RESUMO
Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum are strongly associated with periodontitis, and their evaluations are relevant to understand their role in the etiology and progression of periodontal diseases. In this study, the qualitative and quantitative detection of A. actinomycetemcomitans and F. nucleatum, as well as their genetic diversity, were evaluated in individuals with gingivitis, chronic periodontitis and periodontally healthy. In addition, the biotyping, serotyping, and prevalence of the ltx and cdt genes in A. actinomycetemcomitans were also determined. Subgingival biofilms obtained from gingivitis (70), periodontitis (75) and healthy (95) individuals were analyzed by cultures and PCR. Bacterial typing and presence of ltx and cdt genes in A. actinomycetemcomitans were also verified. DNA from A. actinomycetemcomitans and F. nucleatum was detected respectively, in 65.7% and 57.1% of gingivitis, 80% and 68% of periodontitis, and 57.8% and 37.8% of healthy. A. actinomycetemcomitans from gingivitis were biotypes I, II, IV, V, and X, and serotypes a, c, and e. In periodontitis, biotypes II, VI, and X, and serotypes a, b, and c were found. In healthy subjects, biotypes II and X, and serotypes b and c were found. The LTX and ltxA were observed in strains from gingivitis and periodontitis pockets. Subsequently, our data also showed no direct relationship between ltxA gene expression and leukotoxin gene 530-bp presence. On the other hand, cdt gene predominated during the inflammatory disease process. Our results strongly support a role of A. actinomycetemcomitans and F. nucleatum in advanced stage of periodontal disease.
Assuntos
Aggregatibacter actinomycetemcomitans/isolamento & purificação , Fusobacterium nucleatum/isolamento & purificação , Doenças Periodontais/microbiologia , Adulto , Aggregatibacter actinomycetemcomitans/classificação , Aggregatibacter actinomycetemcomitans/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Estudos Transversais , Exotoxinas/genética , Exotoxinas/metabolismo , Feminino , Fusobacterium nucleatum/classificação , Fusobacterium nucleatum/genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
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.
Assuntos
DNA Bacteriano/genética , Gengiva/microbiologia , Gengivite/microbiologia , Desnutrição/microbiologia , Microbiota/genética , Adolescente , Aggregatibacter actinomycetemcomitans/classificação , Aggregatibacter actinomycetemcomitans/genética , Aggregatibacter actinomycetemcomitans/isolamento & purificação , Argentina , Bacteroides/classificação , Bacteroides/genética , Bacteroides/isolamento & purificação , Campylobacter/classificação , Campylobacter/genética , Campylobacter/isolamento & purificação , Capnocytophaga/classificação , Capnocytophaga/genética , Capnocytophaga/isolamento & purificação , Estudos de Casos e Controles , Criança , Feminino , Fusobacterium nucleatum/classificação , Fusobacterium nucleatum/genética , Fusobacterium nucleatum/isolamento & purificação , Gengivite/fisiopatologia , Humanos , Masculino , Desnutrição/fisiopatologia , Hibridização de Ácido Nucleico , Peptostreptococcus/classificação , Peptostreptococcus/genética , Peptostreptococcus/isolamento & purificação , Porphyromonas gingivalis/classificação , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/isolamento & purificação , Saliva/microbiologiaRESUMO
BACKGROUND: The aim of the study was to assess the bacterial community structures associated with endodontic infections using terminal restriction fragment length polymorphism (T-RFLP), and to investigate the correlation of whole community profiles with the manifestation of particular clinical features. METHODS: Intraradicular samples were collected from 34 subjects and classified into three study groups based on the observed clinical symptoms: acute (n = 16), sub-acute (n = 8), and asymptomatic (n = 10). Genomic DNA was extracted from each sample, submitted to polymerase chain reaction using a fluorescently labeled 16S ribosomal DNA forward primer, and digested with two tetrameric endonucleases (HhaI and MspI). The terminal restriction fragments (T-RFs) were subsequently discriminated in an automated DNA sequencer, and the results were filtered using a statistics-based criterion. RESULTS: Totals of 138 (HhaI) and 145 (MspI) unique T-RFs were detected (means 13.1 and 11.9) and there was high inter-subject variability in the bacterial assemblages. Odds-ratio analysis unveiled the existence of higher order groups of positively associated T-RFs, restating the concept that intricate ecological relationships may take place in the root canal space. A significantly greater T-RF prevalence was detected in acute cases, suggesting a straight correlation between species richness and spontaneous pain. CONCLUSION: Overall, no T-RFLP profile representing a specific bacterial consortium could be associated with the manifestation of symptoms of endodontic origin.
Assuntos
Bactérias/classificação , Cavidade Pulpar/microbiologia , Necrose da Polpa Dentária/microbiologia , Polimorfismo de Fragmento de Restrição/genética , Actinomyces/classificação , Adolescente , Adulto , Bactérias/genética , Bacteroides/classificação , Campylobacter sputorum/classificação , Capnocytophaga/classificação , DNA Bacteriano/genética , Desoxirribonuclease HpaII , Desoxirribonucleases de Sítio Específico do Tipo II , Eubacterium/classificação , Feminino , Flavobacterium/classificação , Fusobacterium nucleatum/classificação , Humanos , Lactobacillus/classificação , Masculino , Pessoa de Meia-Idade , Peptostreptococcus/classificação , Doenças Periapicais/microbiologia , Prevotella/classificação , Selenomonas/classificação , Análise de Sequência de DNA , Veillonella/classificação , Adulto JovemRESUMO
BACKGROUND/AIM: The purpose of this study was to detect bacterial species and to quantify the total number of bacteria from samples of infected root canals before (S1) and after chemo-mechanical preparation using 2% chlorhexidine (CHX) gel as auxiliary chemical substance (S2) and after 7 days of intracanal dressing (S3) to compare microbial changes. METHOD: Twenty-four teeth were selected for this study. Chemo-mechanical preparation was performed using 2% CHX gel, then three different intracanal medicaments [M1: Ca(OH)(2) paste; M2: 2% CHX gel; and M3: Ca(OH)(2) paste plus 2% CHX gel] were used for 7 days. Checkerboard DNA-DNA hybridization was performed to detect 40 bacterial species. Aerobic and anaerobic culture techniques were used to determine the bacterial community by counting the colony-forming units (CFU). RESULTS: The species most frequently identified by checkerboard in S1 were: Fusobacterium nucleatum ssp. polymorphum, Treponema socranskii ssp. socranskii, Parvimonas micra and Enterococcus faecalis. In S2 and S3 a total of eight different species were identified; and only one of them was gram-positive (E. faecalis). Microorganisms were not identified after use of M2 for 7 days. The quantification obtained on agar plates ranged from 4 x 10(5) to 2.6 x 10(6) CFU/ml in S1, mean CFU was reduced by 99.96% in S2, and there was no statistical difference between the CFU in S2 and S3. CONCLUSION: The antibacterial effect of the mechanical preparation supplemented by the use of an antibacterial auxiliary substance greatly reduced the microorganisms in the main root canal.
Assuntos
Bactérias/classificação , Cavidade Pulpar/microbiologia , Necrose da Polpa Dentária/microbiologia , Adolescente , Adulto , Anti-Infecciosos Locais/administração & dosagem , Anti-Infecciosos Locais/uso terapêutico , Bactérias/genética , Hidróxido de Cálcio/administração & dosagem , Hidróxido de Cálcio/uso terapêutico , Campylobacter/classificação , Capnocytophaga/classificação , Clorexidina/administração & dosagem , Clorexidina/uso terapêutico , Contagem de Colônia Microbiana , DNA Bacteriano/análise , Necrose da Polpa Dentária/terapia , Combinação de Medicamentos , Enterococcus faecalis/classificação , Eubacterium/classificação , Fusobacterium nucleatum/classificação , Humanos , Pessoa de Meia-Idade , Hibridização de Ácido Nucleico , Periodontite Periapical/microbiologia , Periodontite Periapical/terapia , Irrigantes do Canal Radicular/administração & dosagem , Irrigantes do Canal Radicular/uso terapêutico , Preparo de Canal Radicular/métodos , Streptococcus/classificação , Treponema/classificaçãoRESUMO
BACKGROUND/AIMS: The aim of this study was to evaluate the composition of the microbiota of primary endodontic infections in 111 selected cases of single-rooted teeth with necrotic pulp. METHODS: Samples were collected from the root canals using #15 Hedströen-type files and two sterile paper points, which were introduced 1 mm short of the apical foramen. The presence, levels, and proportions of 40 different bacterial species in each sample were determined using DNA probes and checkerboard DNA-DNA hybridization techniques. RESULTS: The mean number of species per sample was 22. Enterococcus faecalis (89.3%), Campylobacter gracilis (89.3%), Leptotrichia buccalis (89.3%), Neisseria mucosa (87.5%), Prevotella melaninogenica (86.6%), Fusobacterium nucleatum ssp. vincentii (85.7%), Eubacterium saburreum (75.9%), Streptococcus anginosus (75%), and Veillonella parvula (74.1%) were the most prevalent species. The species found in highest mean counts (over 10(5)) were F. nucleatum ssp. vincentii (13.14 x 10(5)), E. saburreum (5.67 x 10(5)), E. faecalis (5.38 x 10(5)), N. mucosa (4.19 x 10(5)), V. parvula (3.63 x 10(5)), C. gracilis (3.46 x 10(5)), Treponema socranskii (3.34 x 10(5)), Porphyromonas endodontalis (2.96 x 10(5)), Porphyromonas gingivalis (2.85 x 10(5)), Micromonas micros (2.81 x 10(5)), Prevotella nigrescens (2.68 x 10(5)) and Fusobacterium nucleatum ssp. nucleatum (2.64 x 10(5)). Most of these species were also found in high proportions. CONCLUSIONS: Our results suggest that several bacterial species considered to be oral pathogens seem to be implicated in the etiology of primary endodontic infections.
Assuntos
DNA Bacteriano/análise , Necrose da Polpa Dentária/microbiologia , Infecções por Bactérias Gram-Negativas/diagnóstico , Infecções por Bactérias Gram-Positivas/diagnóstico , Hibridização de Ácido Nucleico/métodos , Adolescente , Adulto , Idoso , Campylobacter/classificação , Contagem de Colônia Microbiana , Sondas de DNA , Cavidade Pulpar/microbiologia , Enterococcus faecalis/classificação , Eubacterium/classificação , Feminino , Fusobacterium nucleatum/classificação , Humanos , Leptotrichia/classificação , Masculino , Pessoa de Meia-Idade , Neisseria mucosa/classificação , Peptostreptococcus/classificação , Porphyromonas endodontalis/classificação , Porphyromonas gingivalis/classificação , Prevotella melaninogenica/classificação , Prevotella nigrescens/classificação , Streptococcus anginosus/classificação , Treponema/classificação , Veillonella/classificaçãoRESUMO
BACKGROUND: This 6-month study evaluated the effects of resin-modified glass-ionomer cement (RMGI) and microfilled composite (MC) subgingival restorations on periodontal tissues and subgingival biofilm. METHODS: Fifty-four periodontally healthy patients were assigned as follows: group 1 (N = 18), root exposure (RE) without non-carious cervical lesions (NCCL) treated with coronally positioned flap (CPF); group 2 (N = 18), RE with NCCL treated RMGI restorations plus CPF; group 3 (N = 18), RE with NCCL treated with MC restorations plus CPF. Probing depth (PD), visible local plaque score (PL), and local bleeding on probing (BOP) were assessed at baseline and 6 months after surgeries. Restored and non-restored root recoverage (RR) was assessed at 6 months. Each experimental tooth was subgingivally sampled (baseline and 6 months) and analyzed by checkerboard DNA-DNA hybridization. RESULTS: Clinical results showed no significant differences among the groups regarding PL, BOP, and PD at baseline and 6 months. The RR means were similar among the groups at 6 months. Intragroup analyses revealed that the proportions of 10 periodontopathogens decreased at 6 months for the control group. For the RMGI group, there was a significant decrease in the proportions of nine periodontopathogens. For the MC group, there was a significant increase in the proportions of Fusobacterium nucleatum polymorphum and Gemella morbillorum and a decrease in five periodontopathogens. Intergroup analyses showed an increase in the proportion of F. nucleatum polymorphum for the MC group. CONCLUSIONS: In a 6-month evaluation, well-finished RMGI or MC subgingival restorations did not negatively affect periodontal health. Furthermore, RMGI seems to exert more positive effects on the subgingival biofilm composition than MC.
Assuntos
Biofilmes , Resinas Compostas/química , Restauração Dentária Permanente , Cimentos de Ionômeros de Vidro/química , Periodonto/patologia , Cimentos de Resina/química , Adulto , Idoso , Aggregatibacter actinomycetemcomitans/isolamento & purificação , Bactérias/classificação , Bactérias/isolamento & purificação , Placa Dentária/classificação , Feminino , Seguimentos , Fusobacterium nucleatum/classificação , Hemorragia Gengival/classificação , Humanos , Masculino , Pessoa de Meia-Idade , Bolsa Periodontal/classificação , Periodonto/microbiologia , Porphyromonas gingivalis/isolamento & purificação , Estudos Prospectivos , Staphylococcaceae/classificação , Retalhos Cirúrgicos , Doenças Dentárias/microbiologia , Doenças Dentárias/terapia , Raiz Dentária/microbiologia , Raiz Dentária/patologia , Treponema denticola/isolamento & purificaçãoRESUMO
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.
Assuntos
Fusobacterium nucleatum/fisiologia , Estresse Oxidativo/fisiologia , Adaptação Fisiológica , Proteínas de Bactérias/metabolismo , Técnicas de Tipagem Bacteriana/métodos , Eletroforese em Gel Bidimensional/métodos , Fusobacterium nucleatum/classificação , Fusobacterium nucleatum/genética , Variação Genética , Reação em Cadeia da Polimerase/métodosRESUMO
BACKGROUND: An association between race/ethnicity and the composition of the subgingival microbiota has been found in chronic periodontitis. A study was undertaken to determine the characteristics of the subgingival microbiota of chronic periodontitis in Chileans residing in Santiago. METHODS: Twenty-six subjects (mean age 45 +/- 7 years) with chronic periodontitis, mean probing depth (PD) 2.63 +/- 0.5 mm, mean attachment level (AL) 3.70 +/- 0.77 mm, and without a history of periodontal therapy were selected. Measurements of PD, AL, bleeding on probing, and plaque accumulation were recorded at six sites per tooth. Subgingival plaque samples were taken from the mesial aspect of every tooth and evaluated for the presence, levels, and proportions of 40 bacterial taxa using whole genomic DNA probes and checkerboard DNA-DNA hybridization. The microbial data of the Chileans were compared with data from 115 chronic periodontitis patients from Boston, Massachusetts. Since several clinical and demographic parameters differed between the two populations, significance of differences for each species was determined using analysis of covariance, adjusting for age, plaque level, mean PD, gender, and smoking status. RESULTS: Each of the individual test species was present in at least 25 of the 26 subjects, and 12 subjects (46.1%) harbored all 40 test species. With the exception of Prevotella intermedia, all test species colonized more than 75% of sites, and 25 species colonized > or = 90% of sites including the co-colonizing species of advanced periodontal lesions, termed the red complex, composed of the three species Porphyromonas gingivalis, Tannerella forsythensis (formerly Bacteroides forsythus), and Treponema denticola as well as Fusobacterium nucleatum subspecies, Campylobacter rectus, Peptostreptococcus micros, and Treponerma socranskii. Sixteen of the 40 species differed significantly between Chilean and U.S. subjects. Red, yellow, and other complexes were significantly higher in the Chileans, while the Actinomyces were higher in the U.S. subjects. CONCLUSIONS: The composition of the subgingival plaque differs among different subject populations. Thus, care should be taken when extrapolating the findings of one study to different ethnic groups.
Assuntos
Periodontite/microbiologia , Adulto , Fatores Etários , Idoso , Bacteroides/classificação , Boston , Campylobacter/classificação , Chile , Placa Dentária/microbiologia , Feminino , Fusobacterium nucleatum/classificação , Gengiva/microbiologia , Hemorragia Gengival/microbiologia , Humanos , Masculino , Pessoa de Meia-Idade , Peptostreptococcus/classificação , Perda da Inserção Periodontal/microbiologia , Bolsa Periodontal/microbiologia , Periodontite/etnologia , Porphyromonas gingivalis/classificação , Fatores Sexuais , Fumar , Treponema/classificaçãoRESUMO
OBJECTIVE: To define the subgingival microbial profiles of adult subjects from a previously identified rural community of indigenous Indians in Guatemala, Central America. MATERIALS AND METHODS: A full-mouth periodontal examination was performed in 114 adult subjects from 45 families. Plaque samples were collected from both deep and shallow periodontal pockets and checkerboard DNA-DNA hybridization was employed to identify 17 species previously associated with periodontitis or health. RESULTS: Plaque deposits and gingivitis were universal and widespread, and periodontal pocketing > or =5 mm was highly prevalent (84% of subjects). Streptococcus sanguis, Actinomyces naeslundii genospecies 2 and Fusobacterium nucleatum were significantly more prevalent in shallow sites. At the subject level, Actinomyces naeslundii and Peptostreptococcus micros were significantly more prevalent in periodontally-healthy subjects. Actinobacillus actinomycetemcomitans was not detected in any sample. CONCLUSION: There was no association between periodontal disease status and presence of suspected periodontal pathogens. These latter results conflict somewhat with those from treated populations. However, in this population where extensive plaque deposits and gingivitis are universal, the presence of putative pathogens may be more reflective of the local environment.
Assuntos
Bactérias/classificação , Etnicidade , Gengiva/microbiologia , Indígenas Centro-Americanos , Actinomyces/classificação , Adulto , Aggregatibacter actinomycetemcomitans/classificação , DNA Bacteriano/análise , DNA Bacteriano/genética , Placa Dentária/microbiologia , Índice de Placa Dentária , Feminino , Fusobacterium nucleatum/classificação , Gengivite/microbiologia , Guatemala , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Hibridização de Ácido Nucleico , Peptostreptococcus/classificação , Perda da Inserção Periodontal/microbiologia , Índice Periodontal , Bolsa Periodontal/microbiologia , Periodontite/microbiologia , População Rural , Estatística como Assunto , Streptococcus sanguis/classificaçãoRESUMO
BACKGROUND: Fusobacterium nucleatum is the most frequently isolated bacterium in periodontal disease and plays an important role in serious infections in other parts of the body. Arbitrarily primed-polymerase chain reaction (AP-PCR) was used to construct primers for specific identification and subtyping of F. nucleatum. Subtypes may differ in virulence and, hence, are important as periodontal pathogens. Subtypes also may differ in antibiotic susceptibility; therefore, knowing the subtypes may influence choice of treatment. METHODS: We analyzed 70 DNA samples of F. nucleatum isolated from patients with periodontal disease (PD) (N = 32) or AIDS-related PD (N = 8) and from healthy carriers (N = 30). From 90 AP-PCR primers screened, five amplification products were selected, cloned in pCR II vector, and sequenced. These sequences were used to design new pairs of specific primers. Sequences were compared to GenBank entries with BLAST and showed no significant matches. RESULTS: Three primer pairs produced bands of approximately 1 Kb (primer 5059S) or 0.5 Kb (primers FN5047 or M1211) with all F. nucleatum DNAs tested. PCR amplification using primer pair M8171 produced a 1 Kb band with isolates from 7 (22%) PD and 5 (63%) PD-AIDS patients and 9 (30%) healthy controls. Using the same primer pair, 2 other bands of approximately 0.5 Kb and 0.4 Kb were observed with DNA from isolates from 2 (6%) PD and all PD-AIDS patients, but were not observed with DNA samples from healthy controls (P<0.0001). All the primer pairs produced no or different amplicon profiles with DNA samples from bacterial species other than F. nucleatum. CONCLUSIONS: Our results suggest that PCR primer pairs 5059S, FN5047 or M1211 can be used to specifically identify F. nucleatum isolates and distinguish them from other bacteria. The primer pair M8171 could also be used to differentiate F. nucleatum isolated from periodontal patients or healthy individuals. These specific primers can be used in PCR analysis for specific identification of F. nucleatum and to distinguish it from other bacteria associated with human periodontitis. These approaches appear promising in facilitating laboratory identification, molecular subtyping, and taxonomy of putative periodontopathogens.