Nora Silva

The subgingival microbiome in health and periodontitis and its relationship with community biomass and inflammation

The goals of this study were to better understand the ecology of oral subgingival communities in health and periodontitis and elucidate the relationship between inflammation and the subgingival microbiome. Accordingly, we used 454-pyrosequencing of 16S rRNA gene libraries and quantitative PCR to characterize the subgingival microbiome of 22 subjects with chronic periodontitis. Each subject was sampled at two sites with similar periodontal destruction but differing in the presence of bleeding, a clinical indicator of increased inflammation. Communities in periodontitis were also compared with those from 10 healthy individuals. In periodontitis, presence of bleeding was not associated with different α-diversity or with a distinct microbiome, however, bleeding sites showed higher total bacterial load. In contrast, communities in health and periodontitis largely differed, with higher diversity and biomass in periodontitis. Shifts in community structure from health to periodontitis resembled ecological succession, with emergence of newly dominant taxa in periodontitis without replacement of primary health-associated species. That is, periodontitis communities had higher proportions of Spirochetes, Synergistetes, Firmicutes and Chloroflexi, among other taxa, while the proportions of Actinobacteria, particularly Actinomyces, were higher in health. Total Actinomyces load, however, remained constant from health to periodontitis. Moreover, an association existed between biomass and community structure in periodontitis, with the proportion of specific taxa correlating with bacterial load. Our study provides a global-scale framework for the ecological events in subgingival communities that underline the development of periodontitis. The association, in periodontitis, between inflammation, community biomass and community structure and their role in disease progression warrant further investigation.

Characterization of progressive periodontal lesions in chronic periodontitis patients: levels of chemokines, cytokines, matrix metalloproteinase‐13, periodontal pathogens and inflammatory cells

BACKGROUND AND AIMS Periodontitis is an infection with an episodic nature of tissue support destruction. The aim of this work was to determine the levels of chemokines, cytokines, matrix metalloproteinase-13, periodontal pathogens and inflammatory cells in periodontal sites characterized by active periodontal connective tissue destruction. MATERIAL AND METHOD Fifty-six patients with moderate or advanced severity of chronic periodontitis were selected. Periodontitis was characterized by at least six sites with probing depth > or =5 mm, clinical attachment level > or =3 mm and radiographic bone loss. Periodontitis progression was determined by the tolerance method. Receptor activator for nuclear factor kappa B-ligand (RANK-L), monocyte chemoattractant protein-1 (MCP-1), tumour necrosis factor-alpha (TNF-alpha), IL-1beta, MMP-13, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsithia and inflammatory cells levels were determined. Statistical analysis was performed using the Stata 7.0 software. Data were expressed as mean+/-SD and paired samples t-test and chi(2) tests were used. RESULTS Higher RANK-L, IL-1beta and MMP-13 activity levels were observed in active sites (p<0.05). The proportion of P. gingivalis, A. actinomycetemcomitans, T. forsythia and the number of CD4(+) T were higher in active than in inactive sites (p>0.05). CONCLUSION The detection of periodontopathic bacteria, host matrix metalloproteinases and cytokines in periodontitis patients with lesions undergoing episodic attachment loss could partially explain the mechanisms associated with the destruction of the supporting tissues of the tooth.

Host-Pathogen Interactions in Progressive Chronic Periodontitis

Periodontitis is an infection characterized by the occurrence of supporting tissue destruction with an episodic nature. Disease progression is often determined by the loss of attachment level or alveolar bone, and sequential probing of periodontal attachment remains the most commonly utilized method to diagnose progressive destruction of the periodontium. The tolerance method has been the most extensive clinical method used in recent years to determine site-specific attachment level changes. There is abundant evidence that major tissue destruction in periodontal lesions results from the recruitment of immune cells. Considerable effort has been made to study the host cell and mediator profiles involved in the pathogenesis of chronic periodontitis, but the definition of active sites, where current periodontal breakdown occurs, and consecutive characterization of the mediators involved are still among the main concerns. In the present review, we summarize periodontopathic bacteria and host factors, including infiltrating cell populations, cytokines, and host matrix metalloproteinases, associated with under-going episodic attachment loss that could partly explain the mechanisms involved in destruction of the supporting tissues of the tooth.

