RESUMEN
Oral biofilms can degrade the components in dental resin-based composite restorations, thus compromising marginal integrity and leading to secondary caries. This study investigates the mechanical integrity of the dentin-composite interface challenged with multi-species oral biofilms. While most studies used single-species biofilms, the present study used a more realistic, diverse biofilm model produced directly from plaques collected from donors with a history of early childhood caries. Dentin-composite disks were made using bovine incisor roots filled with Z100(TM) or Filtek(TM) LS (3M ESPE). The disks were incubated for 72 h in paired CDC biofilm reactors, using a previously published protocol. One reactor was pulsed with sucrose, and the other was not. A sterile saliva-only control group was run with sucrose pulsing. The disks were fractured under diametral compression to evaluate their interfacial bond strength. The surface deformation of the disks was mapped using digital image correlation to ascertain the fracture origin. Fracture surfaces were examined using scanning electron microscopy/energy-dispersive X-ray spectroscopy to assess demineralization and interfacial degradation. Dentin demineralization was greater under sucrose-pulsed biofilms, as the pH dropped <5.5 during pulsing, with LS and Z100 specimens suffering similar degrees of surface mineral loss. Biofilm growth with sucrose pulsing also caused preferential degradation of the composite-dentin interface, depending on the composite/adhesive system used. Specifically, Z100 specimens showed greater bond strength reduction and more frequent cohesive failure in the adhesive layer. This was attributed to the inferior dentin coverage by Z100 adhesive, which possibly led to a higher level of chemical and enzymatic degradation. The results suggested that factors other than dentin demineralization were also responsible for interfacial degradation. A clinically relevant in vitro biofilm model was therefore developed, which would effectively allow assessment of the degradation of the dentin-composite interface subjected to multi-species biofilm challenge.
Asunto(s)
Resinas Acrílicas/química , Biopelículas , Resinas Compuestas/química , Dentina/microbiología , Poliuretanos/química , Animales , Biopelículas/efectos de los fármacos , Reactores Biológicos , Bovinos , Preescolar , Recubrimiento Dental Adhesivo , Humanos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Especificidad de la Especie , Espectrometría por Rayos X , Sacarosa/farmacologíaRESUMEN
AIMS: Most studies of biofilm effects on dental materials use single-species biofilms, or consortia. Microcosm biofilms grown directly from saliva or plaque are much more diverse, but difficult to characterize. We used the Human Oral Microbial Identification Microarray (HOMIM) to validate a reproducible oral microcosm model. METHODS AND RESULTS: Saliva and dental plaque were collected from adults and children. Hydroxyapatite and dental composite discs were inoculated with either saliva or plaque, and microcosm biofilms were grown in a CDC biofilm reactor. In later experiments, the reactor was pulsed with sucrose. DNA from inoculums and microcosms was analysed by HOMIM for 272 species. Microcosms included about 60% of species from the original inoculum. Biofilms grown on hydroxyapatite and composites were extremely similar. Sucrose pulsing decreased diversity and pH, but increased the abundance of Streptococcus and Veillonella. Biofilms from the same donor, grown at different times, clustered together. CONCLUSIONS: This model produced reproducible microcosm biofilms that were representative of the oral microbiota. Sucrose induced changes associated with dental caries. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first use of HOMIM to validate an oral microcosm model that can be used to study the effects of complex biofilms on dental materials.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Materiales Dentales/análisis , Placa Dental/microbiología , Saliva/microbiología , Adulto , Reactores Biológicos , Niño , Recuento de Colonia Microbiana , Medios de Cultivo/química , ADN Bacteriano/análisis , Durapatita/análisis , Humanos , Ensayo de Materiales , Análisis de Secuencia por Matrices de Oligonucleótidos , Streptococcus/crecimiento & desarrollo , Sacarosa/química , Veillonella/crecimiento & desarrolloRESUMEN
BACKGROUND AND OBJECTIVE: We previously reported that the interleukin-8 (IL-8) response to Fusobacterum nucleatum was attenuated in the presence of Streptococcus cristatus. Here, we further examined the underlying mechanism(s) involved in the modulating effect of S. cristatus by looking specifically at its impact on the nuclear factor-kappa B (NF-κB) pathway under the toll-like receptor (TLR) signaling background. MATERIAL AND METHODS: OKF6/TERT-2 and KB cells were co-cultured with F. nucleatum and S. cristatus, either alone or in combination. Secretion of IL-8 protein was measured by ELISA. The nuclear translocation of NF-κB was evaluated by confocal microscopy, while DNA-binding activity was quantified using TransAM™ ELISA kits. Western blot analysis was performed to determine whether the anti-inflammatory effect of S. cristatus is related to the modulation of the NF-κB inhibitory protein IκB-α. RESULTS: Incubation with F. nucleatum significantly enhanced the nuclear translocation of NF-κB. Exposure to S. cristatus alone did not cause detectable NF-κB translocation and was able to inhibit the F. nucleatum-induced NF-κB nuclear translocation. The TransAM assay further confirmed that S. cristatus blocked the nuclear translocation of NF-κB in response to F. nucleatum stimulation. In contrast to the nearly complete degradation of IκB-α induced by F. nucleatum alone, the presence of S. cristatus stabilized IκB-α. Pre-incubation with TLR2 and TLR4 antibodies, however, did not affect the epithelial response to either species alone or in combination. CONCLUSION: The mechanism by which S. cristatus attenuates F. nucleatum-induced proinflammatory responses in oral epithelial cells appears to involve blockade of NF-κB nuclear translocation at the level of IκB-α degradation.
