RESUMEN
Recently, it has been suggested that the anti-inflammatory hormone ghrelin (GHRL) and its receptor GHS-R may play a pivotal role in periodontal health and diseases. However, their exact regulation and effects in periodontitis are not known. The aim of this in-vitro study was to investigate the effect of microbial and inflammatory insults on the GHS-R1a expression in human osteoblast-like cells. MG-63 cells were exposed to interleukin (IL)-1ß and Fusobacterium nucleatum in the presence and absence of GHRL for up to 2 d. Subsequently, gene expressions of GHS-R1a, inflammatory mediators and matrix metalloproteinase were analyzed by real-time PCR. GHS-R protein synthesis and NF-κB p65 nuclear translocation were assessed by immunocytochemistry and immunofluorescence microscopy, respectively. IL-1ß and F. nucleatum caused a significant upregulation of GHS-R1a expression and an increase in GHS-R1a protein. Pre-incubation with a MEK1/2 inhibitor diminished the IL-1ß-induced GHS-R1a upregulation. IL-1ß and F. nucleatum also enhanced the expressions of cyclooxygenase 2, CC-chemokine ligand 2, IL-6, IL-8, and matrix metalloproteinase 1, but these stimulatory effects were counteracted by GHRL. By contrast, the stimulatory actions of IL-1ß and F. nucleatum on the GHS-R1a expression were further enhanced by GHRL. Our study provides original evidence that IL-1ß and F. nucleatum regulate the GHS-R/GHRL system in osteoblast-like cells. Furthermore, we demonstrate for the first time that the proinflammatory and proteolytic actions of IL-1ß and F. nucleatum on osteoblast-like cells are inhibited by GHRL. Our study suggests that microbial and inflammatory insults upregulate GHS-R1a, which may represent a protective negative feedback mechanism in human bone.
Asunto(s)
Fusobacterium nucleatum/fisiología , Interleucina-1beta/farmacología , Osteoblastos/química , Receptores de Ghrelina/análisis , Análisis de Varianza , Células Cultivadas , Humanos , Inmunohistoquímica , Microscopía Fluorescente , Osteoblastos/efectos de los fármacos , Osteoblastos/microbiología , Periodontitis/microbiología , Periodontitis/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Ghrelina/fisiología , Estadísticas no Paramétricas , Regulación hacia Arriba/fisiologíaRESUMEN
Abstract: Recently, it has been suggested that the anti-inflammatory hormone ghrelin (GHRL) and its receptor GHS-R may play a pivotal role in periodontal health and diseases. However, their exact regulation and effects in periodontitis are not known. The aim of this in-vitro study was to investigate the effect of microbial and inflammatory insults on the GHS-R1a expression in human osteoblast-like cells. MG-63 cells were exposed to interleukin (IL)-1β and Fusobacterium nucleatum in the presence and absence of GHRL for up to 2 d. Subsequently, gene expressions of GHS-R1a, inflammatory mediators and matrix metalloproteinase were analyzed by real-time PCR. GHS-R protein synthesis and NF-κB p65 nuclear translocation were assessed by immunocytochemistry and immunofluorescence microscopy, respectively. IL-1β and F. nucleatum caused a significant upregulation of GHS-R1a expression and an increase in GHS-R1a protein. Pre-incubation with a MEK1/2 inhibitor diminished the IL-1β-induced GHS-R1a upregulation. IL-1β and F. nucleatum also enhanced the expressions of cyclooxygenase 2, CC-chemokine ligand 2, IL-6, IL-8, and matrix metalloproteinase 1, but these stimulatory effects were counteracted by GHRL. By contrast, the stimulatory actions of IL-1β and F. nucleatum on the GHS-R1a expression were further enhanced by GHRL. Our study provides original evidence that IL-1β and F. nucleatum regulate the GHS-R/GHRL system in osteoblast-like cells. Furthermore, we demonstrate for the first time that the proinflammatory and proteolytic actions of IL-1β and F. nucleatum on osteoblast-like cells are inhibited by GHRL. Our study suggests that microbial and inflammatory insults upregulate GHS-R1a, which may represent a protective negative feedback mechanism in human bone.