Host response mechanisms in periodontal diseases

Periodontal diseases usually refer to common inflammatory disorders known as gingivitis and periodontitis, which are caused by a pathogenic microbiota in the subgingival biofilm, including Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Treponema denticola that trigger innate, inflammatory, and adaptive immune responses. These processes result in the destruction of the tissues surrounding and supporting the teeth, and eventually in tissue, bone and finally, tooth loss. The innate immune response constitutes a homeostatic system, which is the first line of defense, and is able to recognize invading microorganisms as non-self, triggering immune responses to eliminate them. In addition to the innate immunity, adaptive immunity cells and characteristic cytokines have been described as important players in the periodontal disease pathogenesis scenario, with a special attention to CD4+ T-cells (T-helper cells). Interestingly, the T cell-mediated adaptive immunity development is highly dependent on innate immunity-associated antigen presenting cells, which after antigen capture undergo into a maturation process and migrate towards the lymph nodes, where they produce distinct patterns of cytokines that will contribute to the subsequent polarization and activation of specific T CD4+ lymphocytes. Skeletal homeostasis depends on a dynamic balance between the activities of the bone-forming osteoblasts (OBLs) and bone-resorbing osteoclasts (OCLs). This balance is tightly controlled by various regulatory systems, such as the endocrine system, and is influenced by the immune system, an osteoimmunological regulation depending on lymphocyte- and macrophage-derived cytokines. All these cytokines and inflammatory mediators are capable of acting alone or in concert, to stimulate periodontal breakdown and collagen destruction via tissue-derived matrix metalloproteinases, a characterization of the progression of periodontitis as a stage that presents a significantly host immune and inflammatory response to the microbial challenge that determine of susceptibility to develop the destructive/progressive periodontitis under the influence of multiple behavioral, environmental and genetic factors.

Levels of interleukin-21 in patients with untreated chronic periodontitis.

BACKGROUND A growing body of evidence suggested that interleukin (IL)-21 enhances the effector phase during T-cell responses. The aim of our study is to determine the levels of IL-21 in periodontal sites from patients with chronic periodontitis and controls. METHODS The population studied consisted of 34 patients (15 with chronic periodontitis and 19 healthy patients). Twenty samples (10 gingival crevicular fluid [GCF] and 10 gingival biopsies) were collected from each group before the patients with periodontitis received periodontal treatment. Total protein concentrations were measured in all samples; the presence of IL-21 was confirmed by immunohistochemistry and Western blot, and IL-21 levels were quantified through an enzyme-linked immunosorbent assay. Statistical analyses were performed using statistical software. Data were expressed as patient means ± SDs or medians (interquartile ranges) by using the χ(2), Student t, and Mann-Whitney U tests. RESULTS GCF IL-21 was mainly detected in patients with chronic periodontitis (P <0.05). Levels of IL-21 in gingival tissues were significantly higher in patients with chronic periodontitis compared to healthy individuals (P <0.05). The Western blot and immunohistochemical staining confirmed the presence of IL-21 in periodontal tissues and GCF. CONCLUSION IL-21 was highly expressed in patients with chronic periodontitis, especially in gingival biopsies; therefore, IL-21 might play a role in the T-cell response.

Clinical Effects of Lactobacillus rhamnosus in Non-Surgical Treatment of Chronic Periodontitis: A Randomized Placebo-Controlled Trial With 1-Year Follow-Up

BACKGROUND Probiotics are living microorganisms that provide beneficial effects for the host when administered in proper quantities. The aim of this double-masked placebo-controlled parallel-arm randomized clinical trial is to evaluate the clinical effects of a Lactobacillus rhamnosus SP1-containing probiotic sachet as an adjunct to non-surgical therapy. METHODS Twenty-eight systemically healthy volunteers with chronic periodontitis were recruited and monitored clinically at baseline and 3, 6, 9, and 12 months after therapy. Clinical parameters measured included plaque accumulation, bleeding on probing, probing depths (PDs), and clinical attachment loss. Patients received non-surgical therapy, including scaling and root planing (SRP), and were assigned randomly to a test (SRP + probiotic, n = 14) or control (SRP + placebo, n = 14) group. The intake, once a day for 3 months, of an L. rhamnosus SP1 probiotic sachet commenced after the last session of SRP. RESULTS Both test and control groups showed improvements in clinical parameters at all time points evaluated. However, the test group showed greater reductions in PD than the control. Also, at initial visits and after 1-year follow-up, the test group showed a statistically significant reduction in the number of participants with PD ≥6 mm, indicating a reduced need for surgery, in contrast to the placebo group. CONCLUSION The results of this trial indicate that oral administration of L. rhamnosus SP1 resulted in similar clinical improvements compared with SRP alone.

