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1.
New Microbiol ; 41(1): 52-60, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-29505064

RESUMEN

An insertion sequence, IS1598 (IsPg4) has been found in virulent strains of Porphyromonas gingivalis in a murine abscess model. The present study was performed to investigate the effects of genetic rearrangements by IS1598 on the phenotypic characteristics of the virulent strains. For this purpose, we searched for a common insertion site of IS1598 among the virulent strains. Through cloning and database search, a common insertion site was identified beside an nrdD-like gene in the virulent FDC 381, W83 and W50 strains. In this region, predicted promoters of the nrdD-like gene and IS1598 are located in tandem, and accumulation of nrdD-like gene mRNA was 5-fold higher in virulent strains (W83, W50, FDC 381) than avirulent strains (ATCC33277, SU63, SUNY1021, ESO59 without IS1598). The role of the nrdD-like gene in virulence of P. gingivalis was investigated by constructing a nrdD-deficient mutant. In the murine abscess model, the parental W83 strain produced necrotic abscesses, while the nrdD-deficient mutant had almost lost this ability. Insertion of IS1598 into the nrdD-like gene promoter region may be related to the phenotypic differences in virulence among P. gingivalis strains through upregulation of the expression of this gene.


Asunto(s)
Proteínas Bacterianas/metabolismo , Porphyromonas gingivalis/clasificación , Porphyromonas gingivalis/patogenicidad , Ribonucleótido Reductasas/metabolismo , Regulación hacia Arriba/fisiología , Absceso/microbiología , Proteínas Bacterianas/genética , Regulación Enzimológica de la Expresión Génica , Genes Bacterianos , Genoma Bacteriano , Humanos , Mutagénesis Insercional , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribonucleótido Reductasas/genética , Virulencia
2.
Bull Exp Biol Med ; 163(6): 718-721, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-29063339

RESUMEN

Biofilm of the gingival sulcus from 22 patients with type 2 diabetes mellitus and periodontitis, 30 patients with periodontitis not complicated by diabetes mellitus (reference group), and 22 healthy volunteers without signs of gingival disease (control group) was studied by quantitative PCR. Quantitative analysis for the content of P. gingivalis, T. forsythia, A. ctinomycetemcomitans, T. denticola, P. intermedia, F. nucleatum/periodonticum, and P. endodontalis in the dental plaque was performed with a Dentoscreen kit. The presence of other bacterial groups was verified by metagenomic sequencing of the 16S rRNA gene to evaluate some specific features of the etiological factor for periodontitis in type 2 diabetes mellitus. Specimens of the Porphiromonadaceae and Fusobacteriaceae families were characterized by an extremely high incidence in combined pathology. The amount of Sphingobacteriaceae bacteria in the biofilm was shown to decrease significantly during periodontitis. Metagenomic analysis confirmed the pathogenic role of microbiota in combined pathology, as well as the hypothesis on a possible influence of periodontitis on the course and development of type 2 diabetes mellitus.


Asunto(s)
Biopelículas/crecimiento & desarrollo , Periodontitis Crónica/microbiología , Placa Dental/microbiología , Diabetes Mellitus Tipo 2/microbiología , Metagenoma , ARN Ribosómico 16S/genética , Adulto , Aggregatibacter actinomycetemcomitans/clasificación , Aggregatibacter actinomycetemcomitans/genética , Aggregatibacter actinomycetemcomitans/aislamiento & purificación , Estudios de Casos y Controles , Periodontitis Crónica/complicaciones , Periodontitis Crónica/patología , Placa Dental/complicaciones , Placa Dental/patología , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/patología , Femenino , Encía/microbiología , Humanos , Masculino , Persona de Mediana Edad , Porphyromonas gingivalis/clasificación , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/aislamiento & purificación , Treponema denticola/clasificación , Treponema denticola/genética , Treponema denticola/aislamiento & purificación
3.
Anaerobe ; 42: 55-59, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27555373

RESUMEN

The aim of this study was to evaluate the relationship among nutritional status, gingival health and the composition of oral microbiota in children of a public school from a very poor area of San Miguel de Tucuman. Forty-five children ranging in age from 6 to 14 years old, 13 males and 32 females were studied. Twenty of these children were undernourished (Lejarraga-Morasso Table) and twenty-five were eutrophic. A clinical study that included DMF and dmf indexes, Löe Silness Plaque Index and bleeding on probing was performed. For microbiological study, saliva samples without stimulation were taken; aliquots of them were immediately placed in TAE buffer pH 7.6, adding NaOH (N and keeping at -70 °C until processed by checkerboard DNA-DNA hybridization method to check the presence of 40 oral microorganism species. Positive bleeding on probing was present in more than 80% of children, without significant differences between eutrophic and undernourished groups. Same result were obtain for the other clinical indexes (p > 0.05, Two Way ANOVA). Significant differences were found for some oral microorganism species, with a higher percentage of undernourished children harboring them. That was the case of S. gordonii (p < 0.05), Capnocitophaga gingivalis and C. ochraceae (p < 0.01 and p < 0.10, respectively), F. nucleatum ss nucleatum (p < 0.05), P. nigrescens (p < 0.10), Campylobacter gracilis (p < 0,05), and T. denticola (p < 0.10, multiple logistic regression). Significant differences were also found between children groups for E. saborreum (p < 0.001), P. acnes (p < 0.10), G. morbillorum (p < 0.05) and L. buccalis (p < 0.10). Gingivitis and bleeding on probing would not be related to nutritional status in the groups of children studied. There were significant differences for the presence of some of the main periodontal pathogen species between eutrophic and undernourished children. It would be important to study the meaning of significant differences found for the other microorganisms more deeply.


Asunto(s)
ADN Bacteriano/genética , Encía/microbiología , Gingivitis/microbiología , Desnutrición/microbiología , Microbiota/genética , Adolescente , Aggregatibacter actinomycetemcomitans/clasificación , Aggregatibacter actinomycetemcomitans/genética , Aggregatibacter actinomycetemcomitans/aislamiento & purificación , Argentina , Bacteroides/clasificación , Bacteroides/genética , Bacteroides/aislamiento & purificación , Campylobacter/clasificación , Campylobacter/genética , Campylobacter/aislamiento & purificación , Capnocytophaga/clasificación , Capnocytophaga/genética , Capnocytophaga/aislamiento & purificación , Estudios de Casos y Controles , Niño , Femenino , Fusobacterium nucleatum/clasificación , Fusobacterium nucleatum/genética , Fusobacterium nucleatum/aislamiento & purificación , Gingivitis/fisiopatología , Humanos , Masculino , Desnutrición/fisiopatología , Hibridación de Ácido Nucleico , Peptostreptococcus/clasificación , Peptostreptococcus/genética , Peptostreptococcus/aislamiento & purificación , Porphyromonas gingivalis/clasificación , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/aislamiento & purificación , Saliva/microbiología
4.
Anaerobe ; 32: 49-50, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25454723

RESUMEN

Colony PCR of anaerobic black-pigmenting Bacteroidetes species Porphyromonas gingivalis and Prevotella intermedia was modified by addition of bovine serum albumin to reverse the inhibitory action of accumulated heme.


Asunto(s)
Bacteroidetes/clasificación , Bacteroidetes/genética , Reacción en Cadena de la Polimerasa , Bacteroidetes/metabolismo , Hemo/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Porphyromonas gingivalis/clasificación , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/metabolismo , Prevotella intermedia/clasificación , Prevotella intermedia/genética , Prevotella intermedia/metabolismo
5.
Klin Lab Diagn ; 60(12): 56-9, 2015 Dec.
Artículo en Ruso | MEDLINE | ID: mdl-27032255

RESUMEN

The examination was carried out of samplings of 110 patients with periodontitis (observation group) and 60 patients without pathology of periodont (comparison group). The polymerase chain reaction was used to analyze samples of saliva and contents of periodontal recesses for detecting species-specific DNA fragments of Porphymmonas gigngivalis, Streptococcus macacae, S. mutans, S. oralis, S. salivarius, S. sangis, S. sobrinus, Treponema denticola. In patients with periodontitis S. mutans, S. oralis S. sobrinus were reliably more often detected in the content of periodontal recesses and S. mutans, S. sobrinus i in saliva. In the observation group the rate of detection of association S. mutans--S. oralis--S. sangis--S. sobrinus was significantly exceeded (up to 15.6%, X2 = 9.1, p = 0.004). In ten days of effective treatment of periodontitis reliable decreastng of rate of detection of S. wasoralis, S. sobrinus was observed in contents of periodontal recesses but not in of saliva. The detection of S.sobrinus using technique of polymerase chain reaction in contents of periodontal recesses and/or saliva of patients with periodontitis has a diagnostic value. The detection of S.sobrinus in contents of periodontal recesses is significant both in monoculture and in association S. mutans--S. oralis--S. sangis--S.sobrinus. The absence of S. sobrinus in contents of periodontal recesses testifies effectiveness of treatment of main disease (periodontitis).


Asunto(s)
ADN Bacteriano/genética , Bolsa Periodontal/diagnóstico , Periodontitis/diagnóstico , Porphyromonas gingivalis/genética , Streptococcus/genética , Treponema denticola/genética , Adolescente , Adulto , Anciano , Anestésicos Locales , Antibacterianos/uso terapéutico , Estudios de Casos y Controles , Femenino , Encía/microbiología , Humanos , Lidocaína , Lincomicina/uso terapéutico , Masculino , Persona de Mediana Edad , Bolsa Periodontal/tratamiento farmacológico , Bolsa Periodontal/microbiología , Bolsa Periodontal/patología , Periodontitis/tratamiento farmacológico , Periodontitis/microbiología , Periodontitis/patología , Reacción en Cadena de la Polimerasa/métodos , Porphyromonas gingivalis/clasificación , Porphyromonas gingivalis/aislamiento & purificación , Saliva/microbiología , Streptococcus/clasificación , Streptococcus/aislamiento & purificación , Treponema denticola/clasificación , Treponema denticola/aislamiento & purificación
6.
Microb Pathog ; 77: 100-4, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25448131

RESUMEN

The periodontopathogen Porphyromonas gingivalis is represented by a spectrum of phenotypes ranging from commensals to pathogenic lineages. Capsule and fimbriae are considered key virulence factors in this specie, involved in colonization and host defenses evasion. Since these virulence traits may not be expressed by certain strains, we aimed to test the hypothesis that certain clusters or genotypes of P. gingivalis correlate with the production of capsule and fimbriae. Sixteen P. gingivalis isolates were evaluated. Capsule (K) was detected by optical microscopy of negatively stained cells. The presence of fimbriae (F) was determined by TEM. Genotypes were determined by NotI macrorestriction fragments analysis through Pulsed-Field Gel Electrophoresis (PFGE) and Multi-locus sequence typing (MLST) based on seven house-keeping genes. The phenotypes included F(+)K(+) (n = 4), F(-)K(+) (n = 5), F(+)K(-) (n = 5) and F(-)K(-) (n = 2). The analysis of whole genome macrorestriction fragments revealed 14 different clusters. MLST data also revealed extensive genetic diversity; however, PFGE and MLST profiles showed evident differences. There was no association between P. gingivalis clusters and encapsulated and/or fimbriated phenotypes. Genotyping methods were not able to discriminate isolates according to the production of virulence factors such as capsule and major fimbriae, indicating that recombination played a key role in the expression of capsule and fimbriae in P. gingivalis.


Asunto(s)
Cápsulas Bacterianas , Fimbrias Bacterianas/ultraestructura , Variación Genética , Porphyromonas gingivalis/genética , Propiedades de Superficie , ADN Bacteriano/química , ADN Bacteriano/genética , Electroforesis en Gel de Campo Pulsado , Genotipo , Microscopía , Tipificación de Secuencias Multilocus , Fenotipo , Porphyromonas gingivalis/química , Porphyromonas gingivalis/clasificación , Porphyromonas gingivalis/ultraestructura
7.
J Clin Periodontol ; 41(1): 19-30, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24117627

RESUMEN

AIM: Porphyromonas gingivalis can synthesize an extracellular capsule and different serotypes have been described based on capsular antigenicity. On dendritic cells (DCs), the type of capsule present plays a role on the strength of the developed immune response. This study aimed to investigate the T-lymphocyte responses when stimulated with autologous mature DCs exposed to different P. gingivalis K-serotypes. MATERIALS AND METHODS: Naïve CD4(+) T-lymphocytes were obtained from healthy subjects and stimulated with autologous DCs primed with increasing multiplicity of infections of the different P. gingivalis K-serotypes. The Th1, Th2, Th17 and T-regulatory cytokines and transcription factor levels were quantified. RESULTS: Distinct types of response were detected when T-lymphocytes were stimulated by DCs primed with the different P. gingivalis K-serotypes. T-lymphocytes stimulated by K1 or K2-primed DCs elicited higher levels of Th1 and Th17-associated cytokines, T-bet and RORC2 than T-lymphocytes stimulated with DCs primed with the other serotypes. Conversely, the serotypes K3-K5 induced higher levels of Th2-associated cytokines and GATA-3 than the others. CONCLUSIONS: These results demonstrate that DCs primed with the different P. gingivalis K-serotypes elicited distinct T-cell responses. Strains K1 (W83) and K2 (HG184) induced a Th1/Th17 pattern of immune response and K3 (A7A1-28), K4 (ATCC(®49417™) ), and K5 (HG1690) a Th2 response.


Asunto(s)
Cápsulas Bacterianas/inmunología , Células Dendríticas/microbiología , Porphyromonas gingivalis/inmunología , Linfocitos T/inmunología , Técnicas Bacteriológicas , Citocinas/análisis , Células Dendríticas/inmunología , Factores de Transcripción Forkhead/análisis , Factor de Transcripción GATA3/análisis , Humanos , Interferón gamma/análisis , Subunidad alfa del Receptor de Interleucina-2/análisis , Interleucinas/análisis , Activación de Linfocitos/inmunología , Monocitos/inmunología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/análisis , Porphyromonas gingivalis/clasificación , Serotipificación , Proteínas de Dominio T Box/análisis , Linfocitos T/microbiología , Linfocitos T Reguladores/inmunología , Células TH1/inmunología , Células Th17/inmunología , Células Th2/inmunología , Factor de Crecimiento Transformador beta1/análisis , Factores de Necrosis Tumoral/análisis
8.
J Clin Periodontol ; 41(10): 943-51, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25070874

RESUMEN

AIMS: To evaluate the association among serum immunoglobulin G (IgG) responses to Aggregatibacter actinomycetemcomitans (Aa) serotypes a, b and c, Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf) and clinical parameters in Aggressive Periodontitis (AP) subjects. Associations between periodontal pathogens and clinical and immunological parameters were also evaluated. METHODS: Thirty-eight subjects diagnosed with generalized AP (GAP) and localized AP (LAP) were included. Ten healthy controls were also evaluated. Clinical parameters were assessed and percentages of subgingival levels of Aa, Pg and Tf (beyond bacterial load), were determined by quantitative real-time polymerase chain reaction. Serum IgG antibody levels against Aa, Pg and Tf were evaluated by enzyme-linked immunosorbent assay. RESULTS: Percentages of Aa, Pg and Tf were significantly higher in AP than in controls. The response to Aa serotype c was higher in LAP subjects than in controls. There were no differences in microbial composition or antibodies responses between GAP and LAP, except for IgG response to Tf. Pg levels were correlated with probing depth (PD), BoP and CAL in GAP but not in LAP subjects. Tf levels correlated with PD and CAL in GAP subjects. In GAP, the infection levels of Aa and Pg correlated with the corresponding IgG levels to Aa serotype c and Pg. CONCLUSION: Given the evidences that IgG response in AP patients correlated with bacterial infection level in GAP, but not in LAP, and that LAP patients lack a response to Tf, despite harbouring this species, our data suggest a difference in host immune defence between these two forms of aggressive periodontitis.


Asunto(s)
Periodontitis Agresiva/microbiología , Anticuerpos Antibacterianos/sangre , Bacterias Gramnegativas/inmunología , Inmunoglobulina G/sangre , Adulto , Aggregatibacter actinomycetemcomitans/clasificación , Aggregatibacter actinomycetemcomitans/inmunología , Periodontitis Agresiva/clasificación , Periodontitis Agresiva/inmunología , Pérdida de Hueso Alveolar/diagnóstico por imagen , Pérdida de Hueso Alveolar/microbiología , Carga Bacteriana , Bacteroides/clasificación , Bacteroides/inmunología , Estudios Transversales , Femenino , Interacciones Huésped-Patógeno/inmunología , Humanos , Masculino , Pérdida de la Inserción Periodontal/inmunología , Pérdida de la Inserción Periodontal/microbiología , Bolsa Periodontal/inmunología , Bolsa Periodontal/microbiología , Porphyromonas gingivalis/clasificación , Porphyromonas gingivalis/inmunología , Radiografía , Serogrupo , Adulto Joven
9.
J Clin Periodontol ; 41(5): 451-9, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24476556

RESUMEN

AIM: Destructive periodontitis is associated with a Th1-Th17 immune response and activation of RANKL-induced osteoclasts. In addition, Porphyromonas gingivalis K1 and K2 serotypes induce a strong Th1-Th17 response. This study aimed to investigate whether these P. gingivalis serotypes induce higher osteoclasts activation, by increased Th17-associated RANKL production, and an antigen-specific memory T-lymphocyte response. MATERIAL AND METHODS: The RANKL production and TRAP(+) osteoclast induction were quantified on naïve T lymphocytes stimulated with dendritic cells primed with the P. gingivalis serotypes. The T-bet, GATA-3, RORC2 and Foxp3 expression was correlated with RANKL production. The frequency of proliferating memory T lymphocytes in response to P. gingivalis serotypes was determined in both periodontitis and healthy subjects. RESULTS: T lymphocytes stimulated by K1 or K2-primed dendritic cells elicited higher levels of RANKL and TRAP(+) osteoclasts than cells stimulated with the other serotypes. RANKL positively correlated with RORC2. Whereas periodontitis patients had a higher frequency of memory T lymphocytes responding to K1 or K2, healthy subjects had a higher frequency of memory T lymphocytes responding to K4 or K(-) . CONCLUSIONS: P. gingivalis serotypes K1 and K2, but not others, are associated with an increased production of the osteoclastogenesis-related factor RANKL. This important information suggests that these serotypes could elicit a greater bone resorption in vivo and have a role in the periodontitis pathogenesis.


Asunto(s)
Memoria Inmunológica/inmunología , Osteoclastos/inmunología , Porphyromonas gingivalis/inmunología , Ligando RANK/inmunología , Serogrupo , Linfocitos T/inmunología , Fosfatasa Ácida/análisis , Fosfatasa Ácida/inmunología , Animales , Antígenos Bacterianos/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/microbiología , Diferenciación Celular/inmunología , Línea Celular , Periodontitis Crónica/inmunología , Selección Clonal Mediada por Antígenos , Células Dendríticas/inmunología , Factores de Transcripción Forkhead/análisis , Factor de Transcripción GATA3/análisis , Humanos , Isoenzimas/análisis , Isoenzimas/inmunología , Macrófagos/inmunología , Ratones , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/análisis , Osteoclastos/efectos de los fármacos , Porphyromonas gingivalis/clasificación , Ligando RANK/análisis , Proteínas de Dominio T Box/análisis , Linfocitos T/microbiología , Fosfatasa Ácida Tartratorresistente , Células TH1/inmunología , Células Th17/inmunología
10.
J Periodontal Res ; 48(2): 213-20, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22957816

RESUMEN

BACKGROUND AND OBJECTIVES: One of the major disadvantages of DNA-based microbial diagnostics is their inability to differentiate DNA between viable and dead microorganisms, which could be important when studying etiologically relevant pathogens. The aim of this investigation was to optimize a method for the selective detection and quantification of only viable Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis cells by combining quantitative real-time polymerase chain reaction (qPCR) and propidium monoazide (PMA). MATERIAL AND METHODS: Three different concentrations of PMA (10, 50 or 100 µm) were added to suspensions of 10(6) (CFU)/mL of viable/dead A. actinomycetemcomitans and P. gingivalis cells. After DNA isolation, qPCR was carried out using specific primers and probes for the tested bacteria. PMA was further tested with different mixtures containing varying ratios of viable and dead cells. The efficacy of PMA to detect viable/dead cells was tested by analysis of variance. RESULTS: For these specific bacterial pathogens, 100 µm PMA resulted in a significant reduction of qPCR amplification with dead cells (10(6) CFU/mL), while with viable cells no significant inhibition was detected. PMA was also effective in detecting selectively viable cells by qPCR detection, when mixtures of varying ratios of viable and dead bacteria were used. CONCLUSIONS: This study demonstrated the efficiency of PMA for differentiating viable and dead A. actinomycetemcomitans and P. gingivalis cells. This method of PMA-qPCR may be useful for monitoring new antimicrobial strategies and for assessing the pathogenic potential of A. actinomycetemcomitans and P. gingivalis in different oral conditions when using molecular diagnostic methods.


Asunto(s)
Aggregatibacter actinomycetemcomitans/clasificación , Azidas , Colorantes , Viabilidad Microbiana , Porphyromonas gingivalis/clasificación , Propidio/análogos & derivados , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , 2-Propanol/farmacología , Aggregatibacter actinomycetemcomitans/efectos de los fármacos , Antiinfecciosos Locales/farmacología , Carga Bacteriana , Técnicas Bacteriológicas , Cartilla de ADN , Sondas de ADN , ADN Bacteriano/análisis , Humanos , Viabilidad Microbiana/efectos de los fármacos , Porphyromonas gingivalis/efectos de los fármacos
11.
J Clin Periodontol ; 40(3): 218-26, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23294017

RESUMEN

AIM: The microbial differences between peri-implantitis and periodontitis in the same subjects were examined using 16S rRNA gene clone library analysis and real-time polymerase chain reaction. MATERIALS AND METHODS: Subgingival plaque samples were taken from the deepest pockets of peri-implantitis and periodontitis sites in six subjects. The prevalence of bacteria was analysed using a 16S rRNA gene clone library and real-time polymerase chain reaction. RESULTS: A total of 333 different taxa were identified from 799 sequenced clones; 231 (69%) were uncultivated phylotypes, of which 75 were novel. The numbers of bacterial taxa identified at the sites of peri-implantitis and periodontitis were 192 and 148 respectively. The microbial composition of peri-implantitis was more diverse when compared with that of periodontitis. Fusobacterium spp. and Streptococcus spp. were predominant in both peri-implantitis and periodontitis, while bacteria such as Parvimonas micra were only detected in peri-implantitis. The prevalence of periodontopathic bacteria was not high, while quantitative evaluation revealed that, in most cases, prevalence was higher at peri-implantitis sites than at periodontitis sites. CONCLUSIONS: The biofilm in peri-implantitis showed a more complex microbial composition when compared with periodontitis. Common periodontopathic bacteria showed low prevalence, and several bacteria were identified as candidate pathogens in peri-implantitis.


Asunto(s)
Bacterias/clasificación , Periimplantitis/microbiología , Periodontitis/microbiología , Anciano , Pérdida de Hueso Alveolar/microbiología , Bacteroides/clasificación , Bacteroidetes/clasificación , Biopelículas , Placa Dental/microbiología , Femenino , Fusobacterium/clasificación , Biblioteca de Genes , Hemorragia Gingival/microbiología , Bacterias Grampositivas/clasificación , Humanos , Masculino , Persona de Mediana Edad , Peptostreptococcus/clasificación , Bolsa Periodontal/microbiología , Porphyromonas gingivalis/clasificación , Proteobacteria/clasificación , ARN Bacteriano/análisis , ARN Ribosómico 16S/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Streptococcus/clasificación , Treponema denticola/clasificación
12.
Microb Pathog ; 52(1): 17-24, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22001095

RESUMEN

Aminoacylhistidine dipeptidase (EC 3.4.13.3; also Xaa-His dipeptidase, carnosinase, or PepD) catalyzes the cleavage and release of an N-terminal amino acid, which is usually a neutral or hydrophobic residue, from an Xaa-His dipeptide or degraded peptide fragment. PepD enzyme is found extensively in prokaryotes and eukaryotes, and belongs to the metallopeptidase family M20, a part of the metallopeptidase H (MH) clan. Carnosine is a naturally occurring dipeptide (ß-alanyl-l-histidine) present in mammalian tissues that has protective functions in addition to anti-oxidant and free-radical scavenging roles. During bacterial infections, degradation of l-carnosine via carnosinase or PepD-like enzymes may enhance the destructive potential of bacteria, resulting in a pathological impact. This process has been proposed to act in an anti-oxidant manner in vivo. In the present study, the recombinant PepD protein encoded by Porphyromonas gingivalis TDC60 pepD was generated and biochemically characterized. In addition, a recombinant dipeptidase enzyme was found to function not only as an alanine-aminopeptidase, but also as a carnosinase. Furthermore, when carnosine was used as substrate for PepD, the transition metals, Mn(2+), Fe(2+), Co(2+), and Ni(2+) stimulated the hydrolyzing activity of rPepD with ß-alanine and l-histidine. Based on its metal ion specificity, we propose that this enzyme should not only be termed l-aminopeptidase, but also a carnosinase.


Asunto(s)
Proteínas Bacterianas/metabolismo , Carnosina/metabolismo , Dipeptidasas/metabolismo , Metales/metabolismo , Porphyromonas gingivalis/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Carnosina/química , Carnosina/genética , Dipeptidasas/química , Dipeptidasas/genética , Datos de Secuencia Molecular , Filogenia , Porphyromonas gingivalis/química , Porphyromonas gingivalis/clasificación , Porphyromonas gingivalis/genética , Alineación de Secuencia , Especificidad por Sustrato
13.
J Periodontal Res ; 47(3): 354-64, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22181039

RESUMEN

BACKGROUND AND OBJECTIVE: The dog has been used extensively for experimental and microbiological studies on periodontitis and peri-implantitis without detailed knowledge about the predominant flora of the subgingival plaque. This study was designed to evaluate the predominant cultivable bacterial species in dogs and compare them phenotypically and genotypically with corresponding human species. MATERIAL AND METHODS: Four subgingival samples were taken from two upper premolars in each of six Labrador retrievers. The samples from each dog were processed for anaerobic culture. From the samples of each dog, the five or six predominating bacteria based on colony morphology were selected and pure cultured. Each of the strains was characterized by Gram stain, anaerobic/aerobic growth and API-ZYM test. Eighteen strains showing clear-cut phenotypic differences were further classified based on DNA sequencing technology. Cross-reactions of DNA probes from human and dog strains were also tested against a panel of both human and dog bacterial species. RESULTS: Thirty-one strains in the dogs were isolated and characterized. They represented 21 different species, of which six belonged to the genus Porphyromonas. No species was found consistently in the predominant flora of all six dogs. Porphyromonas crevioricanis and Fusobacterium canifelinum were the two most prevalent species in predominant flora in dogs. DNA probes from human and dog species cross-reacted to some extent with related strains from humans and dogs; however, distinct exceptions were found. CONCLUSION: The predominant cultural subgingival flora in dogs shows great similarities with the subgingival bacteria from humans at the genus level, but distinct differences at the species level; however, a genetic relatedness could be disclosed for most strains investigated.


Asunto(s)
Bacterias/clasificación , Placa Dental/microbiología , Perros/microbiología , Animales , Bacterias/genética , Técnicas Bacteriológicas , Bacteroides/clasificación , Campylobacter/clasificación , Campylobacter rectus/clasificación , Sondas de ADN , ADN Bacteriano/análisis , Modelos Animales de Enfermedad , Fusobacterium/clasificación , Fusobacterium nucleatum/clasificación , Genotipo , Bolsa Gingival/microbiología , Gingivitis/microbiología , Humanos , Hibridación de Ácido Nucleico , Peptostreptococcus/clasificación , Fenotipo , Porphyromonas/clasificación , Porphyromonas endodontalis/clasificación , Porphyromonas gingivalis/clasificación , Prevotella intermedia/clasificación , Análisis de Secuencia de ADN , Treponema denticola/clasificación
14.
J Pharmacol Sci ; 113(2): 115-9, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20501967

RESUMEN

Accumulating evidence suggests the involvement of Porphyromonas gingivalis (P. gingivalis), a periodontal pathogen, in cardiovascular diseases. Clinical specimens of aneurysmal tissue and dental plaque collected from patients infected with or without P. gingivalis were analyzed. The number of aneurysms in the distal aorta in the P. gingivalis-infected group was significantly higher than that in the non-infected group. Cellular accumulation of adipocytes in aneurysms was less frequently identified in the infected group. The expression of embryonic myosin heavy chain isoform, a phenotypic marker for proliferative smooth muscle cells, was higher in the P. gingivalis-infected group than the non-infected group. Clinical and histopathological features of aortic aneurysms associated with P. gingivalis infection are different from those present in non-infected patients. The major characteristic of P. gingivalis infection associated with aneurysms is smooth muscle cell proliferation in the distal aorta.


Asunto(s)
Aneurisma de la Aorta/etiología , Infecciones por Bacteroidaceae , Periodontitis Crónica/complicaciones , Periodontitis Crónica/microbiología , Porphyromonas gingivalis/aislamiento & purificación , Aneurisma de la Aorta/microbiología , Aterosclerosis/etiología , Fimbrias Bacterianas/clasificación , Fimbrias Bacterianas/genética , Genotipo , Humanos , Hiperplasia , Miocitos del Músculo Liso/microbiología , Miocitos del Músculo Liso/patología , Cadenas Pesadas de Miosina , Porphyromonas gingivalis/clasificación , Porphyromonas gingivalis/genética
15.
J Clin Microbiol ; 47(10): 3350-2, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19675219

RESUMEN

A predominant kgp biovar colonized subgingival sites and buccal and tongue mucosa in 45 of 56 adults in an isolated community. The presence of biovars 381, W83, and W83v, but not HG66, correlated with the Porphyromonas gingivalis load at diseased sites. Biovars W83 and W83v poorly colonized tongue and buccal mucosa.


Asunto(s)
Adhesinas Bacterianas/metabolismo , Infecciones por Bacteroidaceae/microbiología , Cisteína Endopeptidasas/metabolismo , Variación Genética , Mucosa Bucal/microbiología , Porphyromonas gingivalis/clasificación , Porphyromonas gingivalis/enzimología , Adulto , Anciano , Recuento de Colonia Microbiana , Femenino , Cisteína-Endopeptidasas Gingipaínas , Humanos , Masculino , Persona de Mediana Edad , Porphyromonas gingivalis/aislamiento & purificación , Adulto Joven
16.
J Clin Microbiol ; 47(10): 3073-81, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19675220

RESUMEN

Porphyromonas gingivalis is implicated in the etiology of chronic periodontitis. Genotyping studies suggest that genetic variability exists among P. gingivalis strains; however, the extent of variability remains unclear and regions of variability remain largely unidentified. To assess P. gingivalis strain diversity, we previously used heteroduplex analysis of the ribosomal operon intergenic spacer region (ISR) to type strains in clinical samples and identified 22 heteroduplex types. Additionally, we used ISR sequence analysis to determine the relatedness of P. gingivalis strains to one another and demonstrated a link between ISR sequence phylogeny and the disease-associated phenotype of the strains. In the current study, heteroduplex analysis of the ISR was used to determine the worldwide genetic variability and distribution of P. gingivalis, and microarray-based comparative genomic hybridization (CGH) analysis was used to more comprehensively examine the variability of major heteroduplex type strains by using the entire genome. Heteroduplex analysis of clinical samples from geographically diverse populations identified 6 predominant geographically widespread heteroduplex types (prevalence, > or = 5%) and 14 rare heteroduplex types (prevalence, <2%) which are found in one or a few locations. CGH analysis of the genomes of seven clinically prevalent heteroduplex type strains identified 133 genes from strain W83 that were divergent in at least one of the other strains. The relatedness of the strains to one another determined on the basis of genome content (microarray) analysis was highly similar to their relatedness determined on the basis of ISR sequence analysis, and a striking correlation between the genome contents and disease-associated phenotypes of the strains was observed.


Asunto(s)
Variación Genética , Periodontitis/microbiología , Porphyromonas gingivalis/clasificación , Porphyromonas gingivalis/genética , Análisis por Conglomerados , Hibridación Genómica Comparativa , ADN Bacteriano/genética , ADN Espaciador Ribosómico/genética , Genotipo , Geografía , Análisis Heterodúplex , Humanos , Filogenia , Porphyromonas gingivalis/aislamiento & purificación
17.
Int J Med Microbiol ; 299(6): 427-38, 2009 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-19447073

RESUMEN

Scaling and root planing in conjunction with systemic administration of antibiotics is used for treatment of aggressive periodontitis. The study investigated the changes of the subgingival microbiota in a homogeneous cohort of 12 female Caucasian patients. Plaque samples were obtained from 4 defined deep lesions per patient at baseline and 2, 6, and 12 months after therapy (mechanical plaque removal, oral administration of amoxicillin and metronidazole). Amplification of the 16S rRNA gene, cloning, and sequencing were applied to identify microbial species. Porphyromonas gingivalis strains were typed by multilocus sequence typing. Despite of a favorable clinical outcome, 16S rRNA sequence analysis revealed only minor changes of the microbiota with a temporal reduction of P. gingivalis and of Treponema denticola-like phylotypes. In contrast to T. denticola, T. sokranskii-like phylotypes were not affected. In 4 patients with recurrent colonization by P. gingivalis, the bacterial clones were identical before and after therapy as evidenced by multilocus sequence typing suggesting clonal persistence or reinfection during the course of the study. In summary, despite a favorable clinical outcome, a transient effect on only few bacterial species was observed.


Asunto(s)
Amoxicilina/uso terapéutico , Antibacterianos/uso terapéutico , Bacterias/aislamiento & purificación , Biodiversidad , Raspado Dental , Metronidazol/uso terapéutico , Periodontitis/tratamiento farmacológico , Adulto , Bacterias/clasificación , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Femenino , Encía/microbiología , Humanos , Persona de Mediana Edad , Periodontitis/microbiología , Periodontitis/terapia , Filogenia , Porphyromonas gingivalis/clasificación , Porphyromonas gingivalis/aislamiento & purificación , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Treponema/clasificación , Treponema/aislamiento & purificación
18.
Oral Microbiol Immunol ; 24(5): 423-6, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19702958

RESUMEN

BACKGROUND AND OBJECTIVES: Porphyromonas gingivalis is frequently identified to type by evaluation of fimA polymorphisms and less often by pulsed-field gel electrophoresis (PFGE) because of the technical intricacies of PFGE. To compare these techniques, we genotyped P. gingivalis clinical isolates as to (i) their fimA type and (ii) their whole genome restriction profile (PFGE analysis). MATERIAL AND METHODS: Thirty-two P. gingivalis strains were isolated from 16 unrelated periodontitis patients. Two strains were isolated from each patient. Strains were subjected to a fimA-typing polymerase chain reaction (PCR) assay. Strains that could not be typed by PCR were submitted to sequencing of the entire fimA gene. The PFGE profiles of clinical strains were compared using bioinformatic analysis. RESULTS: Seven of the 32 isolates were not typeable by PCR and so their entire fimA gene was sequenced. The sequencing identified each strain as belonging to a single fimA type. In one case, sequencing of the fimA gene did not agree with the result obtained using fimA PCR typing. With the exception of one patient, each patient presented isolates bearing the same fimA type. However, in three patients, isolates with the same fimA type presented different PFGE pulsotypes. CONCLUSION: The P. gingivalis typing using fimA PCR has limitations in typeability and discriminatory power. A typing technique for P. gingivalis that is easy to perform but that presents adequate typeability and discriminatory power is needed if we want to better understand the epidemiology of periodontal disease.


Asunto(s)
Infecciones por Bacteroidaceae/microbiología , Proteínas Fimbrias/clasificación , Periodontitis/microbiología , Pili Sexual/clasificación , Porphyromonas gingivalis/clasificación , Técnicas de Tipificación Bacteriana , Células Clonales/clasificación , Electroforesis en Gel de Campo Pulsado , Proteínas Fimbrias/genética , Genoma Bacteriano/genética , Genotipo , Humanos , Filogenia , Pili Sexual/genética , Porphyromonas gingivalis/aislamiento & purificación
19.
J Clin Periodontol ; 36(11): 915-21, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19735468

RESUMEN

AIMS: To evaluate the effect of oral infection with three Porphyromonas gingivalis strains on alveolar bone loss (ABL) and its correlation with the mouse immune response. MATERIALS AND METHODS: Mice were orally infected with P. gingivalis strains 381, 33277 and 53977. After 42 days, maxillae were analysed for ABL using micro-computed tomography and the serum for anti-P.gingivalis IgG1 and IgG2a levels. The cytokine response to P. gingivalis was tested using the subcutaneous chamber model. RESULTS: The P. gingivalis 53977-infected group showed the highest ABL, which was significantly different from all other groups (p<0.001). In addition, the humoral response to P. gingivalis 53977 was significantly lower than the response to P. gingivalis 381 and 33277 (p< or =0.01). The IgG2a/IgG1 ratio was higher in the P. gingivalis 33277-infected group (1.6) compared with the P. gingivalis 381-infected group (0.51). Four days post-infection, interleukin (IL)-1beta levels remained significantly higher in the P. gingivalis 53977-infected group only (1198.2+/-260.0, p<0.05), while IL-4 levels remained significantly higher in the P. gingivalis 381-infected group (265.8+/-131.6, p<0.05). CONCLUSIONS: The high levels of ABL induced by P. gingivalis 53977 were inversely correlated with the humoral response to this bacterium. In addition, ABL was correlated with an elevated pro-inflammatory response.


Asunto(s)
Infecciones por Bacteroidaceae/inmunología , Periodontitis/microbiología , Porphyromonas gingivalis/inmunología , Pérdida de Hueso Alveolar/inmunología , Pérdida de Hueso Alveolar/microbiología , Animales , Anticuerpos Antibacterianos/sangre , Femenino , Inmunidad Humoral/inmunología , Inmunoglobulina G/sangre , Interleucina-1beta/análisis , Interleucina-4/análisis , Ratones , Ratones Endogámicos BALB C , Boca/microbiología , Periodontitis/inmunología , Porphyromonas gingivalis/clasificación , Microtomografía por Rayos X
20.
J Clin Periodontol ; 36(10): 823-9, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19682172

RESUMEN

AIM: Capsular polysaccharides play an important role in the virulence of Gram-positive and Gram-negative bacteria. In Porphyromonas gingivalis, six serotypes have been described based on capsular antigenicity and its pathogenicity has been correlated both in vitro and in animal models. This study aimed to investigate the differential response of human dendritic cells (DCs) when stimulated with different P. gingivalis capsular serotypes. MATERIALS AND METHODS: Using different multiplicity of infection (MOI) of the encapsulated strains K1-K6 and the non-encapsulated K(-) strain of P. gingivalis, the mRNA expression levels for interleukin (IL)-1beta, IL-2, IL-5, IL-6, IL-10, IL-12, IL-13, interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha, and TNF-beta in stimulated DCs were quantified by real-time reverse transcription-polymerase chain reaction. RESULTS: All P. gingivalis capsular serotypes induced a T-helper type 1 (Th1) pattern of cytokine expression. K1- and K2-stimulated DCs expressed higher levels of IL-1beta, IL-6, IL-12p35, IL-12p40, and IFN-gamma and at lower MOI than DCs stimulated with the other strains. CONCLUSIONS: These results demonstrate a differential potential of P. gingivalis capsular serotypes to induce DC responses and a higher capacity of strains K1 W83 and K2 HG184 than other K serotypes to trigger cytokine expression.


Asunto(s)
Cápsulas Bacterianas/inmunología , Citocinas/inmunología , Células Dendríticas/inmunología , Porphyromonas gingivalis/inmunología , Antígenos Bacterianos/inmunología , Cápsulas Bacterianas/clasificación , Células Cultivadas , Citocinas/análisis , Humanos , Interferón gamma/análisis , Interleucina-10/análisis , Interleucina-12/análisis , Subunidad p35 de la Interleucina-12/análisis , Subunidad p40 de la Interleucina-12/análisis , Interleucina-13/análisis , Interleucina-1beta/análisis , Interleucina-2/análisis , Interleucina-5/análisis , Interleucina-6/análisis , Linfotoxina-alfa/análisis , Polisacáridos Bacterianos/inmunología , Porphyromonas gingivalis/clasificación , Porphyromonas gingivalis/patogenicidad , Serotipificación , Células TH1/inmunología , Factor de Necrosis Tumoral alfa/análisis , Virulencia
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