Asunto(s)
Humanos , Osteoblastos/química , Fusobacterium nucleatum/fisiología , Interleucina-1beta/farmacología , Receptores de Ghrelina/análisis , Osteoblastos/efectos de los fármacos , Osteoblastos/microbiología , Periodontitis/microbiología , Periodontitis/patología , Inmunohistoquímica , Regulación hacia Arriba/fisiología , Células Cultivadas , Análisis de Varianza , Estadísticas no Paramétricas , Receptores de Ghrelina/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Microscopía FluorescenteRESUMEN
Antimicrobial photodynamic therapy (aPDT) kills several planktonic pathogens. However, the susceptibility of biofilm-derived anaerobic bacteria to aPDT is poorly characterized. Here, we evaluated the effect of Photodithazine (PDZ)-mediated aPDT on Fusobacterium nucleatum and Porphyromonas gingivalis biofilms. In addition, aPDT was tested with metronidazole (MTZ) to explore the potential antimicrobial effect of the treatment. The minimum inhibitory concentration (MIC) of MTZ was defined for each bacterial species. Single-species biofilms of each species were grown on polystyrene plates under anaerobic conditions for five days. aPDT was performed by applying PDZ at concentrations of 50, 75 and 100â¯mg/L, followed by exposure to 50â¯J/cm2 LED light (660â¯nm) with or without MTZ. aPDT exhibited a significant reduction in bacterial viability at a PDZ concentration of 100â¯mg/L, with 1.12 log10 and 2.66 log10 reductions for F. nucleatum and P. gingivalis in biofilms, respectively. However, the antimicrobial effect against F. nucleatum was achieved only when aPDT was combined with MTZ at 100× MIC. Regarding P. gingivalis, the combination of PDZ-mediated aPDT at 100â¯mg/L with MTZ 100× MIC resulted in a 5 log10 reduction in the bacterial population. The potential antimicrobial effects of aPDT in combination with MTZ for both single pathogenic biofilms were confirmed by live/dead staining. These results suggest that localized antibiotic administration may be an adjuvant to aPDT to control F. nucleatum and P. gingivalis biofilms.
Asunto(s)
Antiinfecciosos/farmacología , Biopelículas/efectos de los fármacos , Fusobacterium nucleatum/fisiología , Fármacos Fotosensibilizantes/farmacología , Porphyromonas gingivalis/fisiología , Antiinfecciosos/química , Biopelículas/efectos de la radiación , Fusobacterium nucleatum/aislamiento & purificación , Glucosamina/análogos & derivados , Glucosamina/química , Humanos , Luz , Metronidazol/farmacología , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Fármacos Fotosensibilizantes/química , Porphyromonas gingivalis/aislamiento & purificación , Saliva/microbiologíaRESUMEN
BACKGROUND: The aim of this study is to characterize and evaluate the host response caused by three different models of experimental periodontitis in mice. METHODS: C57BL/6 wild-type female mice were distributed into six experimental groups and sacrificed at 7, 15, and 30 days after the induction of periodontal disease: 1) group C: no treatment control group; 2) group L: periodontal disease induced by ligature; 3) group G-Pg: oral gavage with Porphyromonas gingivalis (Pg); 4) group G-PgFn: oral gavage with Fusobacterium nucleatum + Pg; 5) group I-Pg: heat-killed Pg injected into the palatal mucosa between the molars; and 6) group I-V: phosphate-buffered saline injected into the palatal mucosa. The samples were used to analyze the immune-inflammatory process in the gingival tissue via descriptive histologic and real-time polymerase chain reaction analyses. The alveolar bone loss was evaluated using microcomputed tomography. The data were analyzed using the Kruskal-Wallis test, followed by a post hoc Dunn test and analysis of variance, followed by a Tukey test using a 5% significance level. RESULTS: Only the ligature model displayed significant alveolar bone loss in the initial period (7 days), which was maintained with time. The group injected with heat-killed Pg displayed significant alveolar bone loss starting from day 15, which continued to progress with time (P <0.05). A significant increase (P <0.05) in the gene expression of proinflammatory cytokines (interleukin-6 and -1ß) and proteins involved in osteoclastogenesis (receptor activator of nuclear factor-κB ligand and osteoprotegerin) was observed in the ligature group on day 7. CONCLUSION: The ligature and injection of heat-killed Pg models were the most representative of periodontal disease in humans, whereas the oral gavage models were not effective at inducing the disease under the experimental conditions.