Genotipificación de Porphyromonas gingivalis en Pacientes con Periodontitis

Resumen Porphyromonas gingivalis es un bacilo Gram-negativo y anaerobio, considerado como el mayor agente causal en la iniciacion y progresion de las periodontitis. Este microorganismo presenta diversos factores de virulencia, como su capacidad de adherencia a los tejidos periodontales y a otras bacterias orales, el Lipopolisacarido (LPS) que induce la respuesta inmune del hospedero y la accion de multiples proteasas. Las bases moleculares de estos mecanismos de virulencia y su relacion con la diversidad genetica no han sido suficientemente comprendidas aun. El proposito de este estudio fue genotipificar aislados de P. gingivalis seleccionados desde pacientes con Periodontitis Cronica y Agresiva utilizando una metodologia basada en la reaccion en cadena de la polimerasa (PCR) para la “Secuencia de Insercion” IS1126. Se tomaron muestras de placa subgingival en 4 sitios periodontalmente afectados (el sitio de mayor profundidad de saco periodontal por cuadrante). Para ello se utilizaron conos de papel esteriles, estos se transportaron y almacenaron en RTF frio hasta su procesamiento en el laboratorio. Se obtuvo, mediante cultivo, bacterias pigmentadas de negro para su posterior identificacion fenotipica, molecular y genotipificacion, de los aislados confirmados mediante PCR como P. gingivalis. Se genotipificaron 35 aislados, provenientes de 6 pacientes, caracterizandose 7 perfiles geneticos diferentes. Cuatro pacientes presentaron un solo genotipo en sus aislados, mientras que 2 pacientes mostraron dos perfiles geneticos distintos. Los aislados de P. gingivalis estudiados presentaron variabilidad genetica a nivel de la secuencia IS1126 y tal vez por el numero de aislados analizados, no se identifico un perfil genetico bacteriano caracteristico de periodontitis cronica o agresiva.

Relación entre enfermedad periodontal, infección bacteriana ascendente y patología placentaria con parto prematuro

Objetivo: Determinar la relacion entre enfermedad periodontal, infeccion bacteriana ascendente y patologia placentaria, con parto prematuro. Pacientes y Metodos: Participaron embarazadas entre 24 y 34 semanas de gestacion, con trabajo de parto prematuro sin causa clinica evidente y membranas intactas o con el diagnostico de rotura prematura de membranas (RPM), sin trabajo de parto y sin corioamnionitis clinica. Todas las embarazadas tuvieron estudio periodontal clinico y evaluacion microbiologica de la placa subgingival, del liquido amniotico (LA) y cervicovaginal. R ecibieron corticoesteroides, antibioticos, tocolisis (casos con membranas intactas) y manejo expectante hasta las 35 semanas (casos con RPM) . Las placentas se enviaron a estudio y se diagnostico corioamnionitis, funisitis y vellositis. Se definio invasion microbiana de la cavidad amniotica ( IMCA) el cultivo positivo del liquido amniotico. Infeccion cervicovaginal (ICV) se diagnostico con vaginosis bacteriana (VB) o cultivo positivo para bacteria patogena u oportunista en cervix o vagina, con incremento significativo de los leucocitos polimorfonucleares. Se considero como infeccion bacteriana ascendente (IBA) la presencia de IMCA por bacterias ascendentes y/o ICV. Resultados: Se incluyeron 59 pacientes, 42 con membranas intactas y 17 con RPM . La frecuencia de la enfermedad periodontal fue 93.2%. La IMCA fue 27.1% Se aislaron bacterias patogenas periodontales del LA en el 18.6% y de la placa subgingival en el 71.2% de los casos. La IBA fue 83.1%. La asociacion IBA con enfermedad periodontal fue 72.9%. El parto prematuro ( 82.1% p=0.03 y con la presencia conjunta de infeccion bacteriana ascendente y enfermedad periodontal 74.4% p=0.03. Los casos con nacimiento prematuro y enfermedad periodontal generalizada tuvieron significativa mayor frecuencia de corioamnionitis y funisitis histologica 69.6% p=0.04. Conclusiones : L a enfermedad periodontal generalizada y la presencia conjunta de infeccion bacteriana ascendente y enfermedad periodontal se asocian con parto prematuro . En estos casos, son frecuentes l os marcadores histologicos placentarios de infeccion ascendente .