Asunto(s)
Fusobacterium nucleatum/fisiología , Interacciones Huésped-Patógeno , Interleucina-8/biosíntesis , Interacciones Microbianas , Streptococcus/fisiología , Línea Celular Transformada , Células Epiteliales/metabolismo , Humanos , Proteínas I-kappa B/metabolismo , Células KB , Inhibidor NF-kappaB alfa , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Transporte de Proteínas , Transducción de Señal , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/inmunologíaRESUMEN
We previously reported that Streptococcus cristatus, an oral commensal, was able to downregulate the interleukin-8 (IL-8) response to Fusobacterium nucleatum, a putative oral pathogen in oral epithelial cells. The aim of this study was to extend the understanding of how S. cristatus regulates cytokine expression in oral epithelial cells on a broad basis, and investigate whether the modulation of a Toll-like receptor (TLR) pathway was involved in this process. KB and TERT-2 cells were co-cultured with F. nucleatum and S. cristatus, either alone or in combination. Total RNA was extracted and pathway-specific focused microarrays were used to profile the transcriptional responses of various cytokine genes and those related to TLR-mediated signal transduction. Reverse transcription-polymerase chain reactions (RT-PCR) and protein assays were performed to confirm the microarray results for selected genes. We found that exposure to either S. cristatus or F. nucleatum alone led to distinct changes in cytokine expression patterns. Fusobacterium nucleatum induced a greater number of gene expression changes than S. cristatus (15% vs. 4%, respectively). The presence of S. cristatus with F. nucleatum attenuated the expression of a number of inflammatory cytokines, and upregulated several anti-inflammatory mediators. The RT-PCR confirmed the messenger RNA attenuation of IL-1α, tumor necrosis factor-α and IL-6 by S. cristatus. Profiling of TLR-signaling-related genes revealed that S. cristatus most significantly impacted the downstream pathways, especially nuclear factor-κB, rather than altering TLRs and their adaptors and interacting proteins. Our data suggest that S. cristatus may attenuate the epithelial proinflammatory cytokine response to F. nucleatum by influencing pathways converging on nuclear factor-κB.
Asunto(s)
Citocinas/inmunología , Fusobacterium nucleatum/inmunología , Mucosa Bucal/inmunología , FN-kappa B/inmunología , Streptococcus/inmunología , Línea Celular , Quimiocinas/genética , Quimiocinas/inmunología , Técnicas de Cocultivo , Citocinas/genética , Células Epiteliales/inmunología , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica/genética , Humanos , Mediadores de Inflamación/inmunología , Interleucina-1alfa/genética , Interleucina-1alfa/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Interleucinas/genética , Interleucinas/inmunología , Células KB/inmunología , Análisis por Micromatrices , Interacciones Microbianas , Mucosa Bucal/citología , FN-kappa B/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Receptores Toll-Like/genética , Receptores Toll-Like/inmunología , Transcripción Genética/genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Regulación hacia Arriba/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/inmunologíaRESUMEN
Metagenomics uses gene expression patterns to understand the taxonomy and metabolic activities of microbial communities. Metaproteomics applies the same approach to community proteomes. Previously, we used a novel three-dimensional peptide separation method to identify over 2000 salivary proteins. This study used those data to carry out the first metaproteomic analysis of the human salivary microbiota. The metagenomic software MEGAN generated a phylogenetic tree, which was checked against the Human Oral Microbiome Database (HOMD). Pathway analyses were performed with the Clusters of Orthologous Groups and MetaCyc databases. Thirty-seven per cent of the peptides were identifiable only at the level of cellular organisms or bacteria. The rest were distributed among five bacterial phyla (61%), archea (0.5%), and viruses (0.8%); 29% were assignable at the genus level, and most belonged to Streptococcus (17%). Eleven per cent of all peptides could be assigned to species. Most taxa were represented in HOMD and they included well-known species such as periodontal pathogens. However, there also were 'exotic' species including aphid endosymbionts; plant, water, and soil bacteria; extremophiles; and archea. The pathway analysis indicated that peptides were linked to translation (37%), followed by glycolysis (19%), amino acid metabolism (8%), and energy production (8%). The taxonomic structure of the salivary metaproteome is very diverse but is dominated by streptococci. 'Exotic' species may actually represent close relatives that have not yet been sequenced. Salivary microbes appear to be actively engaged in protein synthesis, and the pathway analysis is consistent with the metabolism of salivary glycoproteins.
Asunto(s)
Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Técnicas de Tipificación Bacteriana , Proteoma/análisis , Saliva/microbiología , Sistemas de Transporte de Aminoácidos , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/metabolismo , Bases de Datos de Proteínas , Metabolismo Energético , Glucólisis , Humanos , Péptidos/análisis , Filogenia , Biosíntesis de Proteínas , Proteoma/genética , Proteínas y Péptidos Salivales/análisis , Espectrometría de Masas en TándemRESUMEN
OBJECTIVE: In previous studies, we defined groups of subjects with opposite salivary function. Group membership was associated with clinically relevant outcomes. High aggregation-adherence (HAA) groups showed lower levels of caries, supragingival plaque, total streptococci, and Tannerella forsythensis than low high aggregation-adherence (LAA) groups. In this study, we used a proteomic approach to search for biomarkers which could be useful as risk indicators for those outcomes. DESIGN: Clarified resting whole saliva from each of 41 HAA and LAA subjects was separated by preparative isoelectric focusing. Fractions showing the most distinctive protein profiles were pooled into four sets (pI 3-3.5, pI 4-4.7, pI 5.7-7.7, pI 10-11.5). Each pool then was compared by SDS-PAGE. Image analysis software was used to quantify matched bands. Partial least squares analysis (PLS) was used to determine which of the 65 bands from all four pools were the best predictors of group membership, caries, total plaque, total streptococci, and T. forsythensis counts. Those bands were identified by mass spectroscopy (MS-MS). RESULTS: Two bands consistently were strong predictors in separate PLS analyses of each outcome variable. In follow-up univariate analyses, those bands showed the strongest significant differences between the HAA and LAA groups. They also showed significant inverse correlations with caries and all the microbiological variables. MSMS identified those bands as statherin, and a truncated cystatin S missing the first eight N-terminal amino acids. CONCLUSIONS: Levels of statherin and truncated cystatin S may be potential risk indicators for the development of caries and other oral diseases.
Asunto(s)
Caries Dental/microbiología , Saliva/química , Cistatinas Salivales/análisis , Proteínas y Péptidos Salivales/análisis , Adhesión Bacteriana/fisiología , Biopelículas , Biomarcadores/análisis , Western Blotting , Recuento de Colonia Microbiana , Electroforesis en Gel Bidimensional/métodos , Femenino , Humanos , Masculino , Proteómica/métodos , Factores de Riesgo , Saliva/microbiología , Saliva/fisiologíaRESUMEN
BACKGROUND AND OBJECTIVE: Oral epithelial cells may be invaded by a polymicrobial intracellular flora, including pathogens together with commensals. Various oral pathogens can induce the production of interleukin-8, a potent neutrophil chemotractant, in oral epithelial cells. Evidence from the gut suggests that commensal species may modulate inflammatory responses to pathogens. The aim of this study was to examine the interleukin-8 responses of oral epithelial cells to an oral pro-inflammatory species, Fusobacterium nucleatum, in combination with an oral commensal, Streptococcus cristatus. MATERIAL AND METHODS: KB, TERT-2, TR146 and SCC15 cells were cocultured with F. nucleatum and S. cristatus, either alone or in combination, at 37 degrees C in 5% CO2 under various conditions. The mRNA expression of interleukin-8 was analyzed by reverse transcription-polymerase chain reaction and protein secretion was measured by enzyme-linked immunosorbent assay. RESULTS: F. nucleatum alone evoked a potent interleukin-8 response, whereas S. cristatus alone did not induce significant interleukin-8 expression in oral epithelial cells. When present together, S. cristatus attenuated the F. nucleatum-induced interleukin-8 production in the four oral epithelial cell lines to varying degrees. The inhibitory effect of S. cristatus was independent of its viability and its co-aggregation with F. nucleatum, was not related to soluble bacterial products and appeared to require bacterial contact with epithelial cells. Similar effects were seen with several other species of oral streptococci. CONCLUSION: Our data suggest that S. cristatus may exert immunomodulatory effects on the interleukin-8 response of oral epithelial cells to F. nucleatum challenge.