Asunto(s)
Periodontitis/inmunología , Administración Oral , Pérdida de Hueso Alveolar/inmunología , Pérdida de Hueso Alveolar/microbiología , Animales , Coinfección/inmunología , Progresión de la Enfermedad , Femenino , Fusobacterium nucleatum/fisiología , Interacciones Huésped-Patógeno , Mediadores de Inflamación/inmunología , Inyecciones , Interleucina-1beta/análisis , Interleucina-6/análisis , Leucocitos/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Mucosa Bucal/microbiología , Osteoclastos/inmunología , Osteoprotegerina/análisis , Pérdida de la Inserción Periodontal/inmunología , Pérdida de la Inserción Periodontal/microbiología , Periodontitis/microbiología , Porphyromonas gingivalis/fisiología , Ligando RANK/análisis , Distribución Aleatoria , Factores de Tiempo , Microtomografía por Rayos X/métodosRESUMEN
Subinhibitory concentrations (SICs) of antimicrobials may result in alterations in bacterial biology with implications for its potential aggression. This has considerable importance for the resident microbiota. Our aim was to analyze the effects of SICs of antimicrobials on the morphological, biochemical, physiological and molecular characteristics of the resident anaerobic Fusobacterium nucleatum. Fourteen strains were obtained from F. nucleatum ATCC 25586, selected by culturing on SICs of ampicillin, ampicillin/sulbactam, clindamycin, chloramphenicol, levofloxacin, metronidazole and piperacillin/tazobactam and subsequent culturing in the absence of drugs. Antimicrobial susceptibility, bacterial morphology, biochemical profiles and biofilm formation were evaluated. Genotyping and analysis of protein profiles were also performed. The antimicrobial susceptibility patterns showed that most of the derived strains were less sensitive to the antimicrobials, even after culturing them without drugs. Morphological and cell complexity alterations were observed, mainly in strains grown in SICs of ß-lactam; these strains also expressed a reduced ability for biofilm formation. The other strains showed an increase in biofilm formation but no apparent morphological changes. Alterations were observed in the carbohydrate metabolism patterns and in the activity of microbial enzymes. Several proteins were positively or negatively regulated and there was polymorphism in the DNA from all derived strains. Therefore, SICs of antimicrobials induce alterations in F. nucleatum, which directly impact its biology. These results emphasize the risk of inadequate antibioticotherapy, which may have serious implications for clinical microbiology and infectious diseases and also may interfere with the host-bacteria relationship.
Asunto(s)
Antibacterianos/farmacología , Fusobacterium nucleatum/efectos de los fármacos , Proteínas Bacterianas/análisis , Técnicas de Tipificación Bacteriana , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Enzimas/metabolismo , Fusobacterium nucleatum/citología , Fusobacterium nucleatum/genética , Fusobacterium nucleatum/fisiología , Genotipo , Pruebas de Sensibilidad Microbiana , Polimorfismo Genético , Proteoma/análisisRESUMEN
The genus Fusobacterium belongs to the Fusobacteriaceae family and is a Gram-negative obligate anaerobic bacterium found in the human oral microbiota. Even that Fusobacterium nucleatum cannot grow under aerobic conditions, they may exhibit aerotolerance as an adaptive response which could figure as an important virulence factor, during the stages of infection, when these anaerobes are shifted to aerobic conditions. In this regard, little is known about bacterial oxidative stress adaptive response and the influence of this adaptation on the host-bacteria relationship. We aimed to use both techniques 2-DE and Electrospray Ionization Mass Spectrometry (ESI-MS) to characterize proteins in F. nucleatum, after oxidative stress. We related three different proteins which were up-regulated by oxidative stress. As its genome is already sequenced, these proteins were found in data base search, by homology. Thus, by using techniques as ESI-Q/TOF-MS, in addition to 2-DE, the opportunity exists to gain a more holistic view of the bacterial proteome of human pathogens, to achieve a better understanding of species diversity and to elucidate the role of specific proteins in disease. This work represents one of the first studies using genetic and physiological approaches to understand the phenomenon of oxidative stress in F. nucleatum.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Fusobacterium nucleatum/fisiología , Perfilación de la Expresión Génica , Estrés Oxidativo , Proteoma/análisis , Estrés Fisiológico , Electroforesis en Gel Bidimensional , Fusobacterium nucleatum/química , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