Microbiological and clinical effects of probiotics and antibiotics on nonsurgical treatment of chronic periodontitis: a randomized placebo- controlled trial with 9-month follow-up

ABSTRACT Objective The aim of this double-blind, placebo-controlled and parallel- arm randomized clinical trial was to evaluate the effects of Lactobacillus rhamnosus SP1-containing probiotic sachet and azithromycin tablets as an adjunct to nonsurgical therapy in clinical parameters and in presence and levels of Tannerella forsythia, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. Material and Methods Forty-seven systemically healthy volunteers with chronic periodontitis were recruited and monitored clinically and microbiologically at baseline for 3, 6 and 9 months after therapy. Subgingival plaque samples were collected from four periodontal sites with clinical attachment level ≥1 mm, probing pocket depth ≥4 mm and bleeding on probing, one site in each quadrant. Samples were cultivated and processed using the PCR technique. Patients received nonsurgical therapy including scaling and root planing (SRP) and were randomly assigned to a probiotic (n=16), antibiotic (n = 16) or placebo (n = 15) group. L. rhamnosus SP1 was taken once a day for 3 months. Azithromycin 500mg was taken once a day for 5 days. Results All groups showed improvements in clinical and microbiological parameters at all time points evaluated. Probiotic and antibiotic groups showed greater reductions in cultivable microbiota compared with baseline. The placebo group showed greater reduction in number of subjects with P. gingivalis compared with baseline. However, there were no significant differences between groups. Conclusions The adjunctive use of L. rhamnosus SP1 sachets and azithromycin during initial therapy resulted in similar clinical and microbiological improvements compared with the placebo group.

Genotipificación de los genes rgpA y kgp que codifican para las gingipaínas de Porphyromonas gingivalis

Resumen Porphyromonas gingivalis es un microorganismo fuertemente asociado con la etiologia de la periodontitis. Esta bacteria posee varios factores de virulencia, dentro de los que destacan las gingipainas, debido a sus multiples acciones relacionadas con la destruccion de la matriz extracelular del tejido conectivo periodontal, la modulacion del sistema inmune del hospedero y la estimulacion de la expresion de citoquinas pro-inflamatorias. Estas proteinasas tienen afinidades especificas siendo Arg-gingipainas (RgpA y RgpB, codificadas por los genes rgpA y rgpB , respectivamente) y Lys-gingipainas (Kgp, codificada por el gen kgp ). Se ha descrito que existen polimorfismos en los genes que codifican para esta proteinasas. El objetivo del presente estudio fue describir la frecuencia de los genotipos identificados para los genes rgpA y kgp en aislados clinicos de P. gingivalis , obtenidos desde pacientes con periodontitis. Para ello se utilizo amplificacion por PCR de los genes rgpA y kgp , seguido de analisis de restriccion. De un total de 47 aislados provenientes de 4 individuos con periodontitis cronica y 2 con periodontitis agresiva, se genotipificaron 38 aislados para el gen rgpA , exhibiendo la totalidad de estos el patron electroforetico A (100%). Para el gen kgp se genotipificaron 43 aislados, presentando 28 de ellos (65.2%) el perfil electroforetico kgp-I y 15 aislados (34.8%) el perfil kgp-II . En los aislados provenientes de un individuo fue posible apreciar ambos genotipos descritos para el gen kgp . Los resultados indican un predominio del patron electroforetico A ( rgpA ) y que el genotipo kgp-I fue el mas frecuentemente encontrado de los genotipos kgp .