Asunto(s)
Fusobacterium nucleatum/inmunología , Interleucina-8/biosíntesis , Mucosa Bucal/microbiología , Streptococcus/fisiología , Aggregatibacter actinomycetemcomitans/inmunología , Anticuerpos Antibacterianos/inmunología , Canavanina/inmunología , Línea Celular , Técnicas de Cocultivo , Eikenella corrodens/inmunología , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Humanos , Factores Inmunológicos/inmunología , Células KB , Boca/microbiología , Mucosa Bucal/inmunología , Porphyromonas gingivalis/inmunología , Prevotella intermedia/inmunología , Streptococcus gordonii/inmunología , Streptococcus intermedius/inmunología , Streptococcus mitis/inmunología , Streptococcus mutans/inmunología , Streptococcus oralis/inmunología , Streptococcus sanguis/inmunología , Streptococcus sobrinus/inmunologíaRESUMEN
INTRODUCTION: Fusobacterium nucleatum coaggregates with a diverse range of bacterial species, and binds to host tissues and proteins such as immunoglobulin. These interactions may support the attachment of a variety of organisms to oral surfaces and can facilitate the invasion of soft tissues. We hypothesized that coaggregation with streptococci and immunoglobulin binding may occur by a common adhesin sensitive to l-arginine. METHODS: Repeated mixing of F. nucleatum with non-immune secretory immunoglobulin A (S-IgA) and recovery of non-agglutinating cells isolated a spontaneous mutant (isolate 21) of F. nucleatum that was defective in S-IgA binding. Wild-type and mutant F. nucleatum were compared by coaggregation and adhesion assays. RESULTS: Isolate 21 exhibited significantly reduced S-IgA binding and coaggregation with oral streptococci but not with Porphyromonas gingivalis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the mutant was deficient compared to wild-type for a single protein of approximately 360 kilodaltons. The corresponding protein was isolated from wild-type F. nucleatum protein preparations by coprecipitation with arginine-agarose beads. This protein was able to bind both Streptococcus cristatus and S-IgA. Mass spectrometry analysis indicated that this protein was closely related to putative autotransporter proteins in other F. nucleatum strains and was a 100% match to the deduced amino acid sequence of a 10,638-base-pair open reading frame in the incomplete genome sequence of F. nucleatum ATCC 10,953. Peptides identified by MS-MS analysis spanned most of the predicted amino acid sequence, suggesting that the mature protein is not subject to postsecretory cleavage. CONCLUSION: Coaggregation represents a novel function within the autotransporter class of proteins, which are often associated with virulence.
Asunto(s)
Adhesión Bacteriana/fisiología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Fusobacterium nucleatum/fisiología , Inmunoglobulina A Secretora/metabolismo , Estreptococos Viridans/fisiología , Adhesinas Bacterianas/química , Adhesinas Bacterianas/metabolismo , Secuencia de Aminoácidos , Arginina/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Unión Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
OBJECTIVE: We have shown that buccal epithelial cells (BEC) from humans can contain a polymicrobial intracellular flora. Members of that flora can induce proinflammatory responses. However, our subjects all had healthy oral mucosa. This might reflect tolerance of bacterial invasion by live BEC. Alternatively, inflammation might not occur if invaded cells were mostly dead, and thus unable to mount a response. This study addressed that issue, by determining the vital status of BEC and the bacteria associated with them. DESIGN: Initial experiments indicated that BEC were anomalously permeable to the DNA stain propidium iodide. We used that property to develop a protocol that combined the DNA stains SYTO 9 and propidium iodide (indicators of bacterial viability) with the esterase substrate calcein blue AM (an indicator of BEC viability), and Annexin V Alexa Fluor 647 conjugate (an apoptosis marker). That protocol was applied to BEC collected from 36 human subjects. RESULTS: On average, 70% of BEC displayed calcein blue staining, with no binding of Annexin V, 25% showed signs of apoptosis, and 5% did not stain with calcein blue. The mean percent of BEC with live cell-associated bacteria was 29%. Collectively, 25% of total BEC displayed calcein blue staining and live (SYTO 9 stained) bacteria. Only 1% of total BEC were negative for calcein blue and associated with live bacteria. CONCLUSIONS: Our findings suggest that live BEC are tolerant of bacterial invasion. This may be due to complex interactions between members of the polymicrobial flora and their host BEC.
Asunto(s)
Células Epiteliales/microbiología , Mucosa Bucal/citología , Adulto , Apoptosis/fisiología , Recuento de Colonia Microbiana , Colorantes Fluorescentes/análisis , Humanos , Microscopía Fluorescente/métodos , Compuestos Orgánicos/análisis , Streptococcus mitis/fisiologíaRESUMEN
Previously, we reported that intracellular Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis were present within buccal epithelial cells from human subjects, as lesser components of a polymicrobial flora. In this study, we further characterized that intracellular flora by using the same double-labeling techniques to identify Fusobacterium nucleatum, Prevotella intermedia, oral Campylobacter species, Eikenella corrodens, Treponema denticola, Gemella haemolysans, Granulicatella adiacens, and total streptococci within buccal epithelial cells. All those species were found within buccal cells. In every case, species recognized by green-labeled species-specific probes were accompanied by other bacteria recognized only by a red-labeled universal probe. Streptococci appeared to be a major component of the polymicrobial intracellular flora, being present at a level from one to two logs greater than the next most common species (G. adiacens). This is similar to what is observed in oral biofilms, where diverse species interact in complex communities that often are dominated by streptococci.
Asunto(s)
Mucosa Bucal/microbiología , Streptococcus/aislamiento & purificación , Campylobacter rectus/aislamiento & purificación , Recuento de Colonia Microbiana , Eikenella corrodens/aislamiento & purificación , Células Epiteliales/microbiología , Fusobacterium nucleatum/aislamiento & purificación , Humanos , Hibridación Fluorescente in Situ , Microscopía Confocal , Mucosa Bucal/citología , Prevotella intermedia/aislamiento & purificación , Staphylococcaceae/aislamiento & purificación , Streptococcaceae/aislamiento & purificación , Streptococcus/clasificación , Treponema denticola/aislamiento & purificaciónRESUMEN
Previously, we used in situ hybridization and confocal microscopy to detect the periodontal pathogens Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis within buccal epithelial cells taken directly from the mouth. This study tested the hypothesis that the intracellular flora of buccal cells is polymicrobial. Mixtures containing a red fluorescent universal probe paired with green fluorescent versions of either A. actinomycetemcomitans-, P. gingivalis-, or T. forsythensis-specific probes were hybridized with buccal cells collected from each of 38 healthy humans. We verified co-localization of probe pairs within cells by generating three-dimensional reconstructions. Intracellular bacteria were detected in every subject. Each cell that was labeled with a species-specific probe also contained bacteria recognized only by the universal probe. Bacteria labeled with specific probes often occupied smaller regions within larger masses of bacteria. Those findings suggest that future studies of invasion by oral bacteria may need to include microbial consortia.
Asunto(s)
Aggregatibacter actinomycetemcomitans/crecimiento & desarrollo , Bacteroides/crecimiento & desarrollo , Mucosa Bucal/microbiología , Porphyromonas gingivalis/crecimiento & desarrollo , Adulto , Recuento de Colonia Microbiana , ADN Bacteriano/análisis , Ecosistema , Femenino , Humanos , Hibridación Fluorescente in Situ , Masculino , Microscopía Confocal , Mucosa Bucal/citologíaRESUMEN
OBJECTIVE: Previously, we screened 149 subjects and established four groups high or low for salivary killing of oral bacteria, and for aggregation and live and dead adherence of oral bacteria (as a combined factor). Caries scores were significantly lower in both High Aggregation-Adherence groups. Subsequently, we found that supragingival total biofilm DNA, total streptococci and two major streptococcal rRNA variants also were significantly lower in the High Aggregation-Adherence groups. In this study, we looked at the effects of those differences in salivary function on three periodontal pathogens. DESIGN: Quantitative PCR was used to determine levels of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythensis (formerly Bacteroides forsythus) in stored DNA extracts of overnight supragingival biofilm collected from buccal upper central incisors (UC), lingual lower central incisors (LC) and buccal upper and lower first molars (BM) and lingual upper and lower first molars (LM) of subjects in the four groups. RESULTS: A. actinomycetemcomitans and P. gingivalis were almost completely absent from these samples. T. forsythensis was found in 11 of 35 persons at the buccal molar site. Only two of those subjects were in the High Aggregation-Adherence groups, and that difference was statistically significant. The mean quantity of T. forsythensis also was significantly lower in the High Aggregation-Adherence groups. CONCLUSIONS: The difference between the Low and High Aggregation-Adherence groups might reflect direct interactions of salivary proteins with T. forsythensis. Alternatively, the higher levels of total biofilm and total streptococci seen in the Low Aggregation-Adherence groups might create a favourable environment for early secondary colonization of T. forsythensis.
Asunto(s)
Aggregatibacter actinomycetemcomitans/aislamiento & purificación , Bacteroides/aislamiento & purificación , Biopelículas , Boca/microbiología , Porphyromonas gingivalis/aislamiento & purificación , Salivación/fisiología , Adhesión Bacteriana/fisiología , Recuento de Colonia Microbiana , ADN Bacteriano/análisis , Encía/microbiología , Humanos , Incisivo/microbiología , Diente Molar/microbiología , Reacción en Cadena de la Polimerasa/métodosRESUMEN
BACKGROUND: Conventional polymerase chain reaction (PCR) assays for periodontal pathogens are so sensitive that they detect infections of no clinical significance. Quantitative PCR (qPCR) may provide a solution to this problem. However, most qPCR systems require expensive real-time thermal cyclers. OBJECTIVE: Our goal was to develop qPCR assays which would allow endpoint quantification. MATERIALS AND METHODS: 16S rRNA primers for Bacteroides forsythus, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans were adapted to the Amplifluor qPCR system, which incorporates fluorescein into the PCR product so that endpoint fluorescence is proportional to the original amount of template. DNA dilutions representing known numbers of cells were used as standard curves. Pooled subgingival plaques from the four deepest pockets of 21 severe adult periodontitis patients were assayed. Buccal molar supragingival plaque from 35 dental students provided healthy controls. Endpoint fluorescence was measured with a microplate reader. RESULTS: Optimized standard curves were linear in log-log or semilog fits over a range of 100-10(6) cells. Countable B. forsythus was present in all patients, with counts (as logs) from 2.4 to 7.3 (mean = 5.0), and 11 controls with counts from 2.1 to 4.5 (mean = 3.0). P. gingivalis was present in 11 patients and no controls, with counts from 2.2 to 4.7 (mean = 3.2). A. actinomycetemcomitans was present in two patients, with counts of 1.5 and 3.5. CONCLUSIONS: Amplifluor qPCR assays discriminated between plaque samples differing by one log or more, allowing major infections to be distinguished from minor ones. This approach allows high-throughput qPCR of plaque samples, using equipment available to many laboratories.
Asunto(s)
Aggregatibacter actinomycetemcomitans/aislamiento & purificación , Bacteroides/aislamiento & purificación , Placa Dental/microbiología , Reacción en Cadena de la Polimerasa/métodos , Porphyromonas gingivalis/aislamiento & purificación , Adulto , ADN Bacteriano/análisis , Determinación de Punto Final , Fluorescencia , Humanos , Periodontitis/diagnóstico , Periodontitis/microbiologíaRESUMEN
OBJECTIVE: In a previous study, we screened 149 subjects and established four groups high or low for salivary killing of oral bacteria, and for aggregation and live and dead adherence of oral bacteria (as a combined factor). Caries scores were significantly lower in both High Aggregation-Adherence groups. In this study we looked at the effects of those differences in salivary function on the quantity and diversity of oral biofilm streptococci. DESIGN: Subjects from those four groups were recalled for collection of overnight oral biofilm from buccal upper central incisors, lingual lower central incisors, buccal upper and lower first molars, and lingual upper and lower first molars. At each site, groups were compared for total biofilm (by DNA concentration), total streptococci (by quantitative PCR), and streptococcal diversity (by Streptococcus-specific denaturing gradient gel electrophoresis). RESULTS: Total biofilm DNA and total streptococci were correlated. Both were highest on buccal molar surfaces and lowest on lingual lower central incisors, and both were significantly lower in the High Aggregation-Adherence groups (particularly at the buccal molar site). Fifty distinct bands were observed in denaturing gradient gels. There was great diversity within and between sites. Three major bands were present in almost every person at every site. Densities for two of those bands were significantly lower in both High Aggregation-Adherence groups. Other less-prevalent bands also showed the same pattern. CONCLUSION: These findings are consistent with our caries results in suggesting that differences in salivary function can influence the quantity and composition of streptococci in oral biofilms.
Asunto(s)
Biopelículas , Boca/microbiología , Saliva/fisiología , Streptococcus/aislamiento & purificación , Adhesión Bacteriana , Técnicas de Tipificación Bacteriana , Biomasa , ADN Bacteriano/análisis , Electroforesis en Gel de Poliacrilamida/métodos , Humanos , Inmunidad Innata , Boca/inmunología , Reacción en Cadena de la Polimerasa/métodos , Saliva/inmunología , Estadística como Asunto , Streptococcus/clasificaciónRESUMEN
Salivary proteins have multiple functions and many share similar functions, which may be why it has been difficult to relate variations in their concentrations to oral health and ecology. An alternative is to focus on variations in the major functions of saliva. An hydroxyapatite-coated microplate model has been developed that simultaneously measures saliva-promoted bacterial viability, bacterial aggregation, and live and dead bacterial adherence, while simulating oral temperature and shearing forces from swallowing. That model was applied to resting whole and stimulated parotid saliva from 149 individuals, using representative strains of Streptococcus crista, S. mutans, and Actinobacillus actinomycetemcomitans. Two major factors were defined by multivariate analysis (this was successful only for whole-saliva). One factor was correlated with aggregation, live adherence and dead adherence for all three strains; the other was correlated with total viability of all three strains. Participants were grouped <25th percentile and >75th percentile for each factor. Those groups were compared for clinical indices of oral health. Caries scores were significantly lower in those with high scores for aggregation-adherence, regardless of whether total viability scores were low or high. Live bacteria always predominated on surfaces when live and dead adherence scores were expressed as ratios. However, participants with high scores for aggregation-adherence showed significantly more dead adherent bacteria than those with low scores (these ratios were uncorrelated with total viability). This finding may indicate that extreme differences in the ability to kill bacteria on surfaces can influence caries risk.
Asunto(s)
Adhesión Bacteriana , Caries Dental/microbiología , Placa Dental/microbiología , Enfermedades Periodontales/microbiología , Saliva/microbiología , Saliva/fisiología , Adulto , Aggregatibacter actinomycetemcomitans/fisiología , Análisis de Varianza , Índice CPO , Índice de Placa Dental , Análisis Discriminante , Durapatita , Femenino , Fluorescencia , Humanos , Masculino , Modelos Biológicos , Índice Periodontal , Análisis de Componente Principal , Estadísticas no Paramétricas , Streptococcus mutans/fisiología , Streptococcus sanguis/fisiologíaRESUMEN
The mouth may provide an accessible model for studying bacterial interactions with human cells in vivo. Using fluorescent in situ hybridization and laser scanning confocal microscopy, we found that human buccal epithelial cells from 23 of 24 subjects were infected with intracellular bacteria, including the periodontal pathogens Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis, as well as other species which have yet to be identified. Buccal cell invasion may allow fastidious anaerobes to establish themselves in aerobic sites that otherwise present an unfavorable environment. Exfoliated buccal epithelial cells might provide a protected route for bacterial transmission between different oral sites within and between hosts.
Asunto(s)
Aggregatibacter actinomycetemcomitans/aislamiento & purificación , Mucosa Bucal/microbiología , Porphyromonas gingivalis/aislamiento & purificación , Aggregatibacter actinomycetemcomitans/genética , Aggregatibacter actinomycetemcomitans/crecimiento & desarrollo , Mejilla , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Hibridación Fluorescente in Situ , Masculino , Microscopía Confocal , Reacción en Cadena de la Polimerasa , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/crecimiento & desarrolloRESUMEN
BACKGROUND: Previous longitudinal studies investigating the role of microorganisms in periodontitis have focused on subjects with a high prevalence and severity of disease. The complex profile of microbial species in severe cases of periodontitis might not allow us to differentiate which bacterial species initiate disease or which species simply proliferate after disease progression. This prospective longitudinal study followed a group of 205 subjects who showed a low prevalence and severity of adult periodontitis, and thus allowed us to monitor early microbiological changes in the development of periodontitis. METHODS: Subgingival plaque was collected from proximal surfaces of a posterior sextant at 6-month intervals for 2 years. During the monitoring period, 44 subjects had either attachment loss or attachment gain. Using multiplex polymerase chain reaction (PCR), all plaque samples from those 44 subjects were analyzed for the presence of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis. RESULTS: Both subjects with attachment loss and those with attachment gain had a high prevalence of these 3 periodontal pathogens. The mere presence of any of the 3 species at a site could not predict future attachment loss at that specific site. However, subjects with a persistent presence of B. forsythus at any site across all visits had 5.3 times higher odds of having at least one site in their mouth losing attachment compared to subjects with occasional or no presence of B. forsythus. CONCLUSIONS: The persistence of B. forsythus identified subjects at higher risk, but not which specific sites in those subjects would lose attachment.
Asunto(s)
Infecciones por Bacteroides/clasificación , Bacteroides/clasificación , Pérdida de la Inserción Periodontal/microbiología , Periodontitis/microbiología , Infecciones por Actinobacillus/clasificación , Adulto , Anciano , Aggregatibacter actinomycetemcomitans/clasificación , Análisis de Varianza , Infecciones por Bacteroidaceae/clasificación , Distribución de Chi-Cuadrado , Placa Dental/microbiología , Femenino , Predicción , Humanos , Funciones de Verosimilitud , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Periodontitis/clasificación , Porphyromonas gingivalis/clasificación , Prevalencia , Estudios Prospectivos , Factores de RiesgoRESUMEN
Associations with facultative species might help planktonic oral anaerobes survive when they traverse saliva. This study investigated whether co-aggregation with Streptococcus crista ATCC 51110 enhanced the viability of Fusobacterium nucleatum ATCC 10953. Two tubes each of co-aggregates and Fus. nucleatum ATCC 10953 alone were prepared. One of each set was resuspended in buffer, and the other in clarified saliva from each of 20 donors. After 1 h, cells were stained for viability. The median percentage of viable Fus. nucleatum ATCC 10953 in buffer with Strep. crista (86%) was significantly higher (p=0.04) than in buffer alone (77%). A similar trend was observed for saliva with Strep. crista (81%) and saliva alone (74%), although that difference was not significant (p=0.41). The median percentage of Fus. nucleatum co-aggregated in buffer (44%) was significantly reduced after incubation in saliva (16%) (p<0.0002). No such change was seen when saliva was replaced with purified salivary proline-rich glycoprotein, which can bind both species. For co-aggregate suspensions, there was no difference in the viability of fusobacteria that were or were not in direct contact with Strep. crista. In both cases, viability was significantly reduced in saliva relative to buffer. Strep. crista may enhance the viability of planktonic Fus. nucleatum ATCC 10953, but it is not yet clear whether that requires co-aggregation. Transmission of fusobacteria through saliva could depend on the interplay between protective factors, such as the presence of streptococci, and antimicrobial factors, which kill cells or disassociate co-aggregates.
Asunto(s)
Fusobacterium nucleatum/fisiología , Saliva/fisiología , Streptococcus/fisiología , Antibacterianos/farmacología , Técnicas Bacteriológicas , Tampones (Química) , Recuento de Colonia Microbiana , Colorantes , Medios de Cultivo , Fusobacterium nucleatum/crecimiento & desarrollo , Glicoproteínas/farmacología , Humanos , Boca/microbiología , Péptidos/farmacología , Prolina/farmacología , Dominios Proteicos Ricos en Prolina , Sustancias Protectoras/farmacología , Saliva/microbiología , Proteínas y Péptidos Salivales/farmacología , Streptococcus/clasificación , Streptococcus/crecimiento & desarrollo , Simbiosis/fisiologíaRESUMEN
Dental plaque is being redefined as oral biofilm. Diverse overlapping microbial consortia are present on all oral tissues. Biofilms are structured, displaying features like channels and projections. Constituent species switch back and forth between sessile and planktonic phases. Saliva is the medium for planktonic suspension. Several major functions can be defined for saliva in relation to oral biofilm. It serves as a medium for transporting planktonic bacteria within and between mouths. Bacteria in transit may be vulnerable to negative selection. Salivary agglutinins may prevent reattachment to surfaces. Killing by antimicrobial proteins may lead to attachment of dead cells. Salivary proteins form conditioning films on all oral surfaces. This contributes to positive selection for microbial adherence. Saliva carries chemical messengers which allow live adherent cells to sense a critical density of conspecifics. Growth begins, and thick biofilms may become resistant to antimicrobial substances. Salivary macromolecules may be catabolized, but salivary flow also may clear dietary substrates. Salivary proteins act in ways that benefit both host and microbe. All have multiple functions, and many do the same job. They form heterotypic complexes, which may exist in large micelle-like structures. These issues make it useful to compare subjects whose saliva functions differently. We have developed a simultaneous assay for aggregation, killing, live adherence, and dead adherence of oral species. Screening of 149 subjects has defined high killing/low adherence, low killing/high adherence, high killing/high adherence, and low killing/low adherence groups. These will be evaluated for differences in their flora.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Placa Dental/microbiología , Saliva/fisiología , Antibacterianos/farmacología , Adhesión Bacteriana/fisiología , Fenómenos Fisiológicos Bacterianos , Biopelículas/clasificación , Recuento de Colonia Microbiana , Ecosistema , Humanos , Saliva/microbiología , Proteínas y Péptidos Salivales/fisiologíaRESUMEN
Among putative periodontal pathogens, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis are most convincingly implicated as etiological agents in periodontitis. Therefore, techniques for detection of those three species would be of value. We previously published a description of a multiplex PCR that detects A. actinomycetemcomitans and P. gingivalis. The present paper presents an improvement on that technique, which now allows more sensitive detection of all three periodontal pathogens. Sensitivity was determined by testing serial dilutions of A. actinomycetemcomitans, B. forsythus, and P. gingivalis cells. Primer specificity was tested against (i) all gene sequences from the GenBank-EMBL database, (ii) six A. actinomycetemcomitans, one B. forsythus, and four P. gingivalis strains, (iii) eight different species of oral bacteria, and (iv) supra- and subgingival plaque samples from 20 healthy subjects and subgingival plaque samples from 10 patients with periodontitis. The multiplex PCR had a detection limit of 10 A. actinomycetemcomitans, 10 P. gingivalis, and 100 B. forsythus cells. Specificity was confirmed by the fact that (i) none of our forward primers were homologous to the 16S rRNA genes of other oral species, (ii) amplicons of predicted size were detected for all A. actinomycetemcomitans, B. forsythus, and P. gingivalis strains tested, and (iii) no amplicons were detected for the eight other bacterial species. A. actinomycetemcomitans, B. forsythus, and P. gingivalis were detected in 6 of 20, 1 of 20, and 11 of 20 of supragingival plaque samples, respectively, and 4 of 20, 7 of 20, and 13 of 20 of subgingival plaque samples, respectively, from periodontally healthy subjects. Among patients with periodontitis, the organisms were detected in 7 of 10, 10 of 10, and 7 of 10 samples, respectively. The simultaneous detection of three periodontal pathogens is an advantage of this technique over conventional PCR assays.