This study assessed the prevalence and microbial interactions of Fusobacterium nucleatum and Fusobacterium necrophorum in primary endodontic infections from a Brazilian population and their antimicrobial susceptibility to some antibiotics by the E-test. One hundred ten samples from infected teeth with periapical pathologies were analyzed by culture methods. Five hundred eighty individual strains were isolated; 81.4% were strict anaerobes. F. nucleatum was found in 38 root canals and was associated with Porphyromonas gingivalis, Prevotella spp., and Eubacterium spp. F. necrophorum was found in 20 root canals and was associated with Peptostreptococcus prevotii. The simultaneous presence of F. nucleatum and F. necrophorum was not related to endodontic symptoms (p > 0.05). They were 100% susceptible to amoxicillin, amoxicillin/clavulanate, and cephaclor. Fusobacterium spp. is frequently isolated from primary-infected root canals of teeth with periapical pathologies. Amoxicillin is a useful antibiotic against F. nucleatum and F. necrophorum in endodontic infections and has been prescribed as the first choice in Brazil.
Asunto(s)
Enfermedades de la Pulpa Dental/microbiología , Infecciones por Fusobacterium/microbiología , Fusobacterium necrophorum/aislamiento & purificación , Fusobacterium nucleatum/aislamiento & purificación , Adolescente , Adulto , Amoxicilina/uso terapéutico , Combinación Amoxicilina-Clavulanato de Potasio/uso terapéutico , Anaerobiosis , Antibacterianos/uso terapéutico , Bifidobacterium/aislamiento & purificación , Brasil , Cefaclor/uso terapéutico , Niño , Fístula Dental/microbiología , Cavidad Pulpar/microbiología , Eubacterium/aislamiento & purificación , Fusobacterium necrophorum/efectos de los fármacos , Fusobacterium necrophorum/fisiología , Fusobacterium nucleatum/efectos de los fármacos , Fusobacterium nucleatum/fisiología , Humanos , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Penicilina G/uso terapéutico , Peptostreptococcus/aislamiento & purificación , Absceso Periapical/microbiología , Porphyromonas gingivalis/aislamiento & purificación , Prevotella/aislamiento & purificación , Staphylococcaceae/aislamiento & purificación , Adulto JovenRESUMEN
AIM: The purpose of this study was to investigate the effect of oxidative stress on physiological and genetic characteristics of Fusobacterium nucleatum and its interference on this microbial identification methods. METHODS AND RESULTS: Fus. nucleatum ssp. nucleatum ATCC 25586 (wt-strain) and an oxidative-stress-adapted strain derived from the wt-strain (aero-strain) were employed in the study. Cell-free crude protein extracts were obtained from both strains and differentially expressed proteins were identified by two-dimensional electrophoresis. Bacterium identification was performed by conventional biochemical tests, automated Rapid ID 32A system and specific PCR analysis. Genetic diversity between wt- and aero-strain was assessed by arbitrarily-primed (AP)-PCR. There were significant changes in the protein profile of aero-strain. The identification of the wt-strain was confirmed by all methods employed. Similar results were obtained for aero-strain when conventional biochemical tests and PCR were used. However, aero-strain was identified as Fusobacterium varium when submitted to Rapid ID 32A system. According to AP-PCR analysis, no significant genetic alteration was detected in aero-strain. CONCLUSIONS: The adaptive response of Fus. nucleatum to oxidative stress is associated with changes on its biology, which may lead to misidentification of the organism, according to the conventional identification methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Oxidative stress may act as a cause of adaptive response in Fus. nucleatum with consequences to its biology, such as alterations on biochemical and physiological profile.
