the mechanism and damage patterns of aPDT with this class of PS.

14.
Arch Oral Biol ; 89: 77-83, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29482049

RESUMO

OBJECTIVE: Fluoride is widely used as an anti-caries agent, e.g. in toothpastes and mouth rinses. However, the nature of the anti-caries action is not entirely clear. Mechanisms suspected to explain the cariostatic effect include inhibitory effects on acid formation by bacteria, inhibition of extracellular polysaccharide (EPS) production, inhibition of enamel demineralization and enhancement of remineralizaton or combination thereof. The aim of this study was to examine with the supragingival Zurich in vitro biofilm model the effect of fluoride in NaF formulation, on the microbiota and on demineralization. METHODS: Biofilms consisting of Actinomyces oris, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Veillonella dispar and Streptococcus sobrinus, were grown anaerobically on sintered hydroxyapatite or bovine enamel disks, exposed to 200, 400, and 1400 ppm of NaF, or 0.1% chlorhexidine (positive control). The biofilms were harvested after 64 h and CFUs were assessed for total bacteria. Demineralization of enamel disks was measured by quantitative light-induced fluorescence. RESULTS: NaF did not affect the bacterial numbers. No enamel mineral loss was observed at 1400 and 400 ppm of fluoride, whereas the pH of the surrounding medium was increased to 5.5 and 5.0, respectively, compared to the untreated control (pH 4.5 and mineral loss ΔF of -32%). At 1400 ppm NaF the biofilm's EPS volume was also significantly reduced. CONCLUSIONS: Administration of NaF completely prevented demineralization without affecting biofilm composition and growth. This protective effect may be attributed to the observed decrease in acid production or EPS volume, or to a shift in the de/remineralization balance.


Assuntos
Biofilmes/efeitos dos fármacos , Esmalte Dentário/efeitos dos fármacos , Microbiota/efeitos dos fármacos , Boca/microbiologia , Fluoreto de Sódio/farmacologia , Desmineralização do Dente/prevenção & controle , Animais , Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Cariostáticos/farmacologia , Bovinos , Clorexidina/administração & dosagem , Clorexidina/farmacologia , Cárie Dentária/microbiologia , Cárie Dentária/prevenção & controle , Esmalte Dentário/microbiologia , Relação Dose-Resposta a Droga , Durapatita , Fluoretos/administração & dosagem , Fluoretos/farmacologia , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Antissépticos Bucais/farmacologia , Fosfatos , Saliva/metabolismo , Fluoreto de Sódio/administração & dosagem , Calcificação de Dente , Desmineralização do Dente/tratamento farmacológico , Desmineralização do Dente/microbiologia , Cremes Dentais/farmacologia
15.
Sci Rep ; 7(1): 4409, 2017 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-28667274

RESUMO

Anaeroglobus geminatus is a relatively newly discovered putative pathogen, with a potential role in the microbial shift associated with periodontitis, a disease that causes inflammatory destruction of the periodontal tissues, and eventually tooth loss. This study aimed to introduce A. geminatus into a polymicrobial biofilm model of relevance to periodontitis, and monitor the proteomic responses exerted to the rest of the biofilm community. A. geminatus was grown together with another 10-species in a well-established "subgingival" in vitro biofilm model. Its effects on the other species were quantitatively evaluated by qPCR and label-free proteomics. A. geminatus caused a significant increase in P. intermedia numbers, but not the other species in the biofilm. Whole cell proteome profiling of the biofilms by LC-MS/MS identified a total of 3213 proteins. Label-free quantitative proteomics revealed that 187 proteins belonging to the other 10 species were differentially abundant when A. geminatus was present in the biofilm. The species with most up-regulated and down-regulated proteins were P. intermedia and S. oralis, respectively. Regulated proteins were of primarily of ribosomal origin, and other affected categories involved proteolysis, carbon metabolism and iron transport. In conclusion, A. geminatus can be successfully grown in a polymicrobial biofilm community, causing quantitative proteomic shifts commensurate with increased virulence properties.


Assuntos
Biofilmes , Firmicutes/crescimento & desenvolvimento , Firmicutes/metabolismo , Proteoma , Proteômica , Cromatografia Líquida , Biologia Computacional/métodos , Firmicutes/classificação , Firmicutes/genética , Ontologia Genética , Proteômica/métodos , Transdução de Sinais , Espectrometria de Massas em Tandem
16.
Mol Oral Microbiol ; 32(5): 404-418, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28382776

RESUMO

As a member of subgingival multispecies biofilms, Tannerella forsythia is commonly associated with periodontitis. The bacterium has a characteristic cell surface (S-) layer modified with a unique O-glycan. Both the S-layer and the O-glycan were analyzed in this study for their role in biofilm formation by employing an in vitro multispecies biofilm model mimicking the situation in the oral cavity. Different T. forsythia strains and mutants with characterized defects in cell surface composition were incorporated into the model, together with nine species of select oral bacteria. The influence of the T. forsythia S-layer and attached glycan on the bacterial composition of the biofilms was analyzed quantitatively using colony-forming unit counts and quantitative real-time polymerase chain reaction, as well as qualitatively by fluorescence in situ hybridization and confocal laser scanning microscopy. This revealed that changes in the T. forsythia cell surface did not affect the quantitative composition of the multispecies consortium, with the exception of Campylobacter rectus cell numbers. The localization of T. forsythia within the bacterial agglomeration varied depending on changes in the S-layer glycan, and this also affected its aggregation with Porphyromonas gingivalis. This suggests a selective role for the glycosylated T. forsythia S-layer in the positioning of this species within the biofilm, its co-localization with P. gingivalis, and the prevalence of C. rectus. These findings might translate into a potential role of T. forsythia cell surface structures in the virulence of this species when interacting with host tissues and the immune system, from within or beyond the biofilm.


Assuntos
Biofilmes , Membrana Celular/genética , Mutação , Tannerella forsythia/genética , Tannerella forsythia/metabolismo , Campylobacter rectus/isolamento & purificação , Campylobacter rectus/fisiologia , Gengiva/microbiologia , Glicosilação , Interações Microbianas , Boca/microbiologia , Doenças Periodontais/microbiologia , Periodontite/microbiologia , Porphyromonas gingivalis/isolamento & purificação , Porphyromonas gingivalis/fisiologia , Prevotella intermedia/isolamento & purificação , Prevotella intermedia/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Treponema denticola/isolamento & purificação , Treponema denticola/fisiologia , Virulência
17.
Clin Oral Investig ; 21(4): 1029-1036, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-27193939

RESUMO

OBJECTIVE: The aim of this study is to assess the effects of ultrasonic tip distance and orientation on the removal of a multispecies biofilm under standardized conditions in vitro. METHODS: Six-species biofilms were grown on hydroxyapatite discs for 64 h and treated with a magnetostrictive ultrasonic tip (Cavitron) placed either on contact or at 0.25- and 0.5-mm distance. The treatment was performed for 15 s with either the tip at right angle or sideways. Biofilm removal was evaluated by assessing the viable bacteria in each supernatant and compared to respective controls. In the latter, biofilms were mechanically removed and evaluated in supernatants to assess adhering and floating bacteria. Colony-forming units (CFU) were determined by cultivation on solid media. Any remaining biofilm on the treated discs was also visualized after staining with green-fluorescent SYTO® 9 stain using a confocal laser scanning microscope (CLSM). Mann-Whitney U tests and Bonferroni correction were used to analyze the results between the groups. RESULTS: Sideways application of the ultrasonic tip at distances of 0.25 and 0.5 mm removed as many bacteria as present on the control discs compared to the tip on contact (p < 0.05). All other application modes, especially the ultrasonic tip applied perpendicularly on contact, showed no statistical significance in removing biofilm. CONCLUSION: Overall, data indicated that bacterial detachment depended on tip orientation and distance, especially when the tip was applied sideways similar to the clinical setting. CLINICAL RELEVANCE: Biofilm removal by means of ultrasonic debridement remains a crucial aspect in the treatment of periodontal disease. To ensure sufficient biofilm removal, the tip does not necessarily require contact to the surface, but an application parallel to the surface on the side is recommended.


Assuntos
Biofilmes , Placa Dentária/microbiologia , Placa Dentária/terapia , Raspagem Dentária/instrumentação , Terapia por Ultrassom/instrumentação , Durapatita , Técnicas In Vitro , Microscopia Confocal
18.
Clin Oral Investig ; 20(3): 607-13, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26323501

RESUMO

OBJECTIVES: This study aims to assess the potential of a mixture of three antibiotics (TreVitaMix, TVM) as an intracanal dressing to disinfect the outer root surface by applying a new in vitro model. MATERIALS AND METHODS: Fifty freshly extracted bovine roots were endodontically treated. Forty samples were then thoroughly scaled, mounted to petri dishes, gas sterilized, and randomly allocated to four groups (n = 10/group) according to their intracanal medication: sterile saline (NaCl; control, A); the TVM carrier material alone, i.e., propylene glycol (PG; B); TVM (C); and calcium hydroxide (D). In an additional group (E), the cementum was not removed and TVM was placed. Petri dishes were filled with Fastidious Anaerobe Agar, inoculated with Fusobacterium nucleatum suspension and then anaerobically incubated during 48-h intervals at 37 °C up to 192 h. Inhibition zones around the roots were then measured after each incubation period (mm(2)). RESULTS: Only teeth inoculated with the TVM dressing showed inhibition at all time points, whereas the other treatments showed no peri-radicular growing inhibition. Presence of cementum had no negative effect on disinfection (p = 0.9320). CONCLUSION: TVM was able to penetrate through the dentine and inhibit the bacterial growth of F. nucleatum up to 192 h. CLINICAL RELEVANCE: TVM might have the potential to sustainably disinfect the outer root surface in perio-endo lesions and serve as an adjunctive antimicrobial agent.


Assuntos
Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Cefuroxima/farmacologia , Ciprofloxacino/farmacologia , Fusobacterium nucleatum/efeitos dos fármacos , Metronidazol/farmacologia , Raiz Dentária/efeitos dos fármacos , Raiz Dentária/microbiologia , Animais , Bovinos , Desinfecção , Combinação de Medicamentos , Técnicas In Vitro , Irrigantes do Canal Radicular/farmacologia
19.
Clin Oral Implants Res ; 27(7): 890-5, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26461083

RESUMO

OBJECTIVES: Staphylococcus spp. are postulated to play a role in peri-implantitis. This study aimed to develop a "submucosal" in vitro biofilm model, by integrating two staphylococci into its composition. MATERIALS AND METHODS: The standard "subgingival" biofilm contained Actinomyces oris, Fusobacterium nucleatum, Streptococcus oralis, Veillonella dispar, Campylobacter rectus, Prevotella intermedia, Streptococcus anginosus, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola, and was further supplemented with Staphyoccous aureus and/or Staphylococcus epidermidis. Biofilms were grown anaerobically on hydroxyapatite or titanium discs and harvested after 64 h for real-time polymerase chain reaction, to determine their composition. Confocal laser scanning microscopy and fluorescence in situ hybridization were used for identifying the two staphylococci within the biofilm. RESULTS: Both staphylococci established within the biofilms when added separately. However, when added together, only S. aureus grew in high numbers, whereas S. epidermidis was reduced almost to the detection limit. Compared to the standard subgingival biofilm, addition of the two staphylococci had no impact on the qualitative or quantitative composition of the biofilm. When grown individually in the biofilm, S. epidermidis and S. aureus formed small distinctive clusters and it was confirmed that S. epidermidis was not able to grow in presence of S. aureus. CONCLUSIONS: Staphyoccous aureus and S. epidermidis can be individually integrated into an oral biofilm grown on titanium, hence establishing a "submucosal" biofilm model for peri-implantitis. This model also revealed that S. aureus outcompetes S. epidermidis when grown together in the biofilm, which may explain the more frequent association of the former with peri-implantitis.


Assuntos
Biofilmes/crescimento & desenvolvimento , Modelos Biológicos , Peri-Implantite/microbiologia , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus epidermidis/crescimento & desenvolvimento , Gengiva/microbiologia , Humanos , Técnicas In Vitro , Titânio
20.
Virulence ; 6(7): 704-9, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26305580

RESUMO

Periodontitis is an infectious inflammatory disease that destroys the tooth-supporting tissues. It is caused by the formation of subgingival biofilms on the surface of the tooth. Characteristic bacteria associated with subgingival biofilms are the Gram-negative anaerobes Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola, collectively known as the "red complex" species. Inter-epithelial junctions ensure the barrier integrity of the gingival epithelium. This may however be disrupted by the biofilm challenge. The aim of this in vitro study was to investigate the effect of subgingival biofilms on the expression of inter-epithelial junctions by gingival epithelia, and evaluate the relative role of the red complex. Multi-layered human gingival epithelial cultures were challenged with a 10-species in vitro subgingival biofilm model, or its variant without the red complex, for 3 h and 24 h. A low-density array microfluidic card platform was then used for analyzing the expression of 62 genes encoding for tight junctions, gap junctions, adherens junctions, and desmosomes. Although there was a limited effect of the biofilms on the expression of tight, adherens and gap junctions, the expression of a number of desmosomal components was affected. In particular, Desmoglein-1 displayed a limited and transient up-regulation in response to the biofilm. In contrast, Desmocollin-2, Desmoplakin and Plakoglobin were down-regulated equally by both biofilm variants, after 24 h. In conclusion, this subgingival biofilm model may down-regulate selected desmosomal junctions in the gingival epithelium, irrespective of the presence of the "red complex." In turn, this could compromise the structural integrity of the gingival tissue, favoring bacterial invasion and chronic infection.


Assuntos
Biofilmes/crescimento & desenvolvimento , Gengiva/microbiologia , Bactérias Gram-Negativas/fisiologia , Junções Intercelulares/metabolismo , Junções Intercelulares/microbiologia , Células Cultivadas , Desmocolinas/genética , Desmocolinas/metabolismo , Desmoplaquinas/genética , Desmoplaquinas/metabolismo , Regulação para Baixo , Bactérias Gram-Negativas/crescimento & desenvolvimento , Humanos , Junções Intercelulares/genética , Periodontite/microbiologia , Transcriptoma , Regulação para Cima , gama Catenina/genética , gama Catenina/metabolismo
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