Asunto(s)
Fusobacterium nucleatum/fisiología , Estrés Oxidativo/fisiología , Adaptación Fisiológica , Proteínas Bacterianas/metabolismo , Técnicas de Tipificación Bacteriana/métodos , Electroforesis en Gel Bidimensional/métodos , Fusobacterium nucleatum/clasificación , Fusobacterium nucleatum/genética , Variación Genética , Reacción en Cadena de la Polimerasa/métodosRESUMEN
AIM: To test the effect of two commercial brands of grey mineral trioxide aggregate (ProRoot and MTA-Angelus) on cytokine production by M1 and M2 inflammatory macrophages. METHODOLOGY: M1 (from C57BL/6 mice) and M2 peritoneal inflammatory macrophages (from C57BL/6 IL12p40-/- mice) were obtained and cultured in vitro in the presence of MTA. The cellular viability and the production of tumour necrosis factor-alpha, interleukin (IL)-12 and IL-10 in response to stimulation with interferon-gamma and Fusobacterium nucleatum or Peptostreptococcus anaerobius were evaluated. Data were analysed by Mann-Whitney, Kruskal-Wallis and anova tests. RESULTS: The cements did not interfere with cellular viability or with cytokine production by either type of macrophage. However, M2 macrophages produced higher levels of IL-10 when stimulated with F. nucleatum than M1 macrophages (P < 0.05). CONCLUSIONS: The brands of MTA evaluated did not interfere in the cytokine response by M1 or M2 macrophages to the two bacteria tested. However, a difference in cytokine production between the two types of macrophages was found.
Asunto(s)
Compuestos de Aluminio/farmacología , Compuestos de Calcio/farmacología , Interleucina-10/análisis , Interleucina-12/análisis , Macrófagos Peritoneales/efectos de los fármacos , Óxidos/farmacología , Materiales de Obturación del Conducto Radicular/farmacología , Silicatos/farmacología , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Combinación de Medicamentos , Femenino , Fusobacterium nucleatum/fisiología , Interferón gamma/farmacología , Subunidad p40 de la Interleucina-12 , Macrófagos Peritoneales/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Peptostreptococcus/fisiología , Subunidades de Proteína/efectos de los fármacosRESUMEN
The aim of this study was to determine whether microorganisms recovered from infected human root canals were able to survive and translocate to a local lymph node when experimentally inoculated into the root canal system of germ-free mice. The microorganisms isolated from two patients with pulpal necrosis were inoculated in two groups of experimental animals; group I (Gemella morbillorum) and group II (Bifidobacterium adolescentis, Fusobacterium nucleatum, and Clostridium butyricum). G. morbillorum showed the highest frequency of colonization and translocation to the draining lymph node. In group II only F. nucleatum and C. butyricum colonized and translocated when inoculated in tri-association. When the bacteria from group II were inoculated in monoinfection all three species colonized the root canal of germ-free mice and translocated to the draining lymph node, but with different frequencies. We conclude that selective mechanisms occur in which some bacterial species are fit to survive, multiply, and translocate in the germ-free mouse model.
Asunto(s)
Bacterias Anaerobias/crecimiento & desarrollo , Bacterias Anaerobias/fisiología , Traslocación Bacteriana , Cavidad Pulpar/microbiología , Necrosis de la Pulpa Dental/microbiología , Ganglios Linfáticos/microbiología , Animales , Bacteriocinas/análisis , Bifidobacterium/crecimiento & desarrollo , Bifidobacterium/fisiología , Clostridium/crecimiento & desarrollo , Clostridium/fisiología , Modelos Animales de Enfermedad , Fusobacterium nucleatum/crecimiento & desarrollo , Fusobacterium nucleatum/fisiología , Vida Libre de Gérmenes , Cocos Grampositivos/crecimiento & desarrollo , Cocos Grampositivos/fisiología , Humanos , RatonesRESUMEN
Haemagglutination and haemolytic activity of 80 Fusobacterium nucleatum isolates from human and animal origin, on different human blood types was evaluated. All the isolates were able to agglutinate erythrocytes and the most were either alpha-haemolytic or beta-haemolytic. No specificity between haemolysin or haemagglutinin and blood type was observed. Haemagglutination activity was inhibited when D-galactose, D-lactose or D-raffinose were used. Haemagglutination and haemolysis may be important factors in the pathogenesis of human and animal periodontal